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LA1512

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val_pubmed_ORC_4096_1592

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occupancy of a wide range of g - protein - coupled receptors and receptor tyrosine kinases triggers the hydrolysis of phosphatidylinositol 4,5-bisphosphate ( pip2 a ) , aabbreviations : pkc , protein kinase c ; dag , diacylglycerol ; pkd , protein kinase d ; rasgrp , ras guanyl releasing protein ; mrck , mytonic dystrophy kinase - related cdc42-binding kinase ; unc13 , uncoordination gene 13 product ; pip2 , phosphatidylinositol 4,5-bisphosphate . leading to the release of membrane - bound diacylglycerol ( dag ) . dag in turn interacts with the dag recognition domains , termed c1 domains , on multiple families of signaling proteins , including isoforms of protein kinase c , the chimerins , rasgrp , pkd , mrck , unc13 , and dag kinase . the activation of conventional ( cpkcs , i , ii , ) and novel ( npkcs , , , ) protein kinase c isoforms has been studied in great detail and involves both recruitment to membranes and allosteric activation by dag . specific molecular interactions of dag with the lipid environment into which it and the c1 domains insert influence both processes . the physical properties of the membranes and the interplay between the membranes and the alkyl chains of the dag are thus of great importance . the combination of these interactions controls the localization of an isozyme to specific intracellular compartments and correspondingly controls which substrates are accessible . abbreviations : pkc , protein kinase c ; dag , diacylglycerol ; pkd , protein kinase d ; rasgrp , ras guanyl releasing protein ; mrck , mytonic dystrophy kinase - related cdc42-binding kinase ; unc13 , uncoordination gene 13 product ; pip2 , phosphatidylinositol 4,5-bisphosphate . the effective use of a dag - lactone template as a more potent dag surrogate has been well documented in our laboratory . in a recent study , we extended this concept to include a series of dag - lactones containing rigid rods composed of ethynylene - substituted aromatic spacers [ oligo-(p - phenyleneethynylene ) ] as acyl groups . these compounds were designed to achieve various levels of membrane organization and penetration and were investigated for their ability to bind and translocate pkc and isoforms to plasma and internal membranes . some of the dag - lactones were able to induce a substantially prolonged translocation state relative to the parent dag - lactone containing an equivalent flexible acyl chain . more recently , we studied the thermodynamic features of some new dag - lactones with specific small groups attached to the terminus of a rigid 4-(phenylethynyl)benzoyl chain which affected both the self - assembly of the molecules and their interactions with phospholipids . the results suggested that these dag - lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases formed by cholesterol and that the size and charge of the phospholipid headgroups did not seem to affect the dag - lactone interactions with lipids . because different extents of membrane penetration and preference for membrane subdomains might yield different patterns of response in the ability of dag - lactones to interact or translocate different gfp - tagged isozymes , we designed a series of dag - lactones with a rigid 4-[(methylphenyl)ethynyl]phenyl rod ( red ) that is separated from the acylcarbonyl of the dag - lactone core [ 2-(hydroxymethyl)-4-(methylethylidene)-5-oxo-2 - 2,3-dihydrofuryl]methyloxocarbonyl ] ( blue ) by a o(ch2)n spacer of 1 , 2 , or 3 methylene units ( 1ac , figure 1 ) . the rigid rod moiety is indicated in red , the dag - lactone in blue , and the variable linker in black . we evaluated the compounds in both in vitro and in cellular systems : ( 1 ) we determined their binding affinities for a panel of classical and novel pkc isozymes , assayed in two different phospholipid environments , either 100% phosphatidylserine or a lipid mixture corresponding to that of the plasma membrane of cells ; ( 2 ) we compared the kinetics and site of translocation for the pkc isozymes and upon treatment with the compounds ; ( 3 ) we measured the potencies of the compounds for the early response of erk phosphorylation and for the late response of induction of apoptosis in the human prostatic carcinoma cell line lncap ; and ( 4 ) we evaluated the interaction of the compounds with a biomimetic lipid / polydiacetylene membrane by the associated chromatic response . compound 2 was synthesized according to our previously published approach . the preparation of racemic -(4-[2-(methylphenyl)ethynyl]phenoxy)alkanoyl monoesters of dag - lactones ( figure 1 ) with one to three methylene spacers between the lactone and the rigid chain was achieved as depicted in schemes 1 and 2 . the rigid - rod component , 4-[2-(4-methylphenyl)ethynyl]phenol ( 3 ) , was prepared in excellent yield by a modified sonogashira coupling between 4-iodophenol and 4-ethynyltoluene . this phenol derivative was used as a building block for the preparation of all tert - butyl esters shown in scheme 1 . thus , 4a ( n = 1 ) was obtained straightforwardly by treatment of 3 and tert - butyl 2-bromoacetate with cesium carbonate in excellent yield ( 92% ) . unfortunately , because of -elimination , this was not the case when tert - butyl 3-bromopropanoate was used . although the successful alkylation of phenols with -propiolactone as the alkylating agent has been reported , our reaction of 3 with -propiolactone in the presence of t - buok or csco3 led to mixtures of products . on the other hand , michael addition of 3 to tert - butyl acrylate in the presence of catalytic amounts of sodium and hydroquinone gave the desired tert - butyl ester 4b ( n = 2 ) in sufficient quantities to continue the synthesis . despite the fact that several byproducts were obtained , including starting material 3 , the self - addition product of phenol to the triple bond , and cross - reaction between the starting phenol and desired product 4b , successful separation of 4b was achieved by simple column chromatography . the synthesis of the 3-methylene unit derivative was first attempted by reaction between 3 and -butyrolactone , but again , this method failed when applied to our system . starting from 4-bromobutanoic acid , the corresponding tert - butyl 4-bromobutanoate was obtained in quantitative yield , and following nucleophilic displacement with 3 resulted in the desired 3-methylene unit , tert - butyl ester 4c , in 83% yield ( scheme 1 ) . at this point , hydrolysis of tert - butyl esters was necessary to perform the acylation of the dag - lactone moiety . probably because of the presence of the triple bond , acidic hydrolysis of tert - butyl ester produced complex mixtures , even when triethylsilane was added as a carbocation scavenger . however , thermolysis in quinoline at 205 c led cleanly to the desired free acids in quantitative yield which were isolated as crystalline solids ( 5ac , scheme 2 ) . the use of bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride ( bopcl ) and dimethylaminopyridine ( dmap ) allowed the one - pot coupling of the acids with the free hydroxyl group of the monoprotected o - pmp - dag - lactone 6(6 ) ( for n = 1 and 3 ) and o - benzyl - dag - lactone 7(6 ) ( for n = 2 ) in very good yield . removal of the p - methoxyphenyl ( pmp ) protecting group in the presence of ceric ammonium nitrate ( can ) to give the target compounds 1a and 1c resulted in low yields ( 54% and 50% ) due to the presence of the additional phenoxy ether linkage in 8a and 8c . a better strategy was the coupling of the o - benzyl protected dag - lactone 7 that led to the desired dag - lactone 1b in a markedly better yield ( 80% ) after treatment with boron trichloride at low temperature ( scheme 2 ) . enzymeligand interactions were assessed in vitro in terms of the ability of the ligand to displace bound [ 20-h]phorbol 12,13-dibutyrate ( pdbu ) from the recombinant human pkc isozymes in the presence of phosphatidylserine . the partition coefficients ( log p ) were calculated according to the atom - based program moe slogp ( table 1 ) . each of the pkc isozymes showed a similar pattern of dependence on the length of the spacer for compounds 1ac ( table 1 ) . in each case , the affinities increased ( lower ki ) with the distance of the rigid rod from the dag - lactone core , with compound 1c ( n = 3 ) being the most potent . in sharp contrast , however , the most potent of all ligands ( 2 ) lacks a spacer between these two units , suggesting a qualitatively different interaction between it , the enzymes , and the lipid environment . in terms of isozyme specificity , pkc stood out from the other isoforms in showing a 5- to 4-fold lower affinity for compounds 1a and 1b with the shorter spacers . the above assays were carried out under standard conditions in which the pkc isoforms were assayed in the presence of 100 g / ml phosphatidylserine , which provides a highly anionic surface . these conditions maximize the interaction of the pkc isoforms with the phospholipid bilayer . to detect differences in interactions that might depend on a more physiological lipid composition , we also assayed the isoforms in the presence of 100 g / ml of a mixture of phosphatidylcholine / phosphatidylethanolamine / phosphatidylserine / phosphatidylinositol / cholesterol ( 12:35:22:9:21 ) , a plasma membrane lipid ( pml ) mixture designed to mimic that of the inner leaflet of the plasma membrane . we observed ( table 2 , figure 2 ) that the rigid rod compounds were generally several - fold less potent for the classic pkc isoforms in the plasma membrane mimetic membranes as compared to the 100 g / ml phosphatidylserine , whereas for the novel pkc isoforms they were equal or more potent . effect of the lipid environment on the potencies of the rigid rod compounds for pkc isoforms . compounds were assayed with the various pkc isoforms in the presence of either phosphatidylserine ( ps ) or a lipid mixture corresponding to the composition of the inner leaflet of the plasma membrane , as described in the methods section . values represent the ratio of the ki value with the plasma membrane lipid ( pml ) mimetic mixture to that with phosphatidylserine , where the ki values are the average of three independent experiments . activation of the erk pathway , as reflected by erk phosphorylation , is a prominent early biological response to pkc stimulation by dag - lactones and phorbol ester . because of the rapidity of the response , phosphorylation of erk is relatively independent of issues of compound stability , so it provides a convenient measure of overall biological potency . all of the compounds caused phosphorylation of erk in the concentration range of 110 m ( figure 3 ) . similar to the potencies in the binding assays , increasing potency was observed as the length of the spacer was increased , with compounds 1b and 1c being the most potent . likewise , compound 2 , which lacked any spacer , was the most potent of all four rigid rod compounds examined . cells were treated with the indicated compounds at the indicated nanomolar concentrations or with pma ( 100 nm ) for 30 min , and then erk phosphorylation was determined by western blotting . ( a ) representative western blot showing the phosphorylated p42/44 bands indicated by arrows ( two additional independent experiments gave similar results ) . control blots ( data not shown ) confirmed that the amounts of erk did not change as a function of the ligand concentration . this response , which we measure at 2 days after treatment , lies downstream of several pkc isoforms , in particular pkc and pkc , and will be influenced by the stability of the compounds as well as by their potencies and selectivities . as seen in figure 4 , all the compounds induced apoptosis , with an apparent preference for the longer spacers of 1b and 1c . it is noteworthy that compound 1c induced a higher level of apoptosis than did compound 2 , contrasting with the higher potency of 2 in the in vitro binding assays ( table 1 ) . doseresponse of compounds 1ac and 2 for induction of apoptosis in lncap cells relative to 1000 nm pma . data are the mean sem of three experiments ( 2 days of treatment ) . translocation of individual pkc isoforms from cytosol to membrane compartments provides a measure of their activation , can reveal differences in the kinetics of response , and may uncover differences in the distribution of an isoform between various membrane compartments . we examined the pattern of response of pkc ( figure 5 ) and pkc ( figure 6 ) as representatives of the classic and novel pkc isoforms , using green fluorescent protein fusion constructs heterologously expressed in chinese hamster ovary cells . translocation of gfp - pkc in cho cells in response to addition of dag - lactones ( 10 m ) . the redistribution of the fluorescent proteins was monitored with a zeiss mrc 1024 confocal microscope as a function of time after the addition of the compounds . images were captured every 30 s. each panel represents a typical example from four independent experiments . translocation of gfp - pkc in cho cells upon addition of dag - lactones ( 10 m ) . cells expressing gfp - pkc were treated with 10 m of the compounds as indicated . the redistribution of the fluorescent proteins was monitored with a zeiss mrc 1024 confocal microscope as a function of time after the addition of the drugs . images were captured every 30 s. each panel represents a typical example from three independent experiments . for all derivatives , pkc translocated to the plasma membrane , which is the typical response for this isoform . compound 2 was the fastest acting compound on pkc. as the length of the linker increased in the series 1ac the response became more rapid , although the response to 1c remained slower than that to 2 . pkc shows a more variable pattern of response to various ligands , with pma inducing initial translocation to the plasma membrane followed by redistribution to internal membranes and hydrophilic derivatives inducing the initial translocation to the internal membranes ( not shown ) . compound 2 translocated pkc mainly to the plasma membrane and nuclear membrane , but it was not pma - like for pkc ; there was no sequential translocation to the plasma membrane followed by a shift to nuclear membrane . compounds 1ac translocated pkc mainly to the internal membranes , but it was noteworthy that localization was predominantly perinuclear rather than to the nuclear membrane per se . interactions of dag - lactones with membranes are expected to cause perturbations that affect the structural and dynamic properties of the phospholipid bilayers . visualization of these interactions is possible with the use of membranes that contain polydiacetylene ( pda ) patches that are easily perturbed . the pda - based vesicle assay reports on the occurrence of membrane interactions through color and fluorescence transformations of the vesicle - embedded pda patches induced through interactions of membrane - associated molecules with the vesicle bilayers . figure 7 depicts the dose - dependent fluorescence chromatic response ( % fcr ) curves induced by the dag - lactones following addition to biomimetic dimyristoylphosphatidylcholine ( dmpc)/pda vesicles . all four compounds gave rise to increasing fluorescence signals upon higher concentrations , indicating that the molecules exhibited membrane interactions . however , differences were apparent among the compounds with regards to the extent of induced chromatic response ( intensity of fluorescence signal ) and particularly the shape of the doseresponse curves . specifically , figure 7 shows that 1a , 1b , and 1c exhibited a relatively steep initial increase in % fcr with a subsequent moderate rise of the doseresponse curve [ or a slight decrease in % fcr in the case of 1a ( figure 7 , curve i ) , most likely due to severe precipitation ] . compound 2 , on the other hand , induced a very low chromatic response up to approximately 2 mm , followed by a pronounced increase in fluorescence emission , which subsequently exceeded the % fcs values induced by the three other molecules ( figure 7 , iv ) . fluorescence chromatic response ( % fcr ) induced by the dag - lactones in dmpc / pda vesicles : ( i ) 1a ; ( ii ) 1b ; ( iii ) 1c ; ( iv ) 2 . previous studies have shown that the features of the chromatic lipid / pda doseresponse curves are intimately affected by the mechanisms of membrane binding and insertion . accordingly , the pronounced difference between the fluorescence curve induced by 2 and the curves of 1ac indicates that dag - lactone 2 exhibits a distinct lipid binding profile . in particular , the apparent parabolic shape of the chromatic response curve of 2 suggests that a self - assembly process precedes membrane binding and that this self - association might be a precondition for bilayer interactions . compounds 1ac , on the other hand , most likely undergo a simple concentration - dependent bilayer binding , as reflected in the doseresponse curves in figure 7 ( iiii ) . protein kinase c has attracted great interest as a therapeutic target for cancer and other conditions in consequence of its central role in cellular signal transduction , mediating responses to the second messenger dag . enzastaurin , an orally active pkc inhibitor that is relatively selective for pkc among the pkc isoforms , is directed at the catalytic site . like most kinase inhibitors , it faces the imposing challenge of distinguishing among the 500 plus kinases of the mammalian kinome , and it indeed shows off - target activity on several other kinases . bryostatin 1 and pep005 , on the other hand , are natural products , which interact with the dag - phorbol ester responsive c1 domains shared by pkc , rasgrp , and the chimerins . although the mechanistic rationale for the therapeutic activity of c1 directed agents is less clear than for catalytic site directed agents , interest is driven by the demonstrated ability of different ligands for pkc to induce different biological responses , by the ability of bryostatin to antagonize many responses induced by phorbol esters , and by the demonstration that one pkc isoform may have effects antagonistic of another . opportunities for antagonism are further enhanced by the additional classes of c1 domain containing targets . for example , dag kinases will terminate dag signaling , and the chimerins , by inhibiting rac , would be expected to be tumor suppressors . in contrast to kinase inhibitors , a great opportunity for design of selectivity for c1 domain ligands is provided by the nature of the ligand interaction site , where not only the c1 domain but also the lipid environment with which it is associated contributes to ligand recognition . thus , for example , we have described very different selectivity for pma relative to bryostatin between the pkc and pkc isoforms in mouse 3t3 cells and mouse keratinocytes . likewise , using several libraries of dag - lactones differing only in their patterns of side chains , modulating their lipid interactions , we have shown that different compounds were selective for different biological end points . we describe here our initial characterization of a small series of compounds in which we have manipulated the length and nature of the connection of the dag - lactone template and a rigid rod terminus on one side chain . we have previously observed that incorporation of a rigid rod structure into the side chain caused exclusion of the ligand from cholesterol rich subdomains , suggesting that such rigid structural motifs might contribute to differential recognition . lipid rafts rich in cholesterol are proposed to represent specialized areas for signaling complexes in the plasma membrane , and plasma membranes differ from those of internal membranes such as the nuclear membrane in being overall richer in cholesterol . , we observed that the rigid rod compounds were relatively less active on the classic pkc isoforms in membranes containing cholesterol and relatively deficient in phosphatidylserine compared to those constituted solely of phosphatidylserine . we also observed that the potencies of the compounds in vitro depended not only on the log p but also on the particular nature of the linker , emphasizing the importance of the specific interactions with the lipid . at the biological level , we noted differences in induction of the downstream response of erk phosphorylation by the various compounds , as we did for apoptotic response and pkc isoform translocation . the biomimetic lipid / pda vesicle assay showed that dag - lactones exhibited significant membrane interactions ; however , there appears to be a pronounced difference between the mechanism of membrane association between dag - lactone 2 and the other three compounds with variable tethers . specifically , the chromatic doseresponse analysis in figure 6 might suggest that 2 undergoes a distinct self - assembly process ( possibly forming micelles or similar aggregates ) prior to membrane binding ; the consequent aggregates are therefore the likely entities exhibiting affinity to the membrane bilayer rather than the individual molecules . more extensive exploration of the influence of the positioning of rigid rod substituents on the recognition of dag lactones and clarification of the linkage between mechanistic differences and biological consequences should be of considerable interest . enzymeligand interactions were analyzed by competition with [ h]pdbu binding to a single isozyme essentially as described previously . as indicated in the text , the pkc isozymes were assayed in the presence of 100 g / ml phosphatidylserine or else 100 g / ml total lipid , where the lipid composition was chosen to mimic that of the inner leaflet of the plasma membrane . this latter composition was phosphatidylcholine / phosphatidylethanolamine / phosphatidylserine / phosphatidylinositol / cholesterol ( 12:35:22:9:21 ) . the id50 values were determined by least - squares fitting of the theoretical sigmoidal competition curve to the binding data . the ki values were calculated from the id50 values according to the relationship ki = id50/(1 + l / kd ) , where l is the concentration of free [ h]pdbu at the id50 and kd is the dissociation constant for [ h]pdbu under the assay conditions . the octanol / water partition coefficients ( log p ) were calculated according to the atom - based program moe slogp . 1-palmitoyl-2-oleoylphosphatidylcholine , 1-palmitoyl-2-oleoylphosphatidylethanolamine , 1-palmitoyl-2-oleoylphosphatidylserine , and bovine liver phosphatidylinositol were from avanti polar lipids ( alabaster , al ) . the human pkc , pkc , pkc , pkc , and pkc were from invitrogen ( carlsbad , ca ) . androgen - sensitive lncap prostate cancer cells were purchased from the american type culture collection ( manassas , va ) and cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum and antibiotics ( penicillin at 50 units / ml and streptomycin at 0.05 mg / ml ) . cells were plated in 6 cm dishes at about 60% confluency and 24 h later were treated with different concentrations of the test compound and 100 nm pma for 30 min . after treatment , the cells were washed with phosphate buffered saline , total cell lysates were prepared , and the lysates were analyzed by 10% sdspage followed by electrotransfer and immunostaining . the primary antibodies used for immunostaining against phospho - erk ( 9101 ) and total erk ( 9102 ) were from cell signaling ( danvers , ma ) . films were scanned , and densitometry was performed using image j ( developed at national institute of mental health , nih ) . lncap cells plated in 10 cm dishes at about 60% confluency were treated with different concentrations of the test compound and of 1 m pma for 48 h. after treatment , the cells were washed and resuspended in phosphate buffered saline . yo - pro-1 ( invitrogen , carlsbad , ca ) was added to a final concentration of 1 m , and the cells were incubated for 20 min at 4 c in the dark . 7-aminoactinomycin d ( 7-aad ) ( invitrogen , carlsbad , ca ) was then added at a 5 g / ml final concentration 10 min before analysis by flow cytometry using the facscalibur system ( becton dickinson , mountain view , ca ) . the yo - pro-1 positive cells were considered apoptotic and were expressed as % of total cells . cho cells were purchased from the american type culture collection ( manassas , va ) and cultured in f12-k medium supplemented with 10% fetal bovine serum and antibiotics ( penicillin at 50 units / ml and streptomycin at 0.05 mg / ml ) . butler , pa ) were transfected with gfp - tagged pkc or pkc using lipofectamine reagent ( invitrogen , carlsbad , ca ) . cells were visualized with a zeiss lsm 510 confocal microscope ( carl zeiss inc . , thornwood , ny ) with an axiovert 100 m inverted microscope operating with a 25 mw argon laser tuned to 488 nm . cells were imaged with a 63 1.4 na zeiss plan apochromat oil immersion objective and with varying zooms ( 1.42 ) . time - lapse images were collected every 30 s using the zeiss aim software , in which the green emission was collected in a pmt with a lp 505 filter . vesicle emulsions containing dmpc and trcda ( 2:3 molar ratio ) were prepared as previously described . briefly , the lipid constituents were dried together in vacuo followed by addition of deionized water and sonication at 70 c . the vesicle emulsion was then cooled to room temperature and kept at 4 c overnight ; it was then polymerized by irradiation at 254 nm for 30 s. the resulting emulsion exhibits a blue appearance . vesicle samples for experiments were prepared at concentrations of 0.5 mm ( total lipids ) in 50 mm tris - cl , ph 8 . the various compounds at different concentrations were mixed with the vesicles prior to addition of the tris buffer . samples were prepared by adding different volumes of the compounds in dmso solutions to 30 l of the dimyristoylphosphatidylcholine ( dmpc ) and diacetylene 10,12-tricosadiynoic acid ( trcda ) vesicles , followed by addition of 30 l of 50 mm tris base buffer ( ph 8) . fluorescence intensity was measured using a multiwell fluorescence plate reader ( fluoroscan ascent ; thermo , finland ) with a 485/555 nm excitation / emission long path filter set . the fluorescence percentage was calculated as percentage intensity compared to the fully transformed red vesicles . combi - flash column chromatography was performed on silica gel 60 ( 230240 mesh ) employing a teledyne isco instrument , and analytical tlc was performed on analtech uniplates silica gel gf . unless otherwise indicated , nmr spectra were determined in cdcl3 ( 99.8% ) with residual chcl3 as the reference peak ( 7.26 and 77.0 ppm ) and were recorded on a varian 400 mhz spectrophotometer . the coupling constants are reported in hertz , and the peak shifts are reported in the ( ppm ) scale ; abbreviations used are s ( singlet ) , d ( doublet ) , dd ( doublet - of - doublets ) , ddd ( doublet - of - doublet - of - doublets ) , t ( triplet ) , q ( quartet ) , and m ( multiplet ) . positive - ion fast - bombardment mass spectra ( fabms ) were obtained on a vg 7070e mass spectrometer at an accelerating voltage of 6 kv and a resolution of 2000 . glycerol was used as the sample matrix , and ionization was effected by a beam of xenon atoms . combustion analyses were performed for all intermediates and final products to confirm that their level of purity was 95% ; they were performed by atlantic microlab . norcross , ga . infrared spectroscopy data were obtained neat with a jasco ft - ir/615 spectrometer . all reaction glassware was oven - dried and cooled to room temperature in an argon atmosphere prior to use . 4-ethynyltoluene ( 5.0 g , 43.04 mmol ) was added to a solution of 4-iodophenol ( 9.2 g , 41.79 mmol ) , pd(pph3)2cl2 ( 590 mg , 0.84 mmol ) , cui ( 320 mg , 1.67 mmol ) , and et3n ( 8.5 ml , 60.9 mmol ) in thf ( 50 ml ) at room temperature under an argon atmosphere . the solids were separated by filtration , and the volatiles were evaporated under reduced pressure . the residue was purified by combi - flash column chromatography ( silica gel ; hexanes / ethyl acetate , 4:1 ) to give 8.3 g ( 95% yield ) of pure 3 as a yellowish solid : mp 124 c ( lit . 103104 c ) ; ftir ( neat ) 3307 cm ; h nmr ( 400 mhz , cdcl3 ) 7.42 ( m , 4 h , ph ) , 7.14 ( dm , j = 7.8 hz , 2 h , ph ) , 6.80 ( dm , j = 8.8 hz , 2 h , ph ) , 4.80 ( bs , 1 h , oh ) , 2.36 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 155.45 , 138.07 , 133.20 , 133.14 , 131.35 , 131.29 , 129.08 , 120.37 , 115.45 , 88.49 , 88.19 , 21.43 . fabms m / z ( relative intensity ) : 209 ( mh , 36 ) , 208 ( m , 100 ) . tert - butyl bromoacetate ( 0.85 ml , 5.58 mmol ) was added to a suspension of 3 ( 1.02 g , 4.90 mmol ) and csco3 ( 2.55 g , 7.76 mmol ) in anhydrous dmf ( 12 ml ) at room temperature under argon . the reaction mixture was stirred for 5 h. et2o ( 50 ml ) was added , and the solution was washed with nahco3 ( 2 15 ml ) and water ( 1 15 ml ) before it was dried ( mgso4 ) and concentrated . the residue was purified by combi - flash column chromatography ( silica gel ; hexanes / etoac , 9:1 ) to give 1.43 g ( 92% yield ) of pure 4a as a yellowish solid : mp 85 c , ftir ( neat ) 1751 cm ; h nmr ( 400 mhz , cdcl3 ) 7.45 ( dm , j = 8.9 hz , 2 h , ph ) , 7.40 ( dm , j = 8.1 hz , 2 h , ph ) , 7.14 ( dm , j = 8.1 hz , 2 h , ph ) , 6.86 ( dm , j = 8.9 hz , 2 h , ph ) , 4.52 ( s , 2 h , h-2 ) , 2.36 ( s , 3 h , ch3 ) , 1.48 ( s , 9 h , c(ch3)3 ) ; c nmr ( 100 mhz , cdcl3 ) 167.69 , 157.74 , 138.07 , 132.95 , 131.33 , 129.05 , 120.37 , 116.52 , 114.60 , 88.44 , 88.40 , 82.52 , 65.65 , 28.01 , 21.47 . fabms m / z ( relative intensity ) : 323 ( mh , 44 ) , 322 ( m , 100 ) . anal . calcd for c21h22o3 : c , 78.23 ; h , 6.88 . found : c , 78.29 ; h , 6.91 . a solution of tert - butyl ester 4a ( 1.34 g , 0.42 mmol ) in quinoline ( 30 ml ) was heated to 205 c for 3 h. the mixture was cooled to room temperature and was poured into 1.2 m hcl ( 30 ml ) at 0 c . the solid free acid was filtered , washed with 1.2 m hcl and water , and dried under vacuum overnight at 100 c . a solution of 6 ( 151 mg , 0.52 mmol ) in ch2cl2 ( 5 ml ) was added to a suspension of free acid 5a ( 151 mg , 0.57 mml ) in ch2cl2 ( 10 ml ) . dmap ( 158 mg , 1.29 mmol ) and bopcl ( 263 mg , 1.38 mmol ) were successively added , and the reaction mixture was stirred for 1 h at room temperature under argon . nh4cl ( ss , 10 ml ) was added , and the mixture was extracted with ch2cl2 ( 3 20 ml ) . the combined organic phases were washed with brine ( 2 10 ml ) , dried ( mgso4 ) , and concentrated . the residue was purified by combi - flash column chromatography ( hexanes / etoac , 4:1 ) to give a 210 mg ( 75% ) of 8a as a foam : ftir ( neat ) 1748 cm ; h nmr ( 400 mhz , cdcl3 ) 7.41 ( m , 4 h , ph ) , 7.15 ( dm , j = 7.9 hz , 2 h , ph ) , 6.766.84 ( m , 6 h , ph ) , 4.68 ( s , 2 h , h-2 ) , 4.68 ( s , 2 h , ch2ococh2oar ) , 3.95 ( d , j = 9.5 hz , 1 h , chahopmp ) , 3.84 ( d , j = 9.5 hz , 1 h , chhbopmp ) , 3.74 ( s , 3 h , och3 ) , 2.89 ( dm , j = 16.7 hz,1 h , h-3a ) , 2.64 ( dm , j = 16.7 hz,1 h , h-3b ) , 2.36 ( s , 3 h , ch3 ) , 2.27 ( br s , 3 h , ch3 ) , 1.86 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 168.39 , 168.27 , 157.40 , 154.46 , 152.34 , 152.13 , 138.16 , 132.96 , 133.04 , 131.21 , 129.07 , 120.27 , 118.02 , 116.87 , 115.57 , 114.68 , 114.46 , 88.67 , 88.24 , 79.10 , 69.99 , 66.23 , 64.91 , 55.65 , 32.67 , 24.60 , 21.45 , 19.99 . fabms m / z ( relative intensity ) : 541 ( mh , 78 ) , 540 ( m , 100 ) . anal . calcd for c33h32o70.8h2o : c , 71.46 ; h , 6.11 . found : c , 71.33 ; h , 5.86 . cerium ammonium nitrate ( can , 247 mg , 0.45 mmol ) was added to a solution of 8a ( 81 mg , 0.15 mmol ) in acetonitrile / water ( 4:1 , 5 ml ) at 0 c . after 10 min , 5% na2s2o3 solution ( 10 ml ) was added and the mixture was extracted with etoac ( 3 20 ml ) . the organic phase was washed with brine ( 10 ml ) , dried ( mgso4 ) , and concentrated . the residue was purified by combi - flash column chromatography ( silica gel ; hexanes / etoac , 1:1 ) to give 35 mg ( 54% yield ) of pure 1a as a white solid : mp 147 c . ftir ( neat ) 3447 , 1746 cm ; h nmr ( 400 mhz , cdcl3cd3od ) 7.30 ( dm , j = 8.9 hz , 2 h , ph ) , 7.24 ( dm , j = 8.2 hz , 2 h , ph ) , 7.00 ( dm , j = 8.2 hz , 2 h , ph ) , 6.71 ( dm , j = 8.9 hz , 2 h , ph ) , 4.55 ( s , 2 h , h-2 ) , 4.18 ( abq , j = 11.7 hz , 2 h , ch2ococh2oar ) , 3.44 ( mab , 2 h , ch2oh ) , 2.59 ( dm , j = 16.5 hz , 1 h , h-3a ) , 2.42 ( dm , j = 16.5 hz,1 h , h-3b ) , 2.21 ( s , 3 h , ch3 ) , 2.09 ( br s , 3 h , ch3 ) , 1.70 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 169.54 , 168.47 , 157.23 , 152.19 , 138.02 , 132.77 , 131.05 , 128.84 , 119.97 , 118.50 , 116.65 , 114.29 , 88.40 , 87.93 , 81.14 , 65.88 , 64.68 , 64.11 , 31.56 , 24.23 , 21.08 , 19.63 . fabms m / z ( relative intensity ) : 435 ( mh , 99 ) , 434 ( m , 100 ) . a solution of 3 ( 1.05 g , 5.04 mmol ) in tert - butyl acrylate ( 5 ml ) was treated with sodium ( 3.5 mg , cat . ) and hydroquinone ( 1 mg , cat . ) . the mixture was stirred at room temperature for 2 h. et2o ( 20 ml ) and a drop of acetic acid were added , and the solution was washed with water ( 2 10 ml ) , dried ( mgso4 ) , and concentrated . the residue was purified by combi - flash column chromatography ( silica gel ; hexanes / etoac , 9:1 ) to give 550 mg ( 33% yield ) of pure 4b as a yellowish solid : mp 74 c . ftir ( neat ) 1731 cm ; h nmr ( 400 mhz , cdcl3 ) 7.42 ( m , 4 h , ph ) , 7.14 ( dm , j = 7.8 hz , 2 h , ph ) , 6.87 ( dm , j = 8.9 hz , 2 h , ph ) , 4.23 ( t , j = 6.5 hz , 2 h , h-3 ) , 2.71 ( t , j = 6.5 hz , 2 h , h-2 ) , 2.36 ( s , 3 h , ch3 ) , 1.47 ( s , 9 h , c(ch3)3 ) ; c nmr ( 100 mhz , cdcl3 ) 170.06 , 158.51 , 137.91 , 132.87 , 131.26 , 129.00 , 120.42 , 115.75 , 114.55 , 88.59 , 88.19 , 80.92 , 63.72 , 35.62 , 28.02 , 21.40 . fabms m / z ( relative intensity ) : 337 ( mh , 41 ) , 336 ( m , 100 ) . calcd for c22h24o3 : c , 78.24 ; h , 7.19 . found : c , 78.48 ; h , 7.21 . tert - butyl ester 4b was treated in the same way as that described for 4a and the corresponding free acid was reacted with 7 as described for the synthesis of 8a to give 180 mg ( 70% yield ) of 8b as a colorless oil . ftir ( neat ) 1744 cm ; h nmr ( 400 mhz , cdcl3 ) 7.427.25 ( m , 9 h , ph ) , 7.11 ( dm , j = 7.9 hz , 2 h , ph ) , 6.80 ( dm , j = 8.9 hz , 2 h , ph ) , 4.50 ( s , 2 h , och2ph ) , 4.25 ( s , 2 h , ch2ococh2ch2oar ) , 4.18 ( t , j = 6.3 hz , 2 h , h-3 ) , 3.49 ( abq , j = 9.9 hz , 2 h , ch2obn ) , 2.77 ( t , j = 6.3 hz , 2 h , h-2 ) , 2.78 ( dm , j = 16.4 hz , 1 h , h-3a ) , 2.62 ( dm , j = 16.4 hz,1 h , h-3b ) , 2.33 ( s , 3 h , ch3 ) , 2.20 ( t , j = 2.0 hz , 3 h , ch3 ) , 1.76 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 170.26 , 168.83 , 158.25 , 151.28 , 138.05 , 137.44 , 132.96 , 131.31 , 129.05 , 128.43 , 127.86 , 127.64 , 120.39 , 118.74 , 116.07 , 114.51 , 88.48 , 79.94 , 73.65 , 71.72 , 66.19 , 63.20 , 34.40 , 32.72 , 24.47 , 21.47 , 19.89 . fabms m / z ( relative intensity ) : 539 ( mh , 31 ) , 538 ( m , 35 ) . calcd for c34h34o60.6h2o : c , 74.38 ; h , 6.46 . found : c , 74.05 ; h , 6.16 . a solution of 8b ( 141 mg , 0.26 mmol ) in anhydrous ch2cl2 ( 5 ml ) was treated with 1.0 m bcl3 solution in ch2cl2 ( 1 ml , 1.05 mmol ) at 78 c for 15 min . nahco3 ( ss , 5 ml ) was added , and the mixture was allowed to reach room temperature . the aqueous layer was extracted with ch2cl2 ( 3 20 ml ) , and the combined organic layers were dried ( mgso4 ) and concentrated . the residue was purified by combi - flash column chromatography ( silica gel ; hexanes / etoac , 1:1 ) to give 93 mg ( 80% yield ) of pure 1b as a white solid : mp 125 c . ftir ( neat ) 3472 , 1731 cm ; h nmr ( 400 mhz , cdcl3 ) 7.457.38 ( m , 4 h , ph ) , 7.14 ( dm , j = 8.0 hz , 2 h , ph ) , 6.84 ( dm , j = 8.9 hz , 2 h , ph ) , 4.28 ( abq , j = 11.8 hz , 2 h , ch2ococh2ch2oar ) , 4.23 ( t , j = 6.3 hz , 2 h , h-3 ) , 3.66 ( abq , j = 12.1 hz , 2 h , ch2oh ) , 2.83 ( t , j = 6.3 hz , 2 h , h-2 ) , 2.78 ( dm , j = 16.5 hz , 1 h , h-3a ) , 2.64 ( dm , j = 16.4 hz,1 h , h-3b ) , 2.35 ( s , 3 h , ch3 ) , 2.23 ( t , j = 2.0 hz , 3 h , ch3 ) , 1.80 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 170.66 , 168.91 , 158.18 , 152.14 , 138.06 , 132.98 , 131.30 , 129.05 , 120.34 , 118.59 , 116.14 , 114.49 , 88.43 , 88.39 , 80393 , 65.66 , 64.82 , 63.22 , 34.42 , 32.01 , 24.53 , 21.45 , 19.96 ; fabms m / z ( relative intensity ) : 449 ( mh , 85 ) , 448 ( m , 100 ) . anal . calcd for c27h28o60.3h2o : c , 71.49 ; h , 6.35 . found : c , 71.30 ; h , 6.18 . this compound was prepared similarly as described for 4a from tert - butyl 4-bromobutanoate ( 302 mg , 2.43 mmol ) and phenol 3 ( 253 mg , 2.21 mmol ) . after combi - flash column chromatography ( silica gel ; hexanes / ethyl acetate , 9:1 ) 351 mg ( 83% yield ) of pure 4c was obtained as a yellowish solid : mp 68 c . ftir ( neat ) 1723 cm ; h nmr ( 400 mhz , cdcl3 ) 7.45 ( dm , j = 8.3 hz , 2 h , ph ) , 7.40 ( dm , j = 7.8 hz , 2 h , ph ) , 7.14 ( dm , j = 7.8 hz , 2 h , ph ) , 6.85 ( dm , j = 8.3 hz , 2 h , ph ) , 4.01 ( t , j = 6.2 hz , 1 h , h-4 ) , 2.43 ( t , j = 7.3 hz , 1 h , h-2 ) , 2.36 ( s , 3 h , ch3 ) , 2.07 ( m , 2 h , h-3 ) , 1.45 ( s , 9 h , c(ch3)3 ) ; c nmr ( 100 mhz , cdcl3 ) 172.39 , 158.78 , 137.92 , 132.90 , 131.27 , 129.02 , 120.47 , 115.52 , 114.45 , 88.65 , 88.14 , 80.34 , 31.91 , 28.07 , 24.66 , 21.43 ; fabms m / z ( relative intensity ) : 351 ( mh , 16 ) , 350 ( m , 41 ) . anal . calcd for c23h26o30.1h2o : c , 78.48 ; h , 7.50 . found : c , 78.32 ; h , 7.47 . tert - butyl ester 4c ( 342 mg , 0.98 mmol ) was treated in the same manner as described for 4a , and the corresponding free acid was reacted with 6 ( 202 mg , 0.69 mmol ) as described for the synthesis of 8a to give 224 mg ( 57% yield ) of 8c as a white solid : mp 117 c ; ftir ( neat ) 1744 cm ; h nmr ( 400 mhz , cdcl3 ) 7.447.39 ( m , 4 h , ph ) , 7.14 ( dm , j = 7.9 hz , 2 h , ph ) , 6.82 ( m , 6 h , ph ) , 4.34 ( abq , j = 11.8 hz , 2 h , ch2ococh2ch2ch2oar ) , 3.99 ( t , j = 6.0 hz , 2 h , h-4 ) , 3.98 ( abq , j = 9.5 hz , 2 h , ch2opmp ) , 3.76 ( s , 3 h , och3 ) , 2.96 ( dm , j = 16.4 hz , 1 h , h-3a ) , 2.75 ( dm , j = 16.4 hz , 1 h , h-3b ) , 2.55 ( t , j = 7.3 hz , 2 h , h-2 ) , 2.36 ( s , 3 h , ch3 ) , 2.28 ( br s , 3 h , ch3 ) , 2.09 ( m , 2 h , h-3 ) , 1.87 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 172.55 , 168.63 , 158.61 , 154.43 , 152.28 , 151.82 , 138.00 , 132.96 , 131.30 , 129.05 , 120.45 , 118.44 , 115.61 , 114.68 , 114.44 , 88.60 , 88.23 , 79.41 , 70.22 , 66.52 , 65.88 , 55.69 , 32.88 , 30.53 , 24.60 , 24.45 , 21.47 , 19.97 . fabms m / z ( relative intensity ) : 569 ( mh , 100 ) , 568 ( m , 86 ) . starting from 8c , compound 1c was obtained as described for 1a in 50% yield : mp 115116 ; ftir ( neat ) 3428 , 1714 cm ; h nmr ( 400 mhz , cdcl3 ) 7.447.38 ( m , 4 h , ph ) , 7.13 ( dm , j = 7.8 hz , 2 h , ph ) , 6.84 ( dm , j = 8.9 hz , 2 h , ph ) , 4.23 ( abq , j = 11.8 hz , 2 h , ch2ococh2ch2ch2oar ) , 4.00 ( t , j = 6.0 hz , 2 h , h-4 ) , 3.65 ( m , 2 h , ch2oh ) , 2.80 ( dm , j = 16.5 hz , 1 h , h-3a ) , 2.62 ( dm , j = 16.5 hz , 1 h , h-3b ) , 2.56 ( t , j = 7.3 hz , 2 h , h-2 ) , 2.35 ( s , 3 h , ch3 ) , 2.25 ( br s , 3 h , ch3 ) , 2.10 ( m , 2 h , h-3 ) , 1.85 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 172.91 , 168.90 , 158.57 , 151.94 , 138.01 , 132.95 , 131.29 , 129.04 , 120.41 , 118.70 , 115.75 , 114.43 , 88.55 , 88.25 , 80.97 , 66.54 , 65.52 , 64.80 , 32.09 , 30.55 , 24.57 , 24.43 , 21.45 , 19.96 ; fabms m / z ( relative intensity ) : 463 ( mh , 100 ) , 462 ( m , 84 ) . anal . calcd for c28h30o60.4h2o : c , 71.64 ; h , 6.61 . found : c , 71.58 ; h , 6.41 . this compound was prepared by the same methodology as described in ref ( 7 ) . pure 2 ( 260 mg , 76% ) was obtained as a yellowish solid : mp 167168 c ; ftir ( neat ) 3469 , 2214 , 1723 cm ; h nmr ( 400 mhz , cdcl3 ) 7.96 ( m , 2 h , ph ) , 7.57 ( m , 2 h , ph ) , 7.44 ( m , 2 h , ph ) , 7.18 ( m , 2 h , ph ) , 4.58 ( mab , 2 h , ph - co2ch2 ) , 3.76 ( abq , j = 12.1 hz , 2 h , hoch2 ) , 2.902.70 ( mab , 2 h , h-4a , b ) , 2.38 ( s , 3 h , ch3 ) , 2.25 ( t , j = 2.0 hz , 3 h , ch3 ) , 1.86 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 169.10 , 165.78 , 151.67 , 139.09 , 131.62 , 131.46 , 129.61 , 129.18 , 128.82 , 128.23 , 119.00 , 93.12 , 87.88 , 81.45 , 66.32 , 64.78 , 32.25 , 24.55 , 21.53 , 19.91 . fabms m / z ( relative intensity ) : 405 ( mh , 47 ) . calcd for c25h24o50.3h2o : c , 73.26 ; h , 6.05 . found : c , 73.21 ; h , 5.99 .

diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod that is separated from the exocyclic acylcarbonyl of the dag - lactone core by a spacer unit of variable length were synthesized and studied . binding affinities for a panel of classical and novel pkc isozymes in two different phospholipid environments , one corresponding to the plasma membrane of cells , were determined . the kinetics and site of translocation for the pkc isozymes and upon treatment with the compounds were also studied as well as the early response of erk phosphorylation and the late response of induction of apoptosis in the human prostatic carcinoma cell line lncap . finally , the compounds were evaluated in terms of their interaction with biomimetic lipid / polydiacetylene membranes by the associated chromatic response . the different spatial disposition of the rigid structural motif on the dag - lactones contributes to differential activity .

Introduction Results Discussion Experimental Section

dag in turn interacts with the dag recognition domains , termed c1 domains , on multiple families of signaling proteins , including isoforms of protein kinase c , the chimerins , rasgrp , pkd , mrck , unc13 , and dag kinase . specific molecular interactions of dag with the lipid environment into which it and the c1 domains insert influence both processes . the physical properties of the membranes and the interplay between the membranes and the alkyl chains of the dag are thus of great importance . some of the dag - lactones were able to induce a substantially prolonged translocation state relative to the parent dag - lactone containing an equivalent flexible acyl chain . more recently , we studied the thermodynamic features of some new dag - lactones with specific small groups attached to the terminus of a rigid 4-(phenylethynyl)benzoyl chain which affected both the self - assembly of the molecules and their interactions with phospholipids . the results suggested that these dag - lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases formed by cholesterol and that the size and charge of the phospholipid headgroups did not seem to affect the dag - lactone interactions with lipids . because different extents of membrane penetration and preference for membrane subdomains might yield different patterns of response in the ability of dag - lactones to interact or translocate different gfp - tagged isozymes , we designed a series of dag - lactones with a rigid 4-[(methylphenyl)ethynyl]phenyl rod ( red ) that is separated from the acylcarbonyl of the dag - lactone core [ 2-(hydroxymethyl)-4-(methylethylidene)-5-oxo-2 - 2,3-dihydrofuryl]methyloxocarbonyl ] ( blue ) by a o(ch2)n spacer of 1 , 2 , or 3 methylene units ( 1ac , figure 1 ) . the rigid rod moiety is indicated in red , the dag - lactone in blue , and the variable linker in black . we evaluated the compounds in both in vitro and in cellular systems : ( 1 ) we determined their binding affinities for a panel of classical and novel pkc isozymes , assayed in two different phospholipid environments , either 100% phosphatidylserine or a lipid mixture corresponding to that of the plasma membrane of cells ; ( 2 ) we compared the kinetics and site of translocation for the pkc isozymes and upon treatment with the compounds ; ( 3 ) we measured the potencies of the compounds for the early response of erk phosphorylation and for the late response of induction of apoptosis in the human prostatic carcinoma cell line lncap ; and ( 4 ) we evaluated the interaction of the compounds with a biomimetic lipid / polydiacetylene membrane by the associated chromatic response . the preparation of racemic -(4-[2-(methylphenyl)ethynyl]phenoxy)alkanoyl monoesters of dag - lactones ( figure 1 ) with one to three methylene spacers between the lactone and the rigid chain was achieved as depicted in schemes 1 and 2 . the rigid - rod component , 4-[2-(4-methylphenyl)ethynyl]phenol ( 3 ) , was prepared in excellent yield by a modified sonogashira coupling between 4-iodophenol and 4-ethynyltoluene . on the other hand , michael addition of 3 to tert - butyl acrylate in the presence of catalytic amounts of sodium and hydroquinone gave the desired tert - butyl ester 4b ( n = 2 ) in sufficient quantities to continue the synthesis . despite the fact that several byproducts were obtained , including starting material 3 , the self - addition product of phenol to the triple bond , and cross - reaction between the starting phenol and desired product 4b , successful separation of 4b was achieved by simple column chromatography . at this point , hydrolysis of tert - butyl esters was necessary to perform the acylation of the dag - lactone moiety . the use of bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride ( bopcl ) and dimethylaminopyridine ( dmap ) allowed the one - pot coupling of the acids with the free hydroxyl group of the monoprotected o - pmp - dag - lactone 6(6 ) ( for n = 1 and 3 ) and o - benzyl - dag - lactone 7(6 ) ( for n = 2 ) in very good yield . removal of the p - methoxyphenyl ( pmp ) protecting group in the presence of ceric ammonium nitrate ( can ) to give the target compounds 1a and 1c resulted in low yields ( 54% and 50% ) due to the presence of the additional phenoxy ether linkage in 8a and 8c . a better strategy was the coupling of the o - benzyl protected dag - lactone 7 that led to the desired dag - lactone 1b in a markedly better yield ( 80% ) after treatment with boron trichloride at low temperature ( scheme 2 ) . enzymeligand interactions were assessed in vitro in terms of the ability of the ligand to displace bound [ 20-h]phorbol 12,13-dibutyrate ( pdbu ) from the recombinant human pkc isozymes in the presence of phosphatidylserine . each of the pkc isozymes showed a similar pattern of dependence on the length of the spacer for compounds 1ac ( table 1 ) . in each case , the affinities increased ( lower ki ) with the distance of the rigid rod from the dag - lactone core , with compound 1c ( n = 3 ) being the most potent . in sharp contrast , however , the most potent of all ligands ( 2 ) lacks a spacer between these two units , suggesting a qualitatively different interaction between it , the enzymes , and the lipid environment . in terms of isozyme specificity , pkc stood out from the other isoforms in showing a 5- to 4-fold lower affinity for compounds 1a and 1b with the shorter spacers . these conditions maximize the interaction of the pkc isoforms with the phospholipid bilayer . to detect differences in interactions that might depend on a more physiological lipid composition , we also assayed the isoforms in the presence of 100 g / ml of a mixture of phosphatidylcholine / phosphatidylethanolamine / phosphatidylserine / phosphatidylinositol / cholesterol ( 12:35:22:9:21 ) , a plasma membrane lipid ( pml ) mixture designed to mimic that of the inner leaflet of the plasma membrane . we observed ( table 2 , figure 2 ) that the rigid rod compounds were generally several - fold less potent for the classic pkc isoforms in the plasma membrane mimetic membranes as compared to the 100 g / ml phosphatidylserine , whereas for the novel pkc isoforms they were equal or more potent . effect of the lipid environment on the potencies of the rigid rod compounds for pkc isoforms . compounds were assayed with the various pkc isoforms in the presence of either phosphatidylserine ( ps ) or a lipid mixture corresponding to the composition of the inner leaflet of the plasma membrane , as described in the methods section . values represent the ratio of the ki value with the plasma membrane lipid ( pml ) mimetic mixture to that with phosphatidylserine , where the ki values are the average of three independent experiments . activation of the erk pathway , as reflected by erk phosphorylation , is a prominent early biological response to pkc stimulation by dag - lactones and phorbol ester . all of the compounds caused phosphorylation of erk in the concentration range of 110 m ( figure 3 ) . similar to the potencies in the binding assays , increasing potency was observed as the length of the spacer was increased , with compounds 1b and 1c being the most potent . control blots ( data not shown ) confirmed that the amounts of erk did not change as a function of the ligand concentration . this response , which we measure at 2 days after treatment , lies downstream of several pkc isoforms , in particular pkc and pkc , and will be influenced by the stability of the compounds as well as by their potencies and selectivities . it is noteworthy that compound 1c induced a higher level of apoptosis than did compound 2 , contrasting with the higher potency of 2 in the in vitro binding assays ( table 1 ) . doseresponse of compounds 1ac and 2 for induction of apoptosis in lncap cells relative to 1000 nm pma . translocation of individual pkc isoforms from cytosol to membrane compartments provides a measure of their activation , can reveal differences in the kinetics of response , and may uncover differences in the distribution of an isoform between various membrane compartments . we examined the pattern of response of pkc ( figure 5 ) and pkc ( figure 6 ) as representatives of the classic and novel pkc isoforms , using green fluorescent protein fusion constructs heterologously expressed in chinese hamster ovary cells . translocation of gfp - pkc in cho cells in response to addition of dag - lactones ( 10 m ) . the redistribution of the fluorescent proteins was monitored with a zeiss mrc 1024 confocal microscope as a function of time after the addition of the compounds . the redistribution of the fluorescent proteins was monitored with a zeiss mrc 1024 confocal microscope as a function of time after the addition of the drugs . for all derivatives , pkc translocated to the plasma membrane , which is the typical response for this isoform . as the length of the linker increased in the series 1ac the response became more rapid , although the response to 1c remained slower than that to 2 . pkc shows a more variable pattern of response to various ligands , with pma inducing initial translocation to the plasma membrane followed by redistribution to internal membranes and hydrophilic derivatives inducing the initial translocation to the internal membranes ( not shown ) . compound 2 translocated pkc mainly to the plasma membrane and nuclear membrane , but it was not pma - like for pkc ; there was no sequential translocation to the plasma membrane followed by a shift to nuclear membrane . interactions of dag - lactones with membranes are expected to cause perturbations that affect the structural and dynamic properties of the phospholipid bilayers . the pda - based vesicle assay reports on the occurrence of membrane interactions through color and fluorescence transformations of the vesicle - embedded pda patches induced through interactions of membrane - associated molecules with the vesicle bilayers . figure 7 depicts the dose - dependent fluorescence chromatic response ( % fcr ) curves induced by the dag - lactones following addition to biomimetic dimyristoylphosphatidylcholine ( dmpc)/pda vesicles . however , differences were apparent among the compounds with regards to the extent of induced chromatic response ( intensity of fluorescence signal ) and particularly the shape of the doseresponse curves . specifically , figure 7 shows that 1a , 1b , and 1c exhibited a relatively steep initial increase in % fcr with a subsequent moderate rise of the doseresponse curve [ or a slight decrease in % fcr in the case of 1a ( figure 7 , curve i ) , most likely due to severe precipitation ] . compound 2 , on the other hand , induced a very low chromatic response up to approximately 2 mm , followed by a pronounced increase in fluorescence emission , which subsequently exceeded the % fcs values induced by the three other molecules ( figure 7 , iv ) . fluorescence chromatic response ( % fcr ) induced by the dag - lactones in dmpc / pda vesicles : ( i ) 1a ; ( ii ) 1b ; ( iii ) 1c ; ( iv ) 2 . previous studies have shown that the features of the chromatic lipid / pda doseresponse curves are intimately affected by the mechanisms of membrane binding and insertion . accordingly , the pronounced difference between the fluorescence curve induced by 2 and the curves of 1ac indicates that dag - lactone 2 exhibits a distinct lipid binding profile . in particular , the apparent parabolic shape of the chromatic response curve of 2 suggests that a self - assembly process precedes membrane binding and that this self - association might be a precondition for bilayer interactions . enzastaurin , an orally active pkc inhibitor that is relatively selective for pkc among the pkc isoforms , is directed at the catalytic site . bryostatin 1 and pep005 , on the other hand , are natural products , which interact with the dag - phorbol ester responsive c1 domains shared by pkc , rasgrp , and the chimerins . although the mechanistic rationale for the therapeutic activity of c1 directed agents is less clear than for catalytic site directed agents , interest is driven by the demonstrated ability of different ligands for pkc to induce different biological responses , by the ability of bryostatin to antagonize many responses induced by phorbol esters , and by the demonstration that one pkc isoform may have effects antagonistic of another . in contrast to kinase inhibitors , a great opportunity for design of selectivity for c1 domain ligands is provided by the nature of the ligand interaction site , where not only the c1 domain but also the lipid environment with which it is associated contributes to ligand recognition . likewise , using several libraries of dag - lactones differing only in their patterns of side chains , modulating their lipid interactions , we have shown that different compounds were selective for different biological end points . we describe here our initial characterization of a small series of compounds in which we have manipulated the length and nature of the connection of the dag - lactone template and a rigid rod terminus on one side chain . we have previously observed that incorporation of a rigid rod structure into the side chain caused exclusion of the ligand from cholesterol rich subdomains , suggesting that such rigid structural motifs might contribute to differential recognition . lipid rafts rich in cholesterol are proposed to represent specialized areas for signaling complexes in the plasma membrane , and plasma membranes differ from those of internal membranes such as the nuclear membrane in being overall richer in cholesterol . , we observed that the rigid rod compounds were relatively less active on the classic pkc isoforms in membranes containing cholesterol and relatively deficient in phosphatidylserine compared to those constituted solely of phosphatidylserine . we also observed that the potencies of the compounds in vitro depended not only on the log p but also on the particular nature of the linker , emphasizing the importance of the specific interactions with the lipid . at the biological level , we noted differences in induction of the downstream response of erk phosphorylation by the various compounds , as we did for apoptotic response and pkc isoform translocation . the biomimetic lipid / pda vesicle assay showed that dag - lactones exhibited significant membrane interactions ; however , there appears to be a pronounced difference between the mechanism of membrane association between dag - lactone 2 and the other three compounds with variable tethers . more extensive exploration of the influence of the positioning of rigid rod substituents on the recognition of dag lactones and clarification of the linkage between mechanistic differences and biological consequences should be of considerable interest . as indicated in the text , the pkc isozymes were assayed in the presence of 100 g / ml phosphatidylserine or else 100 g / ml total lipid , where the lipid composition was chosen to mimic that of the inner leaflet of the plasma membrane . the id50 values were determined by least - squares fitting of the theoretical sigmoidal competition curve to the binding data . lncap cells plated in 10 cm dishes at about 60% confluency were treated with different concentrations of the test compound and of 1 m pma for 48 h. after treatment , the cells were washed and resuspended in phosphate buffered saline . yo - pro-1 ( invitrogen , carlsbad , ca ) was added to a final concentration of 1 m , and the cells were incubated for 20 min at 4 c in the dark . samples were prepared by adding different volumes of the compounds in dmso solutions to 30 l of the dimyristoylphosphatidylcholine ( dmpc ) and diacetylene 10,12-tricosadiynoic acid ( trcda ) vesicles , followed by addition of 30 l of 50 mm tris base buffer ( ph 8) . tert - butyl ester 4b was treated in the same way as that described for 4a and the corresponding free acid was reacted with 7 as described for the synthesis of 8a to give 180 mg ( 70% yield ) of 8b as a colorless oil . tert - butyl ester 4c ( 342 mg , 0.98 mmol ) was treated in the same manner as described for 4a , and the corresponding free acid was reacted with 6 ( 202 mg , 0.69 mmol ) as described for the synthesis of 8a to give 224 mg ( 57% yield ) of 8c as a white solid : mp 117 c ; ftir ( neat ) 1744 cm ; h nmr ( 400 mhz , cdcl3 ) 7.447.39 ( m , 4 h , ph ) , 7.14 ( dm , j = 7.9 hz , 2 h , ph ) , 6.82 ( m , 6 h , ph ) , 4.34 ( abq , j = 11.8 hz , 2 h , ch2ococh2ch2ch2oar ) , 3.99 ( t , j = 6.0 hz , 2 h , h-4 ) , 3.98 ( abq , j = 9.5 hz , 2 h , ch2opmp ) , 3.76 ( s , 3 h , och3 ) , 2.96 ( dm , j = 16.4 hz , 1 h , h-3a ) , 2.75 ( dm , j = 16.4 hz , 1 h , h-3b ) , 2.55 ( t , j = 7.3 hz , 2 h , h-2 ) , 2.36 ( s , 3 h , ch3 ) , 2.28 ( br s , 3 h , ch3 ) , 2.09 ( m , 2 h , h-3 ) , 1.87 ( s , 3 h , ch3 ) ; c nmr ( 100 mhz , cdcl3 ) 172.55 , 168.63 , 158.61 , 154.43 , 152.28 , 151.82 , 138.00 , 132.96 , 131.30 , 129.05 , 120.45 , 118.44 , 115.61 , 114.68 , 114.44 , 88.60 , 88.23 , 79.41 , 70.22 , 66.52 , 65.88 , 55.69 , 32.88 , 30.53 , 24.60 , 24.45 , 21.47 , 19.97 .

people often perceive others according to their race , gender , or other social category membership ( brewer , 1988 ; fiske and neuberg , 1990 ) . this process of social categorization provides an efficient way to understand others and guides the direction of limited attentional and cognitive resources . in the past few decades , social psychologists have found extensive evidence that social categorization can occur rapidly and without intention , effort , or conscious control , triggering stereotypes ( devine , 1989 ) , prejudice ( fazio et al . , 1995 ) , and ultimately , discrimination ( dovidio et al . , 1997 ) . several dual - process models of person perception have proposed that processing others according to social category membership is the initial stage in person perception , and that only sufficiently motivated perceivers individuate targets or correct for initial categorical judgments in a later stage ( e.g. , brewer , 1988 ; devine , 1989 ; fiske and neuberg , 1990 ) . however , while there is evidence that social categories influence the earliest phases of social perception , others have argued that the initial influence of social categories may not be inevitable ( see van bavel and cunningham , 2011 for a discussion ) . the current paper utilizes electroencephalography ( eeg ) to examine the malleability of early perceptual processes in social categorization during the first few 100 ms of face perception . several recent studies using event - related potentials ( erps ) , which offer precise information about the timing of different cognitive processes as they unfold online , have shown that social categories can influence perceptual processing very quickly ( ito and cacioppo , 2000 ; smith et al . , people differentially process own - race and other - race faces within a few 100 ms of stimulus presentation ( see ito and bartholow , 2009 for a review ) . for instance , target race can modulate erps to faces as early as 122 ms after face onset ( ito and urland , 2003 ) . moreover , these racial biases in perceptual processing persist even when participants attend to another dimension of social categorization ( e.g. , gender ; ito and urland , 2003 ) or attempt to individuate the faces ( ito and urland , 2005 ) . consistent with most dual - process models of person perception , these results have led some researchers to conclude that racial biases in automatic attentional allocation can not be inhibited except under conditions of perceptual load ( ito et al . , 2007 , p. 410 ) or later during subsequent controlled processing ( e.g. , devine , 1989 ) . in contrast , recent developments in the cognitive and neural sciences suggest that human information processing is better characterized in terms of dynamical system models , rather than dual - process models ( see dehaene et al . , 2006 ; cunningham and zelazo , 2007 ; van bavel et al . , in press for recent reviews ) . in a dynamical systems approach , what have generally been considered to be inevitable automatic responses may be influenced by top - down processes . for example , the iterative reprocessing ( ir ) model ( cunningham and zelazo , 2007 ; cunningham et al . , 2007 ) , argues that a sharp distinction between automatic and controlled processes is not accurate ( see also freeman and ambady , 2011 ) . instead , the ir model suggests that goals and contextual features can shape the computations in brain regions involved in ostensibly automatic processes . as such , automatic responses even those occurring within 100200 ms of stimulus onset indeed , several behavioral studies have shown that goals or contextual factors can diminish the automatic activation of attitudes and stereotypes suggesting that automatic biases in social categorization are not inevitable ( see blair , 2002 for a review ) . for example , in a pair of recent studies , people who were assigned to a mixed - race team had relatively positive automatic evaluations toward in - group members on a response - window priming task , regardless of their race , whereas people who were not assigned to a mixed - race team had more positive automatic evaluations toward own - race versus other - race faces ( van bavel and cunningham , 2009 ) . however , because these studies only capture the behavioral consequences of perceptual and cognitive processing , it is difficult to determine the time course of these processes . it is , therefore , unclear whether these manipulations affected initial responses to social categories , altered underlying stereotypic or evaluative associations , or produced controlled processes to correct for initial biases ( see conrey et al . , 2005 ) . the current study was designed to determine if rapid responses to members of different social categories can be modulated by relatively transient , motivational states . prior research has shown that a variety of motivational states , such as approach / avoidance , can affect perception and attention ( e.g. , cacioppo et al . for example , non - black participants who repeatedly used a joystick to approach ( versus avoid ) black target stimuli were subsequently faster to associate the self with blacks and showed less racial bias on the implicit association task ( phills et al . , 2011 ; see also amodio , 2010 ) . therefore , in order to determine if motivational processes can modulate automatic social perception , we placed participants in an approach or avoidant frame during a person perception task while collecting scalp eeg data . specifically , we examined whether approaching other - race faces would attenuate racial biases in early perceptual processing . although the influence of social categories during person perception is widely distributed in the brain ( see cunningham and and van bavel , 2009 ) , social categories appear , in particular , to influence very early components of the face processing network ( see ito and bartholow , 2009 ) . for instance , the core and extended face processing network ( kanwisher et al . , 1997 ) , including the fusiform gyri and amygdala , respectively , have been associated with own - race ( golby et al . , 2001 ; lieberman et al . , 2005 ) and own - group ( van bavel et al . , although the relationship between specific brain regions and erp waves is not perfectly precise ( see luck , 2005 ) , very early erp waves , such as the p100 ( bentin et al . , 1996 ) and n170 ( herrmann et al . , 2005 ) appear to subserve early face processing . the p100 is the first positive going component and peaks around 100 ms following stimulus presentation with a source generated in the ventrolateral prestriate cortex ( martinez et al . , 1999 ; the n170 is a negative going component that peaks around 170 ms after stimulus presentation with a source generated in the fusiform and inferior - temporal gyri ( halgren et al . , 2000 ) . several studies have shown differential responses to race in the n170 ( e.g. , ito and urland , 2005 ; herrmann et al . , 2007 ) . for the purposes of the present research , we were interested in examining whether motivational states might influence these very rapid responses to the race of target faces . if the very early effects of social categories are inevitable , motivational states induced during person perception should not affect rapid biases that is , manipulating an approach / avoidance frame should have no impact on very early erp components , and participants should show racial bias in perceptual processing regardless of motivational state . however , if the earliest effects of social categories are not inevitable , but are sensitive to current motivational states , then biases in early erp components may differ depending on whether people are in an approach versus avoidance frame . specifically , we predict that race - based biases will be attenuated when participants are approaching versus avoiding social stimuli because social categories are less likely to be used in person perception in an approach - motivated state . a person that one approaches is more motivationally relevant than a person that one avoids , and prior studies have shown that altering the motivational relevance of social stimuli through manipulations of processing goals ( cunningham et al . , 2008 ) and group membership ( van bavel et al . , 2008 ) affects the way that people are perceived and evaluated . in particular , increasing motivational relevance during person perception seems to increase the extent to which a target is individuated rather than processed in terms of a category membership ( e.g. , neuberg and fiske , 1987 ; van bavel et al . , thus , if motivational processes can affect initial social categorization , then putting people in the mindset of approaching others should alter the immediate processing of race and attenuate racial bias in early perceptual processing . fourteen white male undergraduate psychology students from the university of toronto completed this study for partial completion of course credit or $ 15 . , participants were informed that they would be completing an experiment designed to examine the neural processing underlying social cognition . participants were asked to respond to the presentation of faces with a joystick as quickly as possible . the experiment employed a block design in which participants approached or avoided blocks of three faces presented in succession . to simulate approach and avoidance toward the faces , participants pulled or pushed a joystick at the onset of each face ( cacioppo et al . at the start of each block an instruction screen appeared for 2 s. during the approach blocks , this instruction screen indicated that participants should pull each face toward them . during the avoidance blocks , this instruction screen indicated that participants should push each face away from them . twenty - four faces of black or white college - aged males were presented randomly during the blocks ( taken from van bavel and cunningham , 2009 ) . faces were fully counterbalanced across conditions to ensure that interactive effects with condition could not be attributed to low - level features of the stimuli . participants saw four runs of 16 blocks of three trials each for a total of 192 trials ( 48 trials per condition ) . in each block , three faces were presented for 1 s each , and 3 s of fixation separated each face . to create and maintain a motivational mental set , two runs included approach blocks and two runs included avoid blocks ( randomized within participants ) . to minimize blink artifacts during experimental trials , runs were separated by a 12 s rest period and participants were encouraged to blink during this period rather than during the task . further , between each set of three faces , participants were instructed to blink if necessary during this period . scalp electroencephalographic data were acquired with a 128-channel ant system ( advanced neuro technology , netherlands ) using a 64-channel acquisition setup , sampled at 512 hz , using an average reference , digitally filtered off - line with a 115 hz bandpass filter . fourteen white male undergraduate psychology students from the university of toronto completed this study for partial completion of course credit or $ 15 . upon arrival at the lab , participants were informed that they would be completing an experiment designed to examine the neural processing underlying social cognition . participants were asked to respond to the presentation of faces with a joystick as quickly as possible . the experiment employed a block design in which participants approached or avoided blocks of three faces presented in succession . to simulate approach and avoidance toward the faces , participants pulled or pushed a joystick at the onset of each face ( cacioppo et al . at the start of each block an instruction screen appeared for 2 s. during the approach blocks , this instruction screen indicated that participants should pull each face toward them . during the avoidance blocks , this instruction screen indicated that participants should push each face away from them . twenty - four faces of black or white college - aged males were presented randomly during the blocks ( taken from van bavel and cunningham , 2009 ) . faces were fully counterbalanced across conditions to ensure that interactive effects with condition could not be attributed to low - level features of the stimuli . participants saw four runs of 16 blocks of three trials each for a total of 192 trials ( 48 trials per condition ) . in each block , three faces were presented for 1 s each , and 3 s of fixation separated each face . to create and maintain a motivational mental set , two runs included approach blocks and two runs included avoid blocks ( randomized within participants ) . to minimize blink artifacts during experimental trials , runs were separated by a 12 s rest period and participants were encouraged to blink during this period rather than during the task . further , between each set of three faces , participants were instructed to blink if necessary during this period . scalp electroencephalographic data were acquired with a 128-channel ant system ( advanced neuro technology , netherlands ) using a 64-channel acquisition setup , sampled at 512 hz , using an average reference , digitally filtered off - line with a 115 hz bandpass filter . after deleting trials with noncephalic artifacts using besa default settings ( megis software , germany ) , each participant 's trials were aggregated based on trial type . on average , trials in each the four stimulus conditions ( black - pull , black - push , white - pull , white - push ) were averaged to create grand average waveforms for each electrode , as well as a grand average aggregating across all trials . because eeg data is not independent , a source modeling analysis was run on the average waveform in order to characterize the whole brain signal ( see smith et al . , 2003 ) , using the full time series for the epoch and all of the electrodes as input using besa 5.2 . the average waveform was used for source modeling to avoid biasing the results toward any particular condition . the resulting pca indicated that one latent source centered in the right occipito - temporal cortex accounted for 90.2% of the observed variance ( see figure 1a ) . the occipito - temporal cortex plays a key role in face processing ( kanwisher et al . , 1997 ) and this region has been associated with own - race ( golby et al . , 2001 ; lieberman et al . , 2005 ) and own - group ( van bavel et al . , 2008 , 2011 ) biases in social perception . an additional loreta source analysis replicated this localization to the right fusiform gyrus ( see figure 1b ) . plotting the latent time courses , we found that this latent variable included both the p100 and n170 components typically found in studies of attention and face processing for each of the four experimental conditions ( figure 2 ) . these results are consistent with previous studies that have shown the p100 ( bentin et al . , 1996 ) and n170 may be associated with processing in the fusiform gyrus ( herrmann et al . , 2005 ) . therefore , in order to test our hypotheses , we analyzed these waveforms as a function of target race and motivational condition . latent time courses for black and white faces in the pull and push conditions . because these values are projected from latent space , values on the y axis are scaled in arbitrary units with respect to underlying microvolts . if the early effects of social categories such as race are inevitable , there should only be a main effect of race on early erp waveforms ( ito et al . , 2007 ) , regardless of motivational state . however , if early processes in person perception are malleable , there should be an interaction between race and motivational state , such that any pattern of racial bias in the avoidance blocks should be attenuated in the approach blocks . to examine this hypothesis , we back projected the latent variable to create individual scores for each participant for each trial type . these scores were subjected to a 2 ( race : black , white ) 2 ( motivational state : approach , avoid ) anova . separate analyses were conducted for the p100 ( mean amplitude between 90 and 110 ms ) and n170 ( mean amplitude between 135 and 200 ms ) components of the waveform . consistent with the prior evidence that people show racial bias during early perceptual process , white faces were associated with a larger p100 than black faces , [ f(1 , 13 ) = 12.68 , p < 0.01 , partial = 0.49 ] ( see figure 3 ) . this pattern replicates previous research showing an own - race bias during very automatic facial processing ( e.g. , ito et al . , 2004 ) . importantly , this own - race effect was qualified by a race motivational state interaction , [ f(1 , 13 ) = 4.32 , p = 0.05 , partial = 0.25 ] . simple effects indicated that the own - race effect was significant for the avoid blocks , [ f(1 , 13 ) = 12.51 , p < 0.01 ] , but not the approach blocks , [ f(1 , 13 ) = 4.07 , p = 0.07 ] . moreover , a contrast pitting all other conditions versus the black - push condition ( 1 , 1 , 1 , 3 ) was significant , [ f(1 , 13 ) = 10.66 , p = 0.01 ] . these results are consistent with the suggestion that approach oriented motivational states can alter the influence of social categories even during the first 100 ms of perceptual processing . when considering the later n170 component , there was no main effect of race , [ f(1 , 13 ) = 1.25 , p = 0.28 , partial = 0.09 ] , nor was there a race motivational state interaction , [ f(1 , 13 ) = 0.62 , p = 0.45 , partial = 0.05 ] . mean amplitudes for the p100 for black and white faces in the pull and push conditions . for these analyses , we averaged data from the right and left occipital electrodes to create two waveforms of interest . the p100 and the n170 mean amplitudes were extracted from the subject level grand averages using the same time windows as above and were subjected to a 2 ( race : black , white ) 2 ( motivational state : approach , avoid ) 2 ( laterality : right , left occipital electrodes ) anova . replicating the results from the latent variable analysis , we found a main effect of race , [ f(1 , 13 ) = 9.20 , p < 0.01 ] , and a race motivational state interaction , [ f(1 , 13 ) = 9.00 , p < 0.01 ] , for the p100 component . specifically , the p100 difference between black and white faces was larger for the avoid blocks ( mwhite = 2.68 ; mblack = 1.22 ) than the approach blocks ( mwhite = 2.04 ; mblack = 1.58 ) . unlike the latent variable analysis , we did find a race motivational state for the n170 [ f(1 , 13 ) = 5.91 , p < 0.03 ] . this analysis suggested that there was a larger n170 response to black faces than white faces ( mwhite = 1.07 ; mwhite = 0.45 ) for the avoid blocks , but not the approach blocks ( mblack = 0.76 ; mwhite = 0.75 ) . however , this effect was not significant when controlling for the p100 effects [ f(1 , 13 ) = 1.55 , p = ns ] . the current research is consistent with the idea that the earliest aspects of social perception are flexible and sensitive to motivational frames . putting people in the mindset of approaching others attenuated racial bias in very early perceptual processing . specifically , when people pushed a joystick away from themselves an experimental manipulation designed to induce an avoidance motivation they showed an own - race bias , such that early perceptual processing ( as indexed by erp activity around 100 ms after stimulus onset ) was stronger to own - race than other - race faces . however , this own - race bias was modulated by motivational state , such that approaching faces by pulling a joystick toward oneself reduced the bias in neural activity . this finding challenges theories suggesting that early biases associated with social categorization are inevitable and only interrupted by controlled processing ( e.g. , devine , 1989 ) or perceptual load ( e.g. , ito et al . , 2007 ) . these models are difficult to reconcile with the current results since the p100 occurs much faster than downstream corrective processes ( amodio et al . , 2008 ) , and there is no reason to believe that the two conditions in the current study ( approach versus avoid ) differ in terms of perceptual demands . instead , this research adds to a growing literature demonstrating that motivational processes can influence the most automatic aspects of social perception and evaluation ( e.g. , cunningham et al . , 2005 ; additionally , this research adds to the literature suggesting that motivational relevance can determine whether top - down processes will override automatic , bottom - up perceptual and attentional effects ( cunningham et al . , 2008 ; by showing that very early processes in person perception are sensitive to motivational states , this research demonstrates that processes once thought to be inevitable may in fact be malleable . as such , dual - process models of social perception may be unable to account for the flexibility of automatic social perception and evaluation ( see cunningham et al . , 2007 ) . addressing the question of the inevitability of attentional biases to social categories requires measures that are highly temporally sensitive . whereas other research has shown that biases associated with social categorization can be eliminated in a number of ways , such as by getting people to focus on a different dimension of categorization ( e.g. , van bavel and cunningham , 2009 ) , the behavioral methods typically used to measure bias do not provide sufficient information to determine if these biases are circumvented during early perceptual processing or rapidly corrected after the fact . by using a measure with exquisite temporal resolution ( i.e. , eeg ) , we were able to determine that very rapid aspects of social categorization are not inevitable and can be altered by modifying motivational states . ( 2007 ) conclusion regarding the inevitability of early attentional biases in social categorization was based on the n170 waveform , while our results were found for the p100 waveform . although they likely differ in important ways , both waveforms are related to early attentional processing ( bentin et al . , 1996 ; clark and hilyard , 1996 ) as well as face processing ( e.g. , liu et al . , 2002 ; herrmann et al . , moreover , a recent study found that people with greater left alpha asymmetries in the prefrontal cortex a correlate of approach motivation had different erp responses to black ( versus white ) faces on the p2 , which peaked approximately 170 ms following stimulus presentation ( amodio , 2010 ) . in that study , p2 responses to black faces were also associated with decreased racial bias on an implicit measure of racial stereotypes . together with the current research , these results suggest that motivation may help one have less prejudiced responses by modulating perception ( see balcetis and dunning , 2006 ) . the current research suggests that very early effects of social categorization can be modulated before they impact subsequent perceptions , evaluations , or behavior . given that post - categorization control often has negative side effects , like rebound ( macrae et al . , 1994 ) and depletion ( richeson and shelton , 2003 ; gordijn et al . , 2004 ) , altering initial processing of social categories by changing motivational states provides a powerful alternative for changing these biases . while preventing the effects of attentional biases associated with social categorization is not necessarily positive ( trawalter et al . , 2008 ) , it does provide an opportunity to overcome ostensibly automatic stereotyping and prejudice . knowing which steps in the processing sequence are malleable is important for understanding what types of interventions will be successful in preventing the downstream consequences of social categorization . 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 . 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 .

research on person categorization suggests that people automatically and inflexibly categorize others according to group memberships , such as race . consistent with this view , research using electroencephalography ( eeg ) has found that white participants tend to show an early difference in processing black versus white faces . yet , new research has shown that these ostensibly automatic biases may not be as inevitable as once thought and that motivational influences may be able to eliminate these biases . it is unclear , however , whether motivational influences shape the initial biases or whether these biases can only be modulated by later , controlled processes . using eeg to examine the time course of biased processing , we manipulated approach and avoidance motivational states by having participants pull or push a joystick , respectively , while viewing white or black faces . consistent with previous work on own - race bias , we observed a greater p100 response to white than black faces ; however , this racial bias was attenuated in the approach condition . these data suggest that rapid social perception may be flexible and can be modulated by motivational states .

Introduction Materials and methods Participants Experimental design Results Discussion Conclusion Conflict of interest statement

people often perceive others according to their race , gender , or other social category membership ( brewer , 1988 ; fiske and neuberg , 1990 ) . this process of social categorization provides an efficient way to understand others and guides the direction of limited attentional and cognitive resources . in the past few decades , social psychologists have found extensive evidence that social categorization can occur rapidly and without intention , effort , or conscious control , triggering stereotypes ( devine , 1989 ) , prejudice ( fazio et al . , 1997 ) . several dual - process models of person perception have proposed that processing others according to social category membership is the initial stage in person perception , and that only sufficiently motivated perceivers individuate targets or correct for initial categorical judgments in a later stage ( e.g. however , while there is evidence that social categories influence the earliest phases of social perception , others have argued that the initial influence of social categories may not be inevitable ( see van bavel and cunningham , 2011 for a discussion ) . the current paper utilizes electroencephalography ( eeg ) to examine the malleability of early perceptual processes in social categorization during the first few 100 ms of face perception . several recent studies using event - related potentials ( erps ) , which offer precise information about the timing of different cognitive processes as they unfold online , have shown that social categories can influence perceptual processing very quickly ( ito and cacioppo , 2000 ; smith et al . , people differentially process own - race and other - race faces within a few 100 ms of stimulus presentation ( see ito and bartholow , 2009 for a review ) . for instance , target race can modulate erps to faces as early as 122 ms after face onset ( ito and urland , 2003 ) . , gender ; ito and urland , 2003 ) or attempt to individuate the faces ( ito and urland , 2005 ) . consistent with most dual - process models of person perception , these results have led some researchers to conclude that racial biases in automatic attentional allocation can not be inhibited except under conditions of perceptual load ( ito et al . in contrast , recent developments in the cognitive and neural sciences suggest that human information processing is better characterized in terms of dynamical system models , rather than dual - process models ( see dehaene et al . , in press for recent reviews ) . in a dynamical systems approach , what have generally been considered to be inevitable automatic responses may be influenced by top - down processes . for example , the iterative reprocessing ( ir ) model ( cunningham and zelazo , 2007 ; cunningham et al . , 2007 ) , argues that a sharp distinction between automatic and controlled processes is not accurate ( see also freeman and ambady , 2011 ) . instead , the ir model suggests that goals and contextual features can shape the computations in brain regions involved in ostensibly automatic processes . as such , automatic responses even those occurring within 100200 ms of stimulus onset indeed , several behavioral studies have shown that goals or contextual factors can diminish the automatic activation of attitudes and stereotypes suggesting that automatic biases in social categorization are not inevitable ( see blair , 2002 for a review ) . for example , in a pair of recent studies , people who were assigned to a mixed - race team had relatively positive automatic evaluations toward in - group members on a response - window priming task , regardless of their race , whereas people who were not assigned to a mixed - race team had more positive automatic evaluations toward own - race versus other - race faces ( van bavel and cunningham , 2009 ) . however , because these studies only capture the behavioral consequences of perceptual and cognitive processing , it is difficult to determine the time course of these processes . it is , therefore , unclear whether these manipulations affected initial responses to social categories , altered underlying stereotypic or evaluative associations , or produced controlled processes to correct for initial biases ( see conrey et al . , 2005 ) . the current study was designed to determine if rapid responses to members of different social categories can be modulated by relatively transient , motivational states . prior research has shown that a variety of motivational states , such as approach / avoidance , can affect perception and attention ( e.g. , cacioppo et al . for example , non - black participants who repeatedly used a joystick to approach ( versus avoid ) black target stimuli were subsequently faster to associate the self with blacks and showed less racial bias on the implicit association task ( phills et al . , 2011 ; see also amodio , 2010 ) . therefore , in order to determine if motivational processes can modulate automatic social perception , we placed participants in an approach or avoidant frame during a person perception task while collecting scalp eeg data . specifically , we examined whether approaching other - race faces would attenuate racial biases in early perceptual processing . although the influence of social categories during person perception is widely distributed in the brain ( see cunningham and and van bavel , 2009 ) , social categories appear , in particular , to influence very early components of the face processing network ( see ito and bartholow , 2009 ) . for instance , the core and extended face processing network ( kanwisher et al . , 1997 ) , including the fusiform gyri and amygdala , respectively , have been associated with own - race ( golby et al . , 2005 ) and own - group ( van bavel et al . , although the relationship between specific brain regions and erp waves is not perfectly precise ( see luck , 2005 ) , very early erp waves , such as the p100 ( bentin et al . the p100 is the first positive going component and peaks around 100 ms following stimulus presentation with a source generated in the ventrolateral prestriate cortex ( martinez et al . , 1999 ; the n170 is a negative going component that peaks around 170 ms after stimulus presentation with a source generated in the fusiform and inferior - temporal gyri ( halgren et al . , 2000 ) . several studies have shown differential responses to race in the n170 ( e.g. , ito and urland , 2005 ; herrmann et al . for the purposes of the present research , we were interested in examining whether motivational states might influence these very rapid responses to the race of target faces . if the very early effects of social categories are inevitable , motivational states induced during person perception should not affect rapid biases that is , manipulating an approach / avoidance frame should have no impact on very early erp components , and participants should show racial bias in perceptual processing regardless of motivational state . however , if the earliest effects of social categories are not inevitable , but are sensitive to current motivational states , then biases in early erp components may differ depending on whether people are in an approach versus avoidance frame . specifically , we predict that race - based biases will be attenuated when participants are approaching versus avoiding social stimuli because social categories are less likely to be used in person perception in an approach - motivated state . a person that one approaches is more motivationally relevant than a person that one avoids , and prior studies have shown that altering the motivational relevance of social stimuli through manipulations of processing goals ( cunningham et al . , 2008 ) affects the way that people are perceived and evaluated . , thus , if motivational processes can affect initial social categorization , then putting people in the mindset of approaching others should alter the immediate processing of race and attenuate racial bias in early perceptual processing . fourteen white male undergraduate psychology students from the university of toronto completed this study for partial completion of course credit or $ 15 . , participants were informed that they would be completing an experiment designed to examine the neural processing underlying social cognition . participants were asked to respond to the presentation of faces with a joystick as quickly as possible . the experiment employed a block design in which participants approached or avoided blocks of three faces presented in succession . to simulate approach and avoidance toward the faces , participants pulled or pushed a joystick at the onset of each face ( cacioppo et al . at the start of each block an instruction screen appeared for 2 s. during the approach blocks , this instruction screen indicated that participants should pull each face toward them . during the avoidance blocks , this instruction screen indicated that participants should push each face away from them . faces were fully counterbalanced across conditions to ensure that interactive effects with condition could not be attributed to low - level features of the stimuli . participants saw four runs of 16 blocks of three trials each for a total of 192 trials ( 48 trials per condition ) . in each block , three faces were presented for 1 s each , and 3 s of fixation separated each face . to create and maintain a motivational mental set , two runs included approach blocks and two runs included avoid blocks ( randomized within participants ) . further , between each set of three faces , participants were instructed to blink if necessary during this period . upon arrival at the lab , participants were informed that they would be completing an experiment designed to examine the neural processing underlying social cognition . participants were asked to respond to the presentation of faces with a joystick as quickly as possible . to simulate approach and avoidance toward the faces , participants pulled or pushed a joystick at the onset of each face ( cacioppo et al . at the start of each block an instruction screen appeared for 2 s. during the approach blocks , this instruction screen indicated that participants should pull each face toward them . during the avoidance blocks , this instruction screen indicated that participants should push each face away from them . faces were fully counterbalanced across conditions to ensure that interactive effects with condition could not be attributed to low - level features of the stimuli . to minimize blink artifacts during experimental trials , runs were separated by a 12 s rest period and participants were encouraged to blink during this period rather than during the task . scalp electroencephalographic data were acquired with a 128-channel ant system ( advanced neuro technology , netherlands ) using a 64-channel acquisition setup , sampled at 512 hz , using an average reference , digitally filtered off - line with a 115 hz bandpass filter . because eeg data is not independent , a source modeling analysis was run on the average waveform in order to characterize the whole brain signal ( see smith et al . the resulting pca indicated that one latent source centered in the right occipito - temporal cortex accounted for 90.2% of the observed variance ( see figure 1a ) . the occipito - temporal cortex plays a key role in face processing ( kanwisher et al . , 1997 ) and this region has been associated with own - race ( golby et al . , 2005 ) and own - group ( van bavel et al . , 2008 , 2011 ) biases in social perception . plotting the latent time courses , we found that this latent variable included both the p100 and n170 components typically found in studies of attention and face processing for each of the four experimental conditions ( figure 2 ) . these results are consistent with previous studies that have shown the p100 ( bentin et al . , 1996 ) and n170 may be associated with processing in the fusiform gyrus ( herrmann et al . , 2005 ) . therefore , in order to test our hypotheses , we analyzed these waveforms as a function of target race and motivational condition . latent time courses for black and white faces in the pull and push conditions . if the early effects of social categories such as race are inevitable , there should only be a main effect of race on early erp waveforms ( ito et al . , 2007 ) , regardless of motivational state . however , if early processes in person perception are malleable , there should be an interaction between race and motivational state , such that any pattern of racial bias in the avoidance blocks should be attenuated in the approach blocks . to examine this hypothesis , we back projected the latent variable to create individual scores for each participant for each trial type . these scores were subjected to a 2 ( race : black , white ) 2 ( motivational state : approach , avoid ) anova . separate analyses were conducted for the p100 ( mean amplitude between 90 and 110 ms ) and n170 ( mean amplitude between 135 and 200 ms ) components of the waveform . consistent with the prior evidence that people show racial bias during early perceptual process , white faces were associated with a larger p100 than black faces , [ f(1 , 13 ) = 12.68 , p < 0.01 , partial = 0.49 ] ( see figure 3 ) . this pattern replicates previous research showing an own - race bias during very automatic facial processing ( e.g. , ito et al . , 2004 ) . importantly , this own - race effect was qualified by a race motivational state interaction , [ f(1 , 13 ) = 4.32 , p = 0.05 , partial = 0.25 ] . simple effects indicated that the own - race effect was significant for the avoid blocks , [ f(1 , 13 ) = 12.51 , p < 0.01 ] , but not the approach blocks , [ f(1 , 13 ) = 4.07 , p = 0.07 ] . moreover , a contrast pitting all other conditions versus the black - push condition ( 1 , 1 , 1 , 3 ) was significant , [ f(1 , 13 ) = 10.66 , p = 0.01 ] . these results are consistent with the suggestion that approach oriented motivational states can alter the influence of social categories even during the first 100 ms of perceptual processing . when considering the later n170 component , there was no main effect of race , [ f(1 , 13 ) = 1.25 , p = 0.28 , partial = 0.09 ] , nor was there a race motivational state interaction , [ f(1 , 13 ) = 0.62 , p = 0.45 , partial = 0.05 ] . mean amplitudes for the p100 for black and white faces in the pull and push conditions . for these analyses , we averaged data from the right and left occipital electrodes to create two waveforms of interest . replicating the results from the latent variable analysis , we found a main effect of race , [ f(1 , 13 ) = 9.20 , p < 0.01 ] , and a race motivational state interaction , [ f(1 , 13 ) = 9.00 , p < 0.01 ] , for the p100 component . specifically , the p100 difference between black and white faces was larger for the avoid blocks ( mwhite = 2.68 ; mblack = 1.22 ) than the approach blocks ( mwhite = 2.04 ; mblack = 1.58 ) . unlike the latent variable analysis , we did find a race motivational state for the n170 [ f(1 , 13 ) = 5.91 , p < 0.03 ] . this analysis suggested that there was a larger n170 response to black faces than white faces ( mwhite = 1.07 ; mwhite = 0.45 ) for the avoid blocks , but not the approach blocks ( mblack = 0.76 ; mwhite = 0.75 ) . however , this effect was not significant when controlling for the p100 effects [ f(1 , 13 ) = 1.55 , p = ns ] . the current research is consistent with the idea that the earliest aspects of social perception are flexible and sensitive to motivational frames . putting people in the mindset of approaching others attenuated racial bias in very early perceptual processing . specifically , when people pushed a joystick away from themselves an experimental manipulation designed to induce an avoidance motivation they showed an own - race bias , such that early perceptual processing ( as indexed by erp activity around 100 ms after stimulus onset ) was stronger to own - race than other - race faces . however , this own - race bias was modulated by motivational state , such that approaching faces by pulling a joystick toward oneself reduced the bias in neural activity . , ito et al . , 2007 ) . these models are difficult to reconcile with the current results since the p100 occurs much faster than downstream corrective processes ( amodio et al . , 2008 ) , and there is no reason to believe that the two conditions in the current study ( approach versus avoid ) differ in terms of perceptual demands . instead , this research adds to a growing literature demonstrating that motivational processes can influence the most automatic aspects of social perception and evaluation ( e.g. , cunningham et al . , 2005 ; additionally , this research adds to the literature suggesting that motivational relevance can determine whether top - down processes will override automatic , bottom - up perceptual and attentional effects ( cunningham et al . , 2008 ; by showing that very early processes in person perception are sensitive to motivational states , this research demonstrates that processes once thought to be inevitable may in fact be malleable . as such , dual - process models of social perception may be unable to account for the flexibility of automatic social perception and evaluation ( see cunningham et al . addressing the question of the inevitability of attentional biases to social categories requires measures that are highly temporally sensitive . whereas other research has shown that biases associated with social categorization can be eliminated in a number of ways , such as by getting people to focus on a different dimension of categorization ( e.g. , van bavel and cunningham , 2009 ) , the behavioral methods typically used to measure bias do not provide sufficient information to determine if these biases are circumvented during early perceptual processing or rapidly corrected after the fact . by using a measure with exquisite temporal resolution ( i.e. , eeg ) , we were able to determine that very rapid aspects of social categorization are not inevitable and can be altered by modifying motivational states . ( 2007 ) conclusion regarding the inevitability of early attentional biases in social categorization was based on the n170 waveform , while our results were found for the p100 waveform . although they likely differ in important ways , both waveforms are related to early attentional processing ( bentin et al . , 1996 ; clark and hilyard , 1996 ) as well as face processing ( e.g. , moreover , a recent study found that people with greater left alpha asymmetries in the prefrontal cortex a correlate of approach motivation had different erp responses to black ( versus white ) faces on the p2 , which peaked approximately 170 ms following stimulus presentation ( amodio , 2010 ) . in that study , p2 responses to black faces were also associated with decreased racial bias on an implicit measure of racial stereotypes . together with the current research , these results suggest that motivation may help one have less prejudiced responses by modulating perception ( see balcetis and dunning , 2006 ) . the current research suggests that very early effects of social categorization can be modulated before they impact subsequent perceptions , evaluations , or behavior . given that post - categorization control often has negative side effects , like rebound ( macrae et al . , 1994 ) and depletion ( richeson and shelton , 2003 ; gordijn et al . , 2004 ) , altering initial processing of social categories by changing motivational states provides a powerful alternative for changing these biases . while preventing the effects of attentional biases associated with social categorization is not necessarily positive ( trawalter et al . , 2008 ) , it does provide an opportunity to overcome ostensibly automatic stereotyping and prejudice . knowing which steps in the processing sequence are malleable is important for understanding what types of interventions will be successful in preventing the downstream consequences of social categorization . 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 . 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 .

metabolism influences brain activity , and metabolic dysfunction is associated with a wide variety of neurological disorders . the cause - and - effect relationship between metabolic and neuronal dysfunction is often unclear , though not in the case of epilepsy and diet . historical observations noted the therapeutic benefits of fasting on epilepsy , but fasting is necessarily a time - limited practice . therapeutic benefits of the metabolic condition of fasting were confirmed over 90 years ago when the high fat , low - carbohydrate ketogenic diet ( kd ) was described as alternative to fasting which still reduced epileptic seizures ( wilder , 1921 ) . in turn , anticonvulsant drugs debuted over the next two decades , such that since then the kd has been used mostly for inoperable and medication - resistant epilepsy , which has been estimated to be 15% up to 45% of cases ( picot et al . although used clinically for many decades , prescribed most often to children , and increasing in popularity over the last two decades , the kd s mechanism of action remains controversial . the kd was designed to produce ketosis without fasting by strictly limiting carbohydrate intake ( wilder , 1921 ) . to make up for lost calories and augment ketosis , when carbohydrate intake is strongly limited ( as during the kd or fasting ) , the liver increases production of the ketone bodies -hydroxybutyrate , acetoacetate , and acetone from circulating fatty acids ( aoki , 1981 ) . because of the -hydroxyl substitution , -hydroxybutyrate is not actually a ketone , ketone bodies are released into the circulation as an alternative energy source to generate atp ( ketolytic metabolism ) within tissues , including the brain and spinal cord . formulation of the kd is calculated using a ratio of fat content to combined protein and carbohydrate content , varying in the clinic from 5:1 to 1:1 depending on a patient s individual needs ( swink et al . , 1997 ; vining , 1999 ) . we found that a kd - fed ad libitum at ratio of 7:1 or 3:1 to rats produced similar changes in blood chemistry ( figure 1 ) . clinically , the trend has been to decrease the ratio where possible and thus make the diet more palatable ( including the more liberal modified atkins diet ; kang et al . , 2007 ; kossoff et al . , 2008b ) but more systematic research is needed . regarding different food types , the kd has now been adapted for widely varying cultures and cuisines in different countries around the world ( e.g. , india , korea , united kingdom , saudi arabia , republic of georgia ; kang et al . , 2007 ; neal et al . , 2008a ; sharma et al . , 2009 ; b. zupec - kania , personal communication ) . understanding the mechanisms by which a diet controls seizures , along with broader opportunities for metabolic therapies , remains an active research topic because of accessibility , efficacy , and economics . ketogenic diets can produce prompt and sustained ketosis and mild hypoglycemia in experimental rodents . here , young male sprague - dawley rats were fed with one of two ketogenic diets for 19 days , or remained fed with normal rodent chow . both kds , with strengths of 3:1 and 7:1 ( bioserv 5140 and 3666 , respectively ) , produced similar and significantly increased blood ketones and reduced blood glucose within 2 days and lasting until the last test day . number of subjects was 1214 . * p < 0.05 , * * p < 0.01 , * * * p < the kd might alleviate seizures and other pathological states partially by providing elevated levels of high - energy molecules ( e.g. , atp , phosphocreatine ) and increased capacity for energy generation ( increased mitochondrial number ; seyfried and mukherjee , 2005 ; bough and rho , 2007 ; masino and geiger , 2008 ) . yet , numerous other changes due to the kd have been hypothesized to underlie increased inhibition and/or decreased excitation in brain , and thus to an anticonvulsant / neuroprotective state . in normal humans fed a kd , electroencephalography and transcranial magnetic stimulation demonstrated increased inhibition in the cerebral cortex , with a magnitude similar to that seen after benzodiazepine administration ( cantello et al . , 2007 ) . with the more extensive investigation possible in experimental animals , a kd was shown to enhance paired - pulse depression , shift the input / output relationship rightward , elevate the threshold for maximal electrical activation , and to block spreading depression - style events in the hippocampus in vivo ( bough et al . , 2003 ) . there have been surprisingly few detailed studies on detailed synaptic effects , likely because of the difficulty in performing such studies in vivo , coupled with the typical glucose - based incubation protocol for in vitro slices ; to date , a kd incubation protocol has not been standardized , although recent work sampling cerebrospinal fluid in kd - fed animals might provide a starting point ( samala et al . , 2011 ) . currently , the major proposed mechanisms for such increased inhibition and/or decreased excitation include increased levels of adenosine , a major inhibitory neuromodulator ( masino and geiger , 2008 ) ; increased levels of -aminobutyric acid ( gaba ) , a major inhibitory neurotransmitter ( yudkoff et al . , 2007 ; omote et al . , 2011 ) ; decreased glutamate , a major excitatory neurotransmitter ( lund et al . , 2009 ; juge et al . , 2010 ) and direct effects of elevated ketone bodies on ion channels ( ma et al . , 2007 ) . increased inhibition or decreased excitability , if sufficiently strong , might not only suppress seizures but also influence normal brain function . many types of normal brain function , as well as recovery from injury , are thought to depend on synaptic plasticity , i.e. , the malleability , either temporary or long - lasting , of the strength of neuronal communication ( davis et al . , 1992 ; goosens and maren , 2002 ) . long - term potentiation ( ltp ) is a sustained increase in synaptic efficacy which can be observed in a number of brain regions including its original discovery site , the hippocampus ( bliss and lmo , 1973 ; bramham and srebro , 1989 ; clugnet and ledoux , 1990 ; bonci and malenka , 1999 ; mahon et al . , 2004 ) . studies have linked metabolism and ltp ( potter et al . , 2010 ) ; we and our collaborators characterized the effects of a kd on hippocampal ltp with the hypothesis that kd - related inhibition or reduced excitation might affect brain plasticity ( koranda et al . , 2011 ) . after 3 weeks on a 7:1 kd , baseline synaptic measurements were taken in the perforant path - dentate gyrus pathway and ltp was induced with tetanic stimulation and the response measured over the next 2 days . the kd had no significant effects on measures of short - term plasticity ( paired - pulse depression , paired - pulse facilitation ) , and did not prevent ltp induction , whereas the magnitude of the potentiation was significantly smaller in kd - fed rats . the ltp magnitude remained lower in these rats out to the longest tested time point ( 48 h ) . as discussed below , cognitive effects of the diet are mixed in animals and overall positive in humans . in addition , it is important to note that 7:1 is a stronger diet ratio than that used clinically , animals used had never had seizures , and another paper looking at the kd on ltp in vivo in anesthetized animals did not find any differences ( thio et al . , 2010 ) . to test the role of adenosine in the kd s ability to reduce seizures , we and our collaborators recently tested the effectiveness of a kd in a transgenic mouse with spontaneous hippocampal electrographic seizures due to adenosine deficiency . these mice overexpress the adenosine - metabolizing enzyme adenosine kinase ( adk ) in brain ( fedele et al . , 2005 ) , and tonic levels of the endogenous inhibitor adenosine are therefore lower than normal . at baseline , seizures recorded with chronically implanted electrodes occur five times per hour , on average ( masino et al . , this antiseizure effect depended on low glucose ( seizures were restored by a peripheral injection of glucose ) , and activation of the adenosine a1 receptor subtype ( a1r ; seizure activity was restored by injection of a selective a1r antagonist ) . together , this evidence suggests that the kd exerts antiseizure effects by restoring adenosine levels and a1r activation via a mechanism related to low glucose . these mice also have spontaneous electrographic seizures in the hippocampus , but the kd has no effect on seizure frequency in a1r knockout mice , and is partially effective in mice heterozygous for the a1r ( masino et al . , 2011 ) . although these models all involve seizures induced by a lack of adenosinergic modulation , the results are likely generalizable : adenosine has been found to be anticonvulsive / antiseizure in virtually every seizure model in which it has been tested ( excepting a1r knockout mice providing further evidence for the primary anticonvulsant role of a1rs ) . adenosine in particular , and a kd in general , might offer more homeostatic upstream bioenergetic regulation of neuronal activity , and possibly long - term benefits on brain homeostasis , than highly specific drug therapies ( boison et al . , 2011 ) . regarding ltp , previous results consistent with the involvement of adenosine in kd effects have shown that adenosine reduces ltp magnitude when present during induction ( mitchell et al . , 1993 ; costenla et al . , 1999 ; de mendonca and ribeiro , 2000 ; fujii et al . ; but see pascual et al . , 2005 ) and , when applied after induction , promotes reversal of existing ltp ( huang et al . , 1999 ; fujii et al . yet , the lack of effects of the kd on input output relationships and short - term plasticity seem to argue against the tonic involvement of adenosine ( koranda et al . , 2011 ) . mechanism aside , the kd can limit excessive neuronal activity ( a class into which the neuronal activity during an ltp induction burst certainly applies ) and perhaps reset baseline activity . compared to in vivo , in vitro paradigms can provide tighter control over experimental variables , allowing for a more thorough characterization of mechanisms . effects of kd feeding on baseline excitability are inconsistent in vitro , however ( stafstrom et al . certainly , the metabolic state established by a kd might be disrupted during tissue preparation for in vitro work . as introduced briefly above , one of the biochemical effects associated with a kd is an abundance of high - energy molecules ( devivo et al . , 1978 ; masino et al . , 2007 ) , as well as increased mitochondrial biogenesis , respiration , and expression of atp synthesis - related proteins ( noh et al . , 2006 ; nylen et al . , 2009 ; balietti et al . , 2010 ) . we modeled key aspects of the kd in vitro by maintaining or increasing intracellular atp while decreasing extracellular glucose in individual ca3 pyramidal neurons in acute hippocampal slices . we varied atp ( 0.55.0 mm ; 2 mm is standard ) in the patch pipet and changed glucose concentration of the bathing solution from 11 mm ( standard ) to either 7 or 3 mm ( kawamura et al . , 2010 ) . note that 3 mm glucose is still a physiological level : in vivo brain concentrations are near 3 mm ( hu and wilson , 1997 ; shram et al . , 1997 ) . moderately lowered extracellular glucose has been reported to attenuate epileptiform activity in brain slices ( kirchner et al . , 2006 ) , whereas experimental studies of pathological hypoglycemia often remove glucose completely from the bathing medium ( aglycemia ; tromba et al . we found that when intracellular atp levels were adequate or high ( 1.05.0 mm ) , reducing extracellular glucose provoked an outward ( inhibitory ) current , with a larger current found with a reduction to 3 mm versus to 7 mm ( figure 2 ) . this outward current was fully reversible on return to 11 mm glucose and had a reversal potential near the equilibrium potential for k , and was blocked by the non - selective k channel antagonist ba ( kawamura et al . , 2010 ) . if intracellular atp levels were low ( 0.5 mm ) , reducing glucose produced a transient inward ( excitatory ) current instead ( figure 2 ) . therefore , moderately low extracellular glucose can inhibit hippocampal neurons that have sufficient or abundant energy stores . furthermore , this inhibition was completely blocked by application of an a1r antagonist and was not present in neurons from a1r knockout mice ( figure 2 ; similar to observations in vivo : masino et al . , 2011 ) implying increased adenosine levels produced the inhibition ( conversely , diabetic hyperglycemia seems to be related to reduced signaling through a1rs ( duarte et al . a similar consistent mechanism was reflected presynaptically ( measured as decreased spontaneous postsynaptic current frequency ) ; an a1r - dependent presynaptic inhibition was produced by adequate / high postsynaptic intracellular atp combined with low extracellular glucose ( kawamura et al . , 2010 ) . ( 2011 ) suggest that a kd can limit seizures ( at least those involving the hippocampus ) through a mechanism dependent on low glucose and abundant high - energy molecules and involving augmentation of adenosine levels . ( a ) peak currents produced by lowering extracellular glucose from 11 to 3 mm depend on intracellular atp concentration ( 0.55 mm ) . ( b ) concentration - dependence of glucose - related outward current ( with 2 mm intracellular atp ) . ( c ) outward current produced in low - glucose buffer reversed completely ( and became slightly inward ) with the a1r antagonist dpcpx . * p < 0.05 , * * p < 0.01 . adapted with permission from kawamura et al . , we manipulated atp only in the patched neuron , suggesting an autocrine mechanism to increase adenosine . loading pyramidal neurons with adenosine + atp versus atp alone , however , suggested that the current was not mediated by direct adenosine release ( kawamura et al . , 2010 ) . cells can release atp by several mechanisms ( dubyak , 2009 ) , and extracellular atp is metabolized rapidly to adenosine ( dunwiddie et al . , 1997 ) . one prominent non - exocytotic atp release mechanism in neurons and glia is atp passage through channels composed of connexins or pannexins ( stout et al . , 2002 ; schock et al . , 2008 ; iwabuchi and kawahara , 2011 ) taken together , our data are consistent with a process by which lowered extracellular glucose promotes release of atp via pannexins . atp is then converted extracellularly to adenosine , which activates a1rs coupled , under these conditions , to katp channels ( kawamura et al . , 2010 ) . this pathway is likely to underlie the a1r - mediated anticonvulsant effect produced by the kd in vivo . certainly , mild hypoglycemia and enhanced adenosine tone can underlie its anticonvulsant effect ( masino et al . , 2011 ) , whereas the in vivo involvement of pannexin channels and atp release remains to be demonstrated directly . altered cognition and affect in children with seizure disorders has always been a concern . regarding pharmacological therapies , several authors have shown that children with epilepsy even those whose seizures were well - controlled with antiepileptic drugs had decreased cognitive function compared to their peers ( devinsky , 1995 ; thompson et al . , 2000 ; the exact mechanism of cognitive decline is unknown : traditional antiepileptic drugs decrease membrane excitability , increase postsynaptic inhibition , or reduce network synchronization to decrease excessive excitability associated with seizure development ( loring , 2005 ) . these neurophysiological mechanisms , if sufficiently strong , will not only suppress seizures but also impair normal brain function . the incidence of cognitive side effects is increased at higher dosing and with polypharmacy which might be necessary for significant seizure control ( loring and kimford , 2001 ) . thus , the cognitive and affective state of a medicated epileptic patient results from a balance of forces including the negative effects of the disease state ( seizures , abnormal interictal brain activity , abnormal sleep ) , the positive effects of the anticonvulsive medication ( seizure control ) , and the negative side effects of the anticonvulsive mediation ( which can include sedation and/or abnormal sleep ) . the kd might offer fewer chronic negative side effects than medication , and given that it has been in use for over 90 years , serious or systematic negative consequences would likely have surfaced by now . in research studies , kds ( albeit at a much stronger ratio than used clinically ) reduced brain mass in juvenile rodents ( cheng et al . , 2004 ; zhao et al . , 2004 ) and kds can affect body growth in children ( who are typically on the diet temporarily ; liu et al . , 2003 ; peterson et al . , 2005 ; neal et al . , 2008b ) but to our knowledge negative kd effects on human brain development and growth have not been quantified . notably , recurrent clinical hypoglycemia can lead to a cumulative cognitive impairment ( langan et al . although this effect might not be directly applicable because the hypoglycemia in these studies was episodic and much more severe than the chronic reduced ( but not abnormal ) glucose levels associated with the kd . overall , positive and negative short- and long - term effects of this strict diet on cognition and mood remain under - examined clinically , particularly in pediatric patients . it is worthwhile to consider that any assessment of cognitive or affective state associated with a kd should occur at multiple time points , as effects of the kd ( including anticonvulsive effects ) clearly evolve . there are limitations to combining data from different laboratories due to differing methodologies , different kds , etc . yet in surveying the research literature , it seems fairly clear that there is a biphasic effect on locomotor behavior : reduced activity characterizes kd onset , whereas increased activity predominate after a few weeks . effects of a kd on locomotion in rodents ( compiled informally from the literature ) are shown in figure 3 . notably , a biphasic pattern over time after diet initiation is found in clinical literature relating to cognition , mood , and vitality . soon after beginning a kd , subjects often complain of lethargy ( vining et al . , 1998 ; lefevre and aronson , 2000 ) ; in children , intolerable drowsiness is a reported side - effect that sometimes leads to cessation of kd treatment ( neal et al . , 2008a ) . yet , after weeks on the diet , subjects report heightened vitality , physical functioning , and alertness ( hallbk et al . , 2007 ; mosek et al . , 2009 ; yancy et al . , these positive effects may be at least partially due to reduced seizure frequency , but similar positive effects are also described in non - epileptic subjects . this delay in beneficial effects is reminiscent of the delay often observed in anticonvulsant effects ( kossoff et al . , 2008a ) . over time , kds produce a biphasic effect on locomotor activity based on this compilation of published rodent data . note that hypoactivity predominated in studies with short diet treatments ( 20 day ) , whereas hyperactivity predominated with longer treatments ( 30 day ) . points were estimated from published graphs and tables in multiple references ( zhao et al . , 2004 ; murphy et al . , 2005 ; ziegler et al . , 2005 ; studies of the kd in epileptic patients rarely characterize mood , which might understandably be poor during the initial lethargic / drowsy stage . several weight - loss studies , however , included affective measures and found positive effects of kd on mood in overweight subjects as early as 2 weeks into diet treatment , and lasting many weeks ( halyburton et al . , 2007 ; mcclernon et al . , 2007 ; brinkworth et al . , 2009 ; yancy et al . , two of these studies provide some evidence against this result simply being a psychological effect of weight loss ( brinkworth et al . , 2009 ; thus , beneficial effects on mood ( as well as weight loss ) await those who conquer the early stage after kd initiation . studies of patients with epilepsy on the kd , including children , have either reported improved cognition anecdotally ( sirven et al . , 1999 ) or reported improvements in more general measures such as attention and social functioning ( kinsman et al . it is difficult to determine if these effects are due to reduced seizures , to concomitantly reduced medications , or a direct action on cognition / attention . investigations in non - epileptic adult subjects ( thus without confounding antiepileptic medications ) have more specifically addressed cognition and the kd . one study found a transient , moderate impairment in one cognitive task ( but not two other tasks ) at 1 week of diet treatment but found no impairments at later time points ( wing et al . , 1995 ) ; two studies examining chronic kd treatments reported improved processing speed and working memory lasting up to 1 year ( halyburton et al . , 2007 ; a minority of animal studies have reported impairments in learning and memory , specifically in a task of spatial reference memory ( su et al . , 2000 ; zhao et al . , other studies , however , have failed to find any detrimental effect of the kd on learning and memory in rodents in various mazes or in fear conditioning ( hori et al . , 1997 ; todorova et al . , 2000 ; silva et al . , 2005 ; we tested normal mice of both sexes in a simple working memory task after feeding on a 7:1 kd at a number of time points , up to 10 weeks , and found no effect of the kd ( though hyperactivity did appear beginning at 2 weeks ( ruskin et al . , 2011a ) . it is worth noting that a kd not only does not impair but in fact reverses age - related deficits in learning and other cognitive measures in aged , but otherwise healthy , dogs and rodents ( pan et al . , 2010 ; xu et al . , 2010 ) taken together , these results largely support the beneficial nature of kd feeding on mood and cognition in patients . there is an overall increase in pain thresholds ( and thus reduced pain ) when glycolytic enzymes are inhibited by exogenous 2-deoxy - d - glucose ( bodnar et al . , 1979 ) . this effect is mediated centrally ( bodnar et al . , 1981 ) , and might involve increased brain / spinal cord inhibition by adenosine , the release of which is stimulated by 2-deoxy - d - glucose ( zhao et al . , 1997 ; minor et al . , 2001 ) . 2-deoxy - d - glucose is also anticonvulsant ( garriga - canut et al . , 2006 ) , and while the mechanisms might not overlap entirely with the kd ( stafstrom et al . , 2009 ; gasior et al second , anticonvulsant drugs such as gabapentin , felbamate , and valproate are useful in treating pain , particularly neuropathic pain and migraine ( johannessen landmark , 2008 ) . these drugs typically act by decreasing neuronal activity or excitability , and it is clear that reducing central activity with adenosine or gaba agonists alleviates pain ( karlsten et al . , 1992 ; malmberg and yaksh , 1993 ; belfrage et al . , 1995 ; malan et al . , 2002 ; gwak et al . , thus , we predicted that the kd , which reduces glucose metabolism and is anticonvulsant , would reduce pain . we fed rats a 7:1 kd in order to test the effects in the hotplate test . in this test , the latency to withdraw a hindpaw from the warm surface indicates the animal s sensitivity to painful heat . in young rats , we found that kd feeding for 34 weeks increased paw withdrawal latency ( i.e. , decreased the sensitivity ) to plate temperatures from 48 to 51c ( ruskin et al . , 2009 ) . in adult rats , the effect seemed to be smaller in magnitude , and was significant only at 49 and 50c . we recently found similar results with a less stringent 3:1 kd ( ruskin et al . , 2011b ) . curiously , another study reported increased thermal pain sensitivity ( tail flick ) after 12 weeks of kd feeding in young rats ( ziegler et al . , 2005 ) ; methodological differences such as rat strain , body part ( paw vs. tail ) , diet composition , and stimulus strength might be factors . the difference in diet treatment length ( 3 vs. 12 weeks ) does not seem to explain the disparity , as subsequently we have found decreased thermal pain sensitivity present after 1011 weeks of feeding with a 3:1 kd ( ruskin et al . thus far the specific mechanism of altered thermal nociception in kd - fed rats is unknown , and could involve hypoglycemia , ketosis , fatty acids , and/or adenosine . one recently published clinical report on kd effects on quality of life reported that beneficial effects on self - reported general bodily pain were at the threshold of statistical significance ( yancy et al . , 2009 ) , suggesting that kd effects on overall pain might be positive . this report , however , was not a dedicated study of pain , but rather a study of overall quality of life ; as such , there was no underlying painful condition to treat . overall , an assessment of pain in kd - treated patients is warranted . a better understanding of the relationship between metabolism and pain could help multiple and comorbid conditions , and the kd might prove uniquely useful against diabetes and diabetes - related neuropathy . , 2009 , 2011 ; garbow et al . , 2011 ; park et al . , 2011 ; poplawski et al . , 2011 ) , clinical studies have found exclusively positive outcomes : after kd treatment , patients with type i or ii diabetes had improved control of blood glucose , and many could have their medications reduced or eliminated ( gumbiner et al . , 1996 ; yancy et al . , 2005 ; westman et al . , 2008 ; dressler et al . , in addition , type i diabetic patients ( and , based on one report , children with epilepsy ) prefer foods that are high in fat and low in carbohydrates ( amari et al . , 2007 ; snell - bergeon et al . , 2009 ) , which might be attempted self - medication . the mixed animal results might result from the use of very strict kds ( garbow et al . , 2011 ; park et al . , 2011 ) , or from the diabetic propensity of many laboratory rodent strains . thus , the kd might benefit diabetic patients both by alleviating neuropathic pain and treating the underlying glycemic control dysfunction . chronic inflammation is typically accompanied by pain due to the release of prostaglandins and the consequent sensitization of sensory neurons ( mense , 1983 ) . some of the most common sources of inflammatory pain are rheumatoid arthritis , chronic inflammatory bowel disease , pancreatitis , back pain , and some cancers . we found that a kd reduced experimental inflammation - induced swelling and plasma extravasation ( ruskin et al . , 2009 ) , and clinical studies describe positive effects of a kd on liver inflammation in non - alcoholic fatty liver disease ( tendler et al . regarding mechanisms linking metabolism to inflammatory pain , reactive oxygen species are a major component of inflammation , and limiting reactive oxygen species should contribute to limiting inflammation . accordingly , ketone - based metabolism should produce fewer free radicals and reactive oxygen species through affecting the mitochondrial co - enzyme q couple and the cytoplasmic glutathione couple ( veech , 2004 ) . indeed , as expected , treatment with ketones reduces the level of reactive oxygen species ( noh et al . , 2006a ; kim et al . , 2007 , 2010 ; maalouf et al . , 2007 ; haces et al . , 2008 ; maalouf and rho , 2008 ) , as does kd feeding ( sullivan et al . , 2004 ) regarding inflammatory pain , by virtue of their high - fat content kds should also activate peroxisome proliferator - activated receptors ( ppars ) . these nuclear receptors bind long - chain polyunsaturated fatty acids , and consequently induce transcriptional changes that culminate in enhanced lipid metabolism ( moya - camarena et al . , 1999 ; 2006 ) . genetic knockout of a major ppar ( the subtype ) augments inflammatory reactions ( cuzzocrea et al . , 2006 ) , whereas synthetic ppar agonists reduce experimentally induced inflammation ( cuzzocrea et al . , 2003 ; loverme et al . , 2005 ) . this latter effect appears to involve reduced transcription of pro - inflammatory genes ( blanquart et al . , 2003 ) and seems to be invoked by the kd ( jeong et al . synthetic ppar agonists are analgesic against inflammatory pain ( loverme et al . , 2006 ) . in addition to these effects , ppar activation augments expression of the enzymes involved in ketogenesis ( cullingford et al . , 2002 ) , promoting the shift to a ketone - based metabolism , in agreement with findings of stronger ketosis with a high - polyunsaturated fat kd ( fuehrlein et al . , 2004 ) . although polyunsaturated fatty acid content of the kd seems not to be important in the diet s anticonvulsant effect ( dell et al . , 2001 ; dahlin et al . , 2007 ) , it might be a crucial characteristic for kd influence on inflammation . it might seem ironic that the kd is discussed here as reducing inflammation , given that other high - fat diets and obesity are definitely linked to chronic inflammation ( thaler and schwartz , 2010 ; ding and lund , 2011 ; laugerette et al . , 2011 ) . those high - fat diets that lead to obesity , including the so - called western diet , include a high amount of fat along with normal amounts of carbohydrate , a crucial difference from the very low - carbohydrate kd which typically leads to weight loss ( gumbiner et al . , 1996 ; halyburton et al . , 2007 ; tendler et al . , 2007 ; westman et al . , 2008 ) . thus , the high - fat - plus - carbohydrate diet promotes fat storage whereas the high fat , low - carbohydrate diet promotes fat metabolism . nevertheless , more clinical work with the kd and inflammation is warranted , particularly regarding long - term effects . it will be crucial to determine which of the mechanisms described above is most important for the kd s alleviation of inflammation . future work on the relationship between the kd s hallmark changes in blood chemistry , ketosis and mild hypoglycemia , and its anti - inflammatory and anti - nociceptive effects should help characterize the pertinent mechanisms . animal studies have found find that the kd protects against seizure - induced neurodegeneration and related sequelae ( such as aberrant neurite sprouting ; muller - schwarze et al . , 1999 ; noh et al . , 2003 , 2005 , 2006b ; linard et al . , 2010 ) . the kd is also neuroprotective against ischemic damage ( tai et al . , 2008 , 2009 ) , hypoglycemic damage ( yamada et al . , 2005 ) , and traumatic brain and spinal injury ( prins et al . , 2009a , b ; prins and hovda , 2009 ; schwartzkroin et al . , 2010 ; streijger et al . , 2011 ) , and improves injury - related deficits in cognition and movement after traumatic brain and spinal injury , respectively ( appelberg et al . , 2009 ; streijger et al . , 2011 ) . ketosis is apparently crucial to these effects as direct application of ketones to in vitro tissue is also protective against hypoglycemia and ischemia ( samoilova et al . , 2010 ) , oxidative stress ( kim et al . , 2007 ) , and excitotoxicity ( massieu et al . , 2003 ; noh et al . the mechanisms are likely to involve reduced reactive oxygen species , reduced tissue excitability , and enhanced production of high - energy molecules . based on evidence for neuroprotection against acute insults , and recognition that metabolic dysfunction accompanies chronic neurological disease positive effects of kd feeding have been found in models of amyotrophic lateral sclerosis ( zhao et al . , 2006 ) , parkinson s disease ( cheng et al . , 2009 ; yang and cheng , 2010 ) , and alzheimer s disease ( van der auwera et al . , 2005 ; mohamed et al . , 2010 ) . in addition , kd feeding reverses aging - related impairments in brain biochemistry in animals ( studzinski et al . . direct application of ketones is also beneficial in models of parkinson s disease ( kashiwaya et al . , 2000 ; huntington s disease , which involves the death of neurons in the caudate and putamen , is thought to involve excitotoxicity and mitochondrial dysfunction ( estrda - sanchez et al . , 2008 ; damiano et al . based on findings reviewed above , we characterized the effects of a strict ( 7:1 ) kd in a rapidly progressing huntington s disease model , the r6/2 mouse ( lifespan less than 16 weeks ) . kd feeding began at 6 weeks of age , when motor impairments are still minor ( ruskin et al . the kd did not increase lifespan or alleviate motor impairments , but , importantly , it did not negatively affect either . however , the kd did delay significantly the onset of progressive weight loss , which is a major problem in patients ( sanberg et al . , 1981 ; lanska et al . , 1988 ) . in addition , the kd reversed a modest working memory impairment in female mice , and working memory is known to be affected in patients with huntington s disease ( lange et al . , 1995 ; lawrence et al . , 1996 ) as well as other neurological disorders and aging . the lack of effect on lifespan or locomotor activity may signal that beneficial effects of a kd might not be similar across neurodegenerative disorders , might depend on the severity or rate of progression , or might differ in different animal models of a disorder ; alternatively , the kd might need to be optimized for strength and composition for different conditions . although it seems paradoxical that the kd , normally associated with weight loss , might maintain , or increase body weight under particular conditions , our data suggest that kd feeding could alleviate huntington s disease - associated cachexia , and , as noted above , in patients a higher body mass is associated with slower disease progression ( myers et al . , 1991 ) . based on this finding , the kd might also deserve consideration for treatment of other cachexias ; for instance , that associated with cancer ( colomer et al . indeed , the kd is beginning to be used as an anti - tumorigenic treatment ( klement and kammerer , 2011 ; seyfried et al . , 2012 ) and if the anti - neurodegenerative effects found in animal models of parkinson s disease , alzheimer s disease , and aging are successfully extended to humans , the kd could also have dual benefits , delaying the primary degenerative condition and alleviating the working memory problems common to these conditions ( halyburton et al . a kd offers known benefits for epilepsy , and it is apparent that the relationship between metabolism and brain function offers primary therapeutic opportunities . basic and clinical research is acutely aware that metabolic dysfunction and comorbidities promulgate lifelong impacts on nervous system function . particularly promising unrealized opportunities for intervention and restoration of metabolic homeostasis occur during development , after injury , and during disease progression all windows with high levels of plasticity and remodeling . 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 .

a link between metabolism and brain function is clear . since ancient times , epileptic seizures were noted as treatable with fasting , and historical observations of the therapeutic benefits of fasting on epilepsy were confirmed nearly 100 years ago . shortly thereafter a high fat , low - carbohydrate ketogenic diet ( kd ) debuted as a therapy to reduce seizures . this strict regimen could mimic the metabolic effects of fasting while allowing adequate caloric intake for ongoing energy demands . today , kd therapy , which forces predominantly ketone - based rather than glucose - based metabolism , is now well - established as highly successful in reducing seizures . cellular metabolic dysfunction in the nervous system has been recognized as existing side - by - side with nervous system disorders although often with much less obvious cause - and - effect as the relationship between fasting and seizures . rekindled interest in metabolic and dietary therapies for brain disorders complements new insight into their mechanisms and broader implications . here we describe the emerging relationship between a kd and adenosine as a way to reset brain metabolism and neuronal activity and disrupt a cycle of dysfunction . we also provide an overview of the effects of a kd on cognition and recent data on the effects of a kd on pain , and explore the relative time course quantified among hallmark metabolic changes , altered neuron function and altered animal behavior assessed after diet administration . we predict continued applications of metabolic therapies in treating dysfunction including and beyond the nervous system .

The Ketogenic Diet and Ketone-Based Metabolism Metabolism, Plasticity, and Synaptic Activity Ketogenic Diet for a Brain Slice: Relaxing in Reduced Glucose? Ketogenic Diets Effect on Cognition and Mood: Negative, then Positive? Nociception and Inflammation: Multiple Mechanisms Likely Attenuating Brain Injury and Neurodegeneration Looking Ahead Conflict of Interest Statement

metabolism influences brain activity , and metabolic dysfunction is associated with a wide variety of neurological disorders . the cause - and - effect relationship between metabolic and neuronal dysfunction is often unclear , though not in the case of epilepsy and diet . historical observations noted the therapeutic benefits of fasting on epilepsy , but fasting is necessarily a time - limited practice . therapeutic benefits of the metabolic condition of fasting were confirmed over 90 years ago when the high fat , low - carbohydrate ketogenic diet ( kd ) was described as alternative to fasting which still reduced epileptic seizures ( wilder , 1921 ) . in turn , anticonvulsant drugs debuted over the next two decades , such that since then the kd has been used mostly for inoperable and medication - resistant epilepsy , which has been estimated to be 15% up to 45% of cases ( picot et al . although used clinically for many decades , prescribed most often to children , and increasing in popularity over the last two decades , the kd s mechanism of action remains controversial . to make up for lost calories and augment ketosis , when carbohydrate intake is strongly limited ( as during the kd or fasting ) , the liver increases production of the ketone bodies -hydroxybutyrate , acetoacetate , and acetone from circulating fatty acids ( aoki , 1981 ) . because of the -hydroxyl substitution , -hydroxybutyrate is not actually a ketone , ketone bodies are released into the circulation as an alternative energy source to generate atp ( ketolytic metabolism ) within tissues , including the brain and spinal cord . formulation of the kd is calculated using a ratio of fat content to combined protein and carbohydrate content , varying in the clinic from 5:1 to 1:1 depending on a patient s individual needs ( swink et al . we found that a kd - fed ad libitum at ratio of 7:1 or 3:1 to rats produced similar changes in blood chemistry ( figure 1 ) . clinically , the trend has been to decrease the ratio where possible and thus make the diet more palatable ( including the more liberal modified atkins diet ; kang et al . understanding the mechanisms by which a diet controls seizures , along with broader opportunities for metabolic therapies , remains an active research topic because of accessibility , efficacy , and economics . yet , numerous other changes due to the kd have been hypothesized to underlie increased inhibition and/or decreased excitation in brain , and thus to an anticonvulsant / neuroprotective state . in normal humans fed a kd , electroencephalography and transcranial magnetic stimulation demonstrated increased inhibition in the cerebral cortex , with a magnitude similar to that seen after benzodiazepine administration ( cantello et al . with the more extensive investigation possible in experimental animals , a kd was shown to enhance paired - pulse depression , shift the input / output relationship rightward , elevate the threshold for maximal electrical activation , and to block spreading depression - style events in the hippocampus in vivo ( bough et al . there have been surprisingly few detailed studies on detailed synaptic effects , likely because of the difficulty in performing such studies in vivo , coupled with the typical glucose - based incubation protocol for in vitro slices ; to date , a kd incubation protocol has not been standardized , although recent work sampling cerebrospinal fluid in kd - fed animals might provide a starting point ( samala et al . increased inhibition or decreased excitability , if sufficiently strong , might not only suppress seizures but also influence normal brain function . many types of normal brain function , as well as recovery from injury , are thought to depend on synaptic plasticity , i.e. , the malleability , either temporary or long - lasting , of the strength of neuronal communication ( davis et al . studies have linked metabolism and ltp ( potter et al . , 2010 ) ; we and our collaborators characterized the effects of a kd on hippocampal ltp with the hypothesis that kd - related inhibition or reduced excitation might affect brain plasticity ( koranda et al . after 3 weeks on a 7:1 kd , baseline synaptic measurements were taken in the perforant path - dentate gyrus pathway and ltp was induced with tetanic stimulation and the response measured over the next 2 days . the kd had no significant effects on measures of short - term plasticity ( paired - pulse depression , paired - pulse facilitation ) , and did not prevent ltp induction , whereas the magnitude of the potentiation was significantly smaller in kd - fed rats . as discussed below , cognitive effects of the diet are mixed in animals and overall positive in humans . in addition , it is important to note that 7:1 is a stronger diet ratio than that used clinically , animals used had never had seizures , and another paper looking at the kd on ltp in vivo in anesthetized animals did not find any differences ( thio et al . to test the role of adenosine in the kd s ability to reduce seizures , we and our collaborators recently tested the effectiveness of a kd in a transgenic mouse with spontaneous hippocampal electrographic seizures due to adenosine deficiency . , 2005 ) , and tonic levels of the endogenous inhibitor adenosine are therefore lower than normal . , this antiseizure effect depended on low glucose ( seizures were restored by a peripheral injection of glucose ) , and activation of the adenosine a1 receptor subtype ( a1r ; seizure activity was restored by injection of a selective a1r antagonist ) . these mice also have spontaneous electrographic seizures in the hippocampus , but the kd has no effect on seizure frequency in a1r knockout mice , and is partially effective in mice heterozygous for the a1r ( masino et al . adenosine in particular , and a kd in general , might offer more homeostatic upstream bioenergetic regulation of neuronal activity , and possibly long - term benefits on brain homeostasis , than highly specific drug therapies ( boison et al . yet , the lack of effects of the kd on input output relationships and short - term plasticity seem to argue against the tonic involvement of adenosine ( koranda et al . mechanism aside , the kd can limit excessive neuronal activity ( a class into which the neuronal activity during an ltp induction burst certainly applies ) and perhaps reset baseline activity . effects of kd feeding on baseline excitability are inconsistent in vitro , however ( stafstrom et al . certainly , the metabolic state established by a kd might be disrupted during tissue preparation for in vitro work . as introduced briefly above , one of the biochemical effects associated with a kd is an abundance of high - energy molecules ( devivo et al . , 2007 ) , as well as increased mitochondrial biogenesis , respiration , and expression of atp synthesis - related proteins ( noh et al . we modeled key aspects of the kd in vitro by maintaining or increasing intracellular atp while decreasing extracellular glucose in individual ca3 pyramidal neurons in acute hippocampal slices . we varied atp ( 0.55.0 mm ; 2 mm is standard ) in the patch pipet and changed glucose concentration of the bathing solution from 11 mm ( standard ) to either 7 or 3 mm ( kawamura et al . moderately lowered extracellular glucose has been reported to attenuate epileptiform activity in brain slices ( kirchner et al . this outward current was fully reversible on return to 11 mm glucose and had a reversal potential near the equilibrium potential for k , and was blocked by the non - selective k channel antagonist ba ( kawamura et al . ( 2011 ) suggest that a kd can limit seizures ( at least those involving the hippocampus ) through a mechanism dependent on low glucose and abundant high - energy molecules and involving augmentation of adenosine levels . ( b ) concentration - dependence of glucose - related outward current ( with 2 mm intracellular atp ) . ( c ) outward current produced in low - glucose buffer reversed completely ( and became slightly inward ) with the a1r antagonist dpcpx . , we manipulated atp only in the patched neuron , suggesting an autocrine mechanism to increase adenosine . atp is then converted extracellularly to adenosine , which activates a1rs coupled , under these conditions , to katp channels ( kawamura et al . altered cognition and affect in children with seizure disorders has always been a concern . regarding pharmacological therapies , several authors have shown that children with epilepsy even those whose seizures were well - controlled with antiepileptic drugs had decreased cognitive function compared to their peers ( devinsky , 1995 ; thompson et al . these neurophysiological mechanisms , if sufficiently strong , will not only suppress seizures but also impair normal brain function . thus , the cognitive and affective state of a medicated epileptic patient results from a balance of forces including the negative effects of the disease state ( seizures , abnormal interictal brain activity , abnormal sleep ) , the positive effects of the anticonvulsive medication ( seizure control ) , and the negative side effects of the anticonvulsive mediation ( which can include sedation and/or abnormal sleep ) . the kd might offer fewer chronic negative side effects than medication , and given that it has been in use for over 90 years , serious or systematic negative consequences would likely have surfaced by now . , 2004 ) and kds can affect body growth in children ( who are typically on the diet temporarily ; liu et al . overall , positive and negative short- and long - term effects of this strict diet on cognition and mood remain under - examined clinically , particularly in pediatric patients . it is worthwhile to consider that any assessment of cognitive or affective state associated with a kd should occur at multiple time points , as effects of the kd ( including anticonvulsive effects ) clearly evolve . effects of a kd on locomotion in rodents ( compiled informally from the literature ) are shown in figure 3 . notably , a biphasic pattern over time after diet initiation is found in clinical literature relating to cognition , mood , and vitality . soon after beginning a kd , subjects often complain of lethargy ( vining et al . , 1998 ; lefevre and aronson , 2000 ) ; in children , intolerable drowsiness is a reported side - effect that sometimes leads to cessation of kd treatment ( neal et al . yet , after weeks on the diet , subjects report heightened vitality , physical functioning , and alertness ( hallbk et al . this delay in beneficial effects is reminiscent of the delay often observed in anticonvulsant effects ( kossoff et al . , 2005 ; studies of the kd in epileptic patients rarely characterize mood , which might understandably be poor during the initial lethargic / drowsy stage . several weight - loss studies , however , included affective measures and found positive effects of kd on mood in overweight subjects as early as 2 weeks into diet treatment , and lasting many weeks ( halyburton et al . studies of patients with epilepsy on the kd , including children , have either reported improved cognition anecdotally ( sirven et al . it is difficult to determine if these effects are due to reduced seizures , to concomitantly reduced medications , or a direct action on cognition / attention . , other studies , however , have failed to find any detrimental effect of the kd on learning and memory in rodents in various mazes or in fear conditioning ( hori et al . , 2005 ; we tested normal mice of both sexes in a simple working memory task after feeding on a 7:1 kd at a number of time points , up to 10 weeks , and found no effect of the kd ( though hyperactivity did appear beginning at 2 weeks ( ruskin et al . it is worth noting that a kd not only does not impair but in fact reverses age - related deficits in learning and other cognitive measures in aged , but otherwise healthy , dogs and rodents ( pan et al . , 1981 ) , and might involve increased brain / spinal cord inhibition by adenosine , the release of which is stimulated by 2-deoxy - d - glucose ( zhao et al . , 2006 ) , and while the mechanisms might not overlap entirely with the kd ( stafstrom et al . , 2009 ; gasior et al second , anticonvulsant drugs such as gabapentin , felbamate , and valproate are useful in treating pain , particularly neuropathic pain and migraine ( johannessen landmark , 2008 ) . these drugs typically act by decreasing neuronal activity or excitability , and it is clear that reducing central activity with adenosine or gaba agonists alleviates pain ( karlsten et al . , thus , we predicted that the kd , which reduces glucose metabolism and is anticonvulsant , would reduce pain . we fed rats a 7:1 kd in order to test the effects in the hotplate test . in adult rats , the effect seemed to be smaller in magnitude , and was significant only at 49 and 50c . , 2005 ) ; methodological differences such as rat strain , body part ( paw vs. tail ) , diet composition , and stimulus strength might be factors . thus far the specific mechanism of altered thermal nociception in kd - fed rats is unknown , and could involve hypoglycemia , ketosis , fatty acids , and/or adenosine . this report , however , was not a dedicated study of pain , but rather a study of overall quality of life ; as such , there was no underlying painful condition to treat . a better understanding of the relationship between metabolism and pain could help multiple and comorbid conditions , and the kd might prove uniquely useful against diabetes and diabetes - related neuropathy . , 2011 ) , clinical studies have found exclusively positive outcomes : after kd treatment , patients with type i or ii diabetes had improved control of blood glucose , and many could have their medications reduced or eliminated ( gumbiner et al . , 2009 ) , which might be attempted self - medication . some of the most common sources of inflammatory pain are rheumatoid arthritis , chronic inflammatory bowel disease , pancreatitis , back pain , and some cancers . we found that a kd reduced experimental inflammation - induced swelling and plasma extravasation ( ruskin et al . , 2009 ) , and clinical studies describe positive effects of a kd on liver inflammation in non - alcoholic fatty liver disease ( tendler et al . regarding mechanisms linking metabolism to inflammatory pain , reactive oxygen species are a major component of inflammation , and limiting reactive oxygen species should contribute to limiting inflammation . accordingly , ketone - based metabolism should produce fewer free radicals and reactive oxygen species through affecting the mitochondrial co - enzyme q couple and the cytoplasmic glutathione couple ( veech , 2004 ) . , 2004 ) regarding inflammatory pain , by virtue of their high - fat content kds should also activate peroxisome proliferator - activated receptors ( ppars ) . these nuclear receptors bind long - chain polyunsaturated fatty acids , and consequently induce transcriptional changes that culminate in enhanced lipid metabolism ( moya - camarena et al . genetic knockout of a major ppar ( the subtype ) augments inflammatory reactions ( cuzzocrea et al . in addition to these effects , ppar activation augments expression of the enzymes involved in ketogenesis ( cullingford et al . , 2002 ) , promoting the shift to a ketone - based metabolism , in agreement with findings of stronger ketosis with a high - polyunsaturated fat kd ( fuehrlein et al . although polyunsaturated fatty acid content of the kd seems not to be important in the diet s anticonvulsant effect ( dell et al . those high - fat diets that lead to obesity , including the so - called western diet , include a high amount of fat along with normal amounts of carbohydrate , a crucial difference from the very low - carbohydrate kd which typically leads to weight loss ( gumbiner et al . thus , the high - fat - plus - carbohydrate diet promotes fat storage whereas the high fat , low - carbohydrate diet promotes fat metabolism . nevertheless , more clinical work with the kd and inflammation is warranted , particularly regarding long - term effects . it will be crucial to determine which of the mechanisms described above is most important for the kd s alleviation of inflammation . future work on the relationship between the kd s hallmark changes in blood chemistry , ketosis and mild hypoglycemia , and its anti - inflammatory and anti - nociceptive effects should help characterize the pertinent mechanisms . , 2005 ) , and traumatic brain and spinal injury ( prins et al . , 2011 ) , and improves injury - related deficits in cognition and movement after traumatic brain and spinal injury , respectively ( appelberg et al . , 2007 ) , and excitotoxicity ( massieu et al . based on evidence for neuroprotection against acute insults , and recognition that metabolic dysfunction accompanies chronic neurological disease positive effects of kd feeding have been found in models of amyotrophic lateral sclerosis ( zhao et al . , 2009 ; yang and cheng , 2010 ) , and alzheimer s disease ( van der auwera et al . in addition , kd feeding reverses aging - related impairments in brain biochemistry in animals ( studzinski et al . , 2000 ; huntington s disease , which involves the death of neurons in the caudate and putamen , is thought to involve excitotoxicity and mitochondrial dysfunction ( estrda - sanchez et al . based on findings reviewed above , we characterized the effects of a strict ( 7:1 ) kd in a rapidly progressing huntington s disease model , the r6/2 mouse ( lifespan less than 16 weeks ) . however , the kd did delay significantly the onset of progressive weight loss , which is a major problem in patients ( sanberg et al . in addition , the kd reversed a modest working memory impairment in female mice , and working memory is known to be affected in patients with huntington s disease ( lange et al . the lack of effect on lifespan or locomotor activity may signal that beneficial effects of a kd might not be similar across neurodegenerative disorders , might depend on the severity or rate of progression , or might differ in different animal models of a disorder ; alternatively , the kd might need to be optimized for strength and composition for different conditions . although it seems paradoxical that the kd , normally associated with weight loss , might maintain , or increase body weight under particular conditions , our data suggest that kd feeding could alleviate huntington s disease - associated cachexia , and , as noted above , in patients a higher body mass is associated with slower disease progression ( myers et al . , 2012 ) and if the anti - neurodegenerative effects found in animal models of parkinson s disease , alzheimer s disease , and aging are successfully extended to humans , the kd could also have dual benefits , delaying the primary degenerative condition and alleviating the working memory problems common to these conditions ( halyburton et al . a kd offers known benefits for epilepsy , and it is apparent that the relationship between metabolism and brain function offers primary therapeutic opportunities . basic and clinical research is acutely aware that metabolic dysfunction and comorbidities promulgate lifelong impacts on nervous system function . particularly promising unrealized opportunities for intervention and restoration of metabolic homeostasis occur during development , after injury , and during disease progression all windows with high levels of plasticity and remodeling . 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 .

fl is the second most common form of nhl , accounting for approximately 30% of nhl cases . the disease is characterized by a slow progression and high response rates to therapy , that is the reason why it is considered the prototype of indolent lymphomas ; median survival is currently around 14 years , with most patients displaying an indolent form of the disease , slowly progressing over many years . nonetheless , most patients eventually develop increasingly resistant disease over time , and in up to 45% of cases , the original indolent subtype transforms into an aggressive subtype , an event that is associated with a poor outcome.1,2,3 the first hit of the oncogenic cascade leading to fl is attributed to the t(14;18 ) chromosomal translocation that occurs in an early b cell stage in the bone marrow . naive b cells , carrying the t(14;18 ) , exit the bone marrow and colonize secondary lymphoid tissue , where they undergo the germinal center reaction but have a survival advantage due to their constitutive expression of bcl2 , which is not normally expressed in the germinal center.4 apart from the t(14;18 ) , recurrent secondary genetic alterations including genomic gains , losses , and mutations ( i.e. alterations in mll2 , epha7 , tnfrsf14 , and ezh2 ) could provide a growth advantage to the neoplastic cells . moreover , the crosstalk between neoplastic b cells and the microenvironment plays an important role in sustaining tumor cell growth and eventually promoting transformation.5 current treatment strategies vary from the classical watch and wait approach to the use of anti cd20 monoclonal antibodies ( labeled or unlabeled with radioimmunoconjugates ) in combination with chemotherapy , while more aggressive treatment approaches including autologous or allogeneic stem cell transplantation are reserved to patients with more resistant disease.6 recently a large bulk of molecular and clinical research has been performed to better understand the molecular mechanisms of lymphomagenesis and to develop non - chemotherapeutic agents active in specific lymphoma subtypes ; in this field infectious agents could represent therapeutic targets for lymphoma treatment toward chemotherapy - free therapeutic approaches . despite huge advances in the comprehension of the genetic anatomy of fl , the potential epidemiologic role of environmental stimuli has not been clearly established ; this is somehow in contrast to the huge bulk of knowledge which has been accumulated in malt lymphomas . nevertheless , a number of biological and clinical observations suggests that interaction with physiological and pathological microbial populations might play a role also in fl . a classical model of the infection - driven lymphoproliferative disorder is helicobacter pylori - induced gastric malt lymphoma where antibiotic therapy allows eradication of both the infectious agent and the clonal b - cell expansion , leading to long - term complete remissions ( cr).7 the identification of this pathogen as the causative agent in gastric malt lymphomas have resulted in substantial progress in understanding the physiopathology of the disease permitting to develop new therapeutic strategies . the list of lymphomas evolving in response to antigen ( bacterial or viral ) has been growing rapidly in recent years , associated in some cases with similar therapeutic success.8 although no such association with infectious agents or other early specific therapeutic target has yet been identified for fl , this concept seems ideally suited to such an indolent disease . this review summarizes current evidence for a role of either physiological and pathological exogenous microbial species in the pathogenesis of fl . so , we underwent an extensive literature search focusing on clinical observations suggesting such correlations , and we tried to underline potential similarities between fl and other indolent lymphoproliferative processes where the role of microbial organisms is clearly established . in recent years , a growing number of exogenous microbial agents have been linked to nhl . the helicobacter pylori ( hp ) , chlamydia psittaci and hepatitis c virus are best - known examples , but other agents have been identified in the pathogenesis of more rare subtypes of nhl ; these associations are important because they have clinical and therapeutic implications and provide novel insights into the mechanisms that govern lymphoma development.9 hp is a proteobacteria epsilon bacterium known to cause stomach ulcers and chronic gastritis . its role in the pathogenesis of the majority of cases of gastric malt lymphoma was demonstrated nearly twenty years ago . hp affects about 50% of the world s population even if only 12% of infected individuals will develop a malignant disease . the pathogenetic role of hp is related to the oncogenic properties of the cytotoxin - associated antigen a ( cag - a ) , a protein that is able to activate the signaling pathway leading to the activation and upregulation of the antiapoptotic molecule bcl2.10,11 three major chromosomal translocations specific of malt lymphomas are reported , i.e. t(11;18 ) which is the most common ( found in nearly 30% of the cases ) , t(14;18 ) and t(1;14 ) . hp eradication using a combination of antibiotics and proton - pump inhibitors ( ppi ) , represents the standard treatment of hp - associated malt - lymphomas , leading to lymphoma regression in about 75% of patients.12 chlamydophila psittaci belongs to the family of chlamydiae and is the second most studied among bacteria having a pathogenetic role in malt - lymphomas . dna from this bacterium has been found in biopsies of malt lymphoma of the ocular adnexa . the finding that c. psittaci infection has been detected in up to approximately 80% of italian patients with ocular adnexa malt lymphoma provided the rationale for the antibiotic treatment of localized lesions . moreover , the eradication of c. psittaci infection with doxycycline for patients with ocular adnexa malt lymphoma resulted in lymphoma regression in approximately 50% of patients.9 epstein - barr virus ( ebv ) is the first human herpes virus found to be associated with the pathogenesis of cancer . ebv has a worldwide distribution , being able to establish a lifelong infection in more than 90% of individuals . primary infection is usually asymptomatic or could cause a benign lymphoproliferative disease , known as infectious mononucleosis . ebv has a successful strategy to reside in the hematopoietic system , including the establishment of a nonpathogenic latent infection of memory b lymphocytes that allows the virus to persist for the lifetime . according to current knowledge , latent antigens encoded by ebv interfere with a number of critical cellular pathways , thereby promoting oncogenesis . although human ebv infection may lead to the development of a variety of hematopoietic and epithelial cancers , most common cases result from the transformation of infected b cells into lymphoproliferative disorders:13 hodgkin lymphomas , diffuse large cell and burkitt lymphomas . moreover , ebv can cause a rare but potentially fatal complication in hematopoietic stem cell transplants , as well as in solid - organ recipients , known as ebv - associated post - transplant lymphoproliferative disease ( ptld).14,15,16 human immunodeficiency virus ( hiv ) is a lentivirus of the retroviridae family that integrates itself into host chromosomal dna . the increased risk for lymphoma appears related to multiple factors , including the transforming properties of the virus itself , the immunosuppression and , most importantly , opportunistic infections associated with other lymphotropic herpes viruses such as ebv and human herpesvirus 8 . the clinical outcome appears to be worse than in similar aggressive lymphomas in the general population . however , following the introduction of highly active antiretroviral therapy , the risk of developing lymphoma in the context of hiv infection has decreased , and the clinical outcome has improved.17 hepatitis c virus ( hcv ) is a small rna virus of the flaviviridae family ; is a hepatotropic and lymphotropic virus responsible for acute hepatitis and chronic liver disease ; the presence of hcv is associated with a spectrum of lymphoproliferative disorders , ranging from polyclonal b - cell expansion to overt malignant lymphoma . indeed , as well as small b - cell clones can be detected in bone marrow or liver biopsies , a higher frequency of lymphoid malignancies has been reported in hcv - positive patients . the association between hcv infection and nhl has been demonstrated by epidemiological studies , in particular in highly endemic geographical areas such as italy , japan , and southern parts of united states . in these countries , together with diffuse large b - cell lymphomas , marginal zone lymphomas are the histotypes most frequently associated with hcv infection . the most convincing argument for a causative link between hcv and lymphoproliferation is represented by studies demonstrating the eradication of the neoplastic clone by the antiviral treatment in hcv - positive patients affected by indolent nhl ( 18 ) . analogous to what has been observed in hp - associated gastric , the role of hcv infection in lymphomagenesis may be related to the chronic antigenic stimulation of b - cell immunologic response by the virus.19,20 adult t - cell leukemia - lymphoma ( atl ) is an aggressive lymphoid proliferation associated with the human lymphotropic virus type i ( htlv - i ) . atl usually occurs in people from htlv - i endemic regions , such as southern japan and the caribbean . htlv - i causes transformation and clonal expansion of t cells , resulting in atl in approximately 1%5% of the infected hosts , with a mean latency period of > 50 years . recently , a worldwide meta - analysis revealed that the combination of zidovudine and ifn- is highly effective in the leukemic subtypes of atl and should be considered as standard first - line therapy in this setting.21 human herpesvirus 8 ( hhv8 ) is a gammaherpesvirus associated with primary effusion lymphoma , a lymphoproliferative disease that is rarely observed in immunocompromised individuals . these neoplastic disorders that result from hhv8 infection are most commonly related to immunodeficiency states , including hiv infection and ebv infection . the lymphoma is characterized by the localization in one of the body cavities ( pleural , pericardial , or peritoneal cavity ) , without lymph node enlargement and lymphadenopathy . given the heterogeneous nature of lymphoma subtypes and their different clinical behavior , it is intriguing to identify the risk factors potentially responsible for the occurrence of nhl , so various occupational , environmental and chemical agents have been claimed by several epidemiological studies . however , although for some factors the correlation seems to exist , definite conclusions have not been drawn . several reports have also investigated the possible association between infection - related conditions and the occurrence of nhl ; in fact , several infectious agents have been identified as causative factors for the development of nhl , most likely due to their induction of dna damage , inflammatory cells proliferation , and cytokine release . to address this issue the international lymphoma epidemiology consortium ( interlymph ) , an open scientific forum for epidemiological research funded in 2001 , undertook the nhl subtypes project ; this is an international group of multidisciplinary specialists , who have worked together with the aim of identifying associations of several risk factors across different lymphoma subtypes.23 regarding fl , in 2013 a large pooled analysis carried on by the interlymph consortium made an attempt to assess associations between medical , hormonal , family history , lifestyle and occupational factors with the risk of developing fl . the incidence rate of fl was reported as higher in western countries , which comprises ~30% of nhl , with a white to black ratio of 2:3 , and relatively rare in developing and far eastern countries . moreover , fl risk was increased in subjects with a first - degree relative with non - hodgkin lymphoma in spray painters among women with sjgren and among cigarette smokers and obese subjects . no specific observation mentioned a link between infection and risk of fl.24 another large retrospective case control study using seer and medicare database investigated the role of infection - related conditions and different nhl subtypes.25 cases were defined as individuals with a seer diagnosis of primary lymphoid malignancy between 1992 and 2005 . the database identified respiratory and skin infections to be associated with an increased risk of nhl in individuals aged more than 66 years . claims for sinusitis , laryngitis and herpes zoster were present in the history of fl patients , sinusitis , laryngitis and herpes zoster were significant at longer latencies . most fl cases carried the t(14;18 ) , which was hypothesized to be transformed by exogenous antigen stimulation , such as from a viral infection.26 antigenic stimulation and/or subclinical immune deficiency , predisposing patients to both infections and lymphoma , were claimed as possible association between infection - related conditions and lymphoma . the gene sequence for the immunoglobulin ( ig ) heavy - chain ( h ) and light - chain ( l ) variable regions are assembled in the early stages of b - cell development in the bone marrow from distinct variable ( v ) , diversity ( d ) and joining ( j ) segments through a process of somatic dna rearrangement known as v(d)j recombination . in later stages , which takes place in the germinal center ( gc ) of the secondary lymphoid tissues , naive b - cells with low - affinity functional surface ig ( sig ) are induced to proliferate . the high proliferation rate is associated with somatic hypermutation of ig genes , a process that introduces a high incidence of mutations within the v region of genes . somatic hypermutation is thought to be a prerequisite for affinity maturation of antibody response . at this stage , normal b cells that are specific for an antigen are induced to operate a selection process that expands the population of b cells with an optimal binding affinity for the antigen . also , b cells carrying the t(14;18 ) exit the bone marrow and colonize the gc of secondary lymphoid tissue ; subsequently they undergo somatic hypermutations of igvh - genes , with mutational patterns very similar to their normal counterpart , but with a survival advantage due to the constitutive expression of bcl2 . recent studies by schneider et al . demonstrated that somatic hypermutations occurring in fl cells could introduce sugar moieties , like high - mannose - terminated glycan , into the variable domain of the surface ig antigen - binding sites , which create potential novel binding sites to mannose - specific lectins . in fl cells , b - cell receptor ( bcr ) expression is retained , despite the characteristic chromosomal translocation t(14;18 ) , because bcr is fundamental for the transduction of the signals that maintain the survival and growth of fl clones . bcr variable - region mannoses in fl are recognized by lectins of common opportunistic bacteria , such as burkholderia cenocepacia and pseudomonas aeruginosa , that are usually found in soil and water ; these lectins represent a potent stimulus for the proliferation of b cells expressing this kind of glycan - terminated glycan.27,28,29 therefore , these studies directly support the potential importance of microbial exposure in the proliferation and survival of fl clones , and they might be a key to a better understanding of the pathogenesis of fl . although an expanding literature has examined several risk factors potentially correlated with the occurrence of fl , the etiology of the proliferative stimulus is generally unknown , and the few relationships observed suggest a complex multifactorial etiology . a recent meta - analysis , selecting more than 20 articles , showed a more than two - times increase in the odds of developing nhl in patients with hbv infection . interestingly , regarding fl subtype , a trend toward statistical risk was observed in countries with a high prevalence of hbv infection while no statistical risk was seen in countries with a low prevalence of hbv infection.30 the conclusion of the study was that was difficult to determine if the increased risk of fl in areas of high prevalence of hbv infections is due simply to a larger number of hbv infections or a true causal relationship . in the latter case , hbv might be responsible for lymphomagenesis through a chronic stimulation of b - cells which may predispose to dna damage and transformation into neoplastic cells , or through an immunologic response to chronic local antigenic stimulation . as opposed to hbv , a case - control study carried on by the interlymph did not show an increased association between hcv infection and fl , that was restricted to other specific b - nhl subtypes like diffuse large b - cell lymphoma ( dlbcl ) , marginal zone lymphoma , and lymphoplasmacytic lymphoma.19 an interesting clinicopathological finding came up from a spanish study that analyzed a retrospective series of 58 patients with a diagnosis of hcv - positive b - cell lymphoproliferative disorder ; eight of them were affected by fl , and at least half of them expressed bcl2 and p53 . interestingly , the authors reported a cohort of 11 patients in which a clonal b cell expansion in the peripheral blood and bone marrow could be revealed , in the absence of conclusive histological evidence of neoplastic infiltration . these expanded clones make up a definite group of hcv - associated monoclonal b - cell lymphocytosis that should be monitored because a 10% risk of evolution to overt lymphoma has been demonstrated.31 currently , the association between ebv and follicular lymphoma is reported only in the form of isolated case reports in patients with various form of immunodeficiency or in the context of transformation to diffuse large cell lymphoma or classical hodgkin lymphoma . mackrides et al . analyzed 382 cases of fl consecutively diagnosed at the university of miami and stanford , in order to provide an estimated prevalence of ebv - positive fl ; all the cases were tested for the expression of ebv - encoded small rna ( eber ) as determined by in situ hybridization . they identified 10 cases of ebv - positive fl ( prevalence=2.6% ) with a significant prevalence of grades 3a3b fl ( 9 out of 10 ) and frequent strong coexpression of cd30 ; all cases demonstrated progression of the disease to a higher grade fl or diffuse large b - cell lymphoma . given the increased incidence of ebv in high - grade fl and the fact that the cases are clinically and morphologically indistinguishable from ebv - negative fl patients , the authors suggested the screening for eber in all high - grade cases.32 recently an intriguing association between coxiella burnetii / q fever and the incidence of b - cell lymphomas was proposed by melenotte et al . in a large scale study.33 starting from the observation of the occurrence of lymphoma in a patient with q fever , they screened over 1000 consecutive patients of the french national referral center for q fever database and examined if there was an association between the two diseases . an excess risk of dlbcl and fl was found in individuals who had q fever compared with the general population and above all patients with a persistent localized infection were found to have a greater risk of lymphoma . these results support the evidence that a novel factor should be added to the list of bacteria that promote human b - cell lymphomas , in particular , fl . the most relevant studies reporting a link between follicular lymphoma and infectious agents are summarized in table 1 . taken in mind the gastric malt as a well - accepted example of antigen - driven neoplastic cell proliferation , portlock et al . explored the association between infectious agents and nhl in a cohort of 56 patients with an untreated advanced non - bulky indolent lymphoma.34 all patients were tested for hp , hcv , borrelia burgdorferi , chlamydia psittaci and small bowel bacterial overgrowth ; in this series , a documented infection was found in 37% of the patients , with a prevalence of hp . starting from the observation of anecdotal lymphoma remissions after antibiotic therapy in a series of patients not requiring chemotherapy , they speculated that a prevention strategy would decrease the risk of future lymphoma progression driven by such antigens . therefore in 2007 , they launched a prospective clinical trial testing the role of prolonged clarithromycin antibiotic therapy as a first treatment in the same category of indolent advanced - stage lymphoma patients . although the small sample size , they reported lymphoma objective responses in 9 of 32 patients ( 28.1% ) with a long treatment - free survival for patients responding to antibiotics.35 the association between infectious agents and fl added new important information about the role played by the antigen stimulation in fl ; moreover , the possibility to treat the neoplastic disease in a simple and efficient way could be considered a step toward developing a lymphoma preventive strategy by reducing the antigen drive . in the last years , several infectious agents , like hepatitis c , human immunodeficiency , and epstein - barr viruses , and helicobacter pylori , chlamydia psittaci , and coxiella burnetii bacteria have been reported as involved in the malignant transformation of b or t lymphocytes , and therefore associated with the pathogenesis of lymphoproliferative disorders . while this hypothesis has been demonstrated for some rare subtypes of nhl , for the majority the evidence is uncertain . regarding fl , based on available data , evidence linking this lymphoma subtype and exogenous infectious agents are weak , and currently , fl can not be considered as an infection - driven disease . however , some clinical , epidemiologic studies and case reports indicate that it is still somehow premature to conclude that exogenous agents have a negligible role in the genesis of fl . it has been estimated that chronic infections caused by viruses , bacteria , and parasites are the causative agents of nearly 1015% of global cancers burden.36,37 these infectious agents promote a cascade of events culminating in chronic inflammatory responses . chronic antigenic stimulation has been postulated as a potential mechanism for carcinogenesis , thus predisposing target tissues to increased cancer susceptibility . in particular , in antigen - driven hematologic malignancies , like hp - associated with malt , the chronic stimulation of the innate immune system causes a clonal expansion of b - lymphocytes , which leads to the production of oxidative reactions ; these events result in genetic alterations , which eventually result in the development of a neoplastic monoclonal lymphoproliferation . as well as for malt lymphomas , also for fl could be postulated an inflammatory response secondary to an infectious - driven chronic antigenic stimulation , inducing t(14:18 ) translocation , leading to the transformation of a germinal center - derived b - cell . apart from hp and other few microorganisms that colonize the gastrointestinal tract , it should be kept in mind that little is known about the complex community of human microbiota which includes more than 10 procaryotic cells per individual . modern next generation sequencing tools for microbiome analysis are becoming widely available and intriguing correlations between the type of bacterial colonization in multiple districts , and some diseases have been established . of note , human microbiota has some well - established differences among different world areas as also observed in several indolent lymphoid disorders . it is , therefore , advisable to further investigate this potential link by performing careful case - control or population analyses aiming at verifying whether specific pathological or nearly physiological microbiota patterns might be responsible for a chronic antigen stimulation in those lymphomas where a clearly responsible microorganism has still not been identified . intestinal microbiota either directly or indirectly through the immune system can lead to aberrant dna replication , particularly in some b lymphocytes which are vulnerable to genetic instability and activation , eventually affecting several pathways associated with lymphomagenesis.38 finally , a chronic antigenic infectious stimulation was shown to be fundamental also in the cell perturbation of the microenvironment in sustaining the neoplastic cell growth . indeed , also this pathway could play a role in the oncogenic cascade leading to fl,39,40 and the encouraging results obtained by a novel panel of inhibitors of the signal transduction of the bcr , has led to further investigate the crosstalk between the downstream bcr signaling cascade and the microenvironment . the btk inhibitor ibrutinib and the pi3k inhibitor idelalisib have demonstrated good safety profile and promising clinical efficacy , affecting the survival of neoplastic b cells by preventing lymphocyte adhesion and homing , and inhibiting the microenvironment signals that commonly sustain the malignant clone.41,42 the pathogenesis of fl is a multistep process in which the t(14;18 ) translocation in a b lymphocyte appears to be fundamental for the initiation of the neoplastic cascade . even if still unclear , infectious agents could play a role as a first hit responsible for the b - cell malignant transformation and growth . precise elucidation of the mechanisms underlying lympho - proliferations may provide important clues for understanding how immune disturbance contributes to the development of this subtype of lymphoma . moreover , the responses shown by bcr inhibitors and by antibacterial treatments , which can have cytoprotection properties like rifaximin , considered a gut microenvironment modulator , provide an intriguing argument for a causative link between infectious agents and b - cell lymphoproliferation .

several infectious agents appear to provide a proliferative signal -- antigen - drive that could be implicated in the pathogenesis of various type of non - hodgkin lymphoma ( nhl ) . a classical model of the infection - driven lymphoproliferative disorder is helicobacter pylori - induced gastric malt lymphoma , where antibiotic therapy allows the eradication of both the infectious agent and the clonal b - cell expansion . following the footsteps of this example , several retrospective studies have found a correlation with other pathogens and b - cell lymphomas , adding new relevant information about pathogenesis and laying the groundwork for chemotherapy - free treatments.although no clear association has been found between infectious agents and follicular lymphoma ( fl ) , a growing number of biological and clinical observations suggests the interaction of physiological and pathological microbial populations also in this subtype of lymphoma . in the last few years , epidemiological studies investigating the association of known risk factors and fl found a potential correlation with viral or bacterial infections ; moreover , recent findings of the stimulation of fl clones support the importance of microbial exposure to lymphomagenesis and disease progression.in the following review we make an attempt to find tangible evidence for a role of either physiological and pathological exogenous microbial species in the pathogenesis of fl , and try to integrate the findings coming from epidemiological , biological and interventional studies to define future novel treatment and prevention strategies for fl .

Introduction Role of Infection in other Lymphomas Epidemiological Evidence Biological Evidence Clinical Evidence Discussion Conclusions

nonetheless , most patients eventually develop increasingly resistant disease over time , and in up to 45% of cases , the original indolent subtype transforms into an aggressive subtype , an event that is associated with a poor outcome.1,2,3 the first hit of the oncogenic cascade leading to fl is attributed to the t(14;18 ) chromosomal translocation that occurs in an early b cell stage in the bone marrow . naive b cells , carrying the t(14;18 ) , exit the bone marrow and colonize secondary lymphoid tissue , where they undergo the germinal center reaction but have a survival advantage due to their constitutive expression of bcl2 , which is not normally expressed in the germinal center.4 apart from the t(14;18 ) , recurrent secondary genetic alterations including genomic gains , losses , and mutations ( i.e. alterations in mll2 , epha7 , tnfrsf14 , and ezh2 ) could provide a growth advantage to the neoplastic cells . moreover , the crosstalk between neoplastic b cells and the microenvironment plays an important role in sustaining tumor cell growth and eventually promoting transformation.5 current treatment strategies vary from the classical watch and wait approach to the use of anti cd20 monoclonal antibodies ( labeled or unlabeled with radioimmunoconjugates ) in combination with chemotherapy , while more aggressive treatment approaches including autologous or allogeneic stem cell transplantation are reserved to patients with more resistant disease.6 recently a large bulk of molecular and clinical research has been performed to better understand the molecular mechanisms of lymphomagenesis and to develop non - chemotherapeutic agents active in specific lymphoma subtypes ; in this field infectious agents could represent therapeutic targets for lymphoma treatment toward chemotherapy - free therapeutic approaches . despite huge advances in the comprehension of the genetic anatomy of fl , the potential epidemiologic role of environmental stimuli has not been clearly established ; this is somehow in contrast to the huge bulk of knowledge which has been accumulated in malt lymphomas . nevertheless , a number of biological and clinical observations suggests that interaction with physiological and pathological microbial populations might play a role also in fl . a classical model of the infection - driven lymphoproliferative disorder is helicobacter pylori - induced gastric malt lymphoma where antibiotic therapy allows eradication of both the infectious agent and the clonal b - cell expansion , leading to long - term complete remissions ( cr).7 the identification of this pathogen as the causative agent in gastric malt lymphomas have resulted in substantial progress in understanding the physiopathology of the disease permitting to develop new therapeutic strategies . the list of lymphomas evolving in response to antigen ( bacterial or viral ) has been growing rapidly in recent years , associated in some cases with similar therapeutic success.8 although no such association with infectious agents or other early specific therapeutic target has yet been identified for fl , this concept seems ideally suited to such an indolent disease . this review summarizes current evidence for a role of either physiological and pathological exogenous microbial species in the pathogenesis of fl . so , we underwent an extensive literature search focusing on clinical observations suggesting such correlations , and we tried to underline potential similarities between fl and other indolent lymphoproliferative processes where the role of microbial organisms is clearly established . in recent years , a growing number of exogenous microbial agents have been linked to nhl . the helicobacter pylori ( hp ) , chlamydia psittaci and hepatitis c virus are best - known examples , but other agents have been identified in the pathogenesis of more rare subtypes of nhl ; these associations are important because they have clinical and therapeutic implications and provide novel insights into the mechanisms that govern lymphoma development.9 hp is a proteobacteria epsilon bacterium known to cause stomach ulcers and chronic gastritis . its role in the pathogenesis of the majority of cases of gastric malt lymphoma was demonstrated nearly twenty years ago . the pathogenetic role of hp is related to the oncogenic properties of the cytotoxin - associated antigen a ( cag - a ) , a protein that is able to activate the signaling pathway leading to the activation and upregulation of the antiapoptotic molecule bcl2.10,11 three major chromosomal translocations specific of malt lymphomas are reported , i.e. t(11;18 ) which is the most common ( found in nearly 30% of the cases ) , t(14;18 ) and t(1;14 ) . hp eradication using a combination of antibiotics and proton - pump inhibitors ( ppi ) , represents the standard treatment of hp - associated malt - lymphomas , leading to lymphoma regression in about 75% of patients.12 chlamydophila psittaci belongs to the family of chlamydiae and is the second most studied among bacteria having a pathogenetic role in malt - lymphomas . dna from this bacterium has been found in biopsies of malt lymphoma of the ocular adnexa . the finding that c. psittaci infection has been detected in up to approximately 80% of italian patients with ocular adnexa malt lymphoma provided the rationale for the antibiotic treatment of localized lesions . moreover , the eradication of c. psittaci infection with doxycycline for patients with ocular adnexa malt lymphoma resulted in lymphoma regression in approximately 50% of patients.9 epstein - barr virus ( ebv ) is the first human herpes virus found to be associated with the pathogenesis of cancer . however , following the introduction of highly active antiretroviral therapy , the risk of developing lymphoma in the context of hiv infection has decreased , and the clinical outcome has improved.17 hepatitis c virus ( hcv ) is a small rna virus of the flaviviridae family ; is a hepatotropic and lymphotropic virus responsible for acute hepatitis and chronic liver disease ; the presence of hcv is associated with a spectrum of lymphoproliferative disorders , ranging from polyclonal b - cell expansion to overt malignant lymphoma . indeed , as well as small b - cell clones can be detected in bone marrow or liver biopsies , a higher frequency of lymphoid malignancies has been reported in hcv - positive patients . the association between hcv infection and nhl has been demonstrated by epidemiological studies , in particular in highly endemic geographical areas such as italy , japan , and southern parts of united states . in these countries , together with diffuse large b - cell lymphomas , marginal zone lymphomas are the histotypes most frequently associated with hcv infection . the most convincing argument for a causative link between hcv and lymphoproliferation is represented by studies demonstrating the eradication of the neoplastic clone by the antiviral treatment in hcv - positive patients affected by indolent nhl ( 18 ) . analogous to what has been observed in hp - associated gastric , the role of hcv infection in lymphomagenesis may be related to the chronic antigenic stimulation of b - cell immunologic response by the virus.19,20 adult t - cell leukemia - lymphoma ( atl ) is an aggressive lymphoid proliferation associated with the human lymphotropic virus type i ( htlv - i ) . recently , a worldwide meta - analysis revealed that the combination of zidovudine and ifn- is highly effective in the leukemic subtypes of atl and should be considered as standard first - line therapy in this setting.21 human herpesvirus 8 ( hhv8 ) is a gammaherpesvirus associated with primary effusion lymphoma , a lymphoproliferative disease that is rarely observed in immunocompromised individuals . given the heterogeneous nature of lymphoma subtypes and their different clinical behavior , it is intriguing to identify the risk factors potentially responsible for the occurrence of nhl , so various occupational , environmental and chemical agents have been claimed by several epidemiological studies . several reports have also investigated the possible association between infection - related conditions and the occurrence of nhl ; in fact , several infectious agents have been identified as causative factors for the development of nhl , most likely due to their induction of dna damage , inflammatory cells proliferation , and cytokine release . to address this issue the international lymphoma epidemiology consortium ( interlymph ) , an open scientific forum for epidemiological research funded in 2001 , undertook the nhl subtypes project ; this is an international group of multidisciplinary specialists , who have worked together with the aim of identifying associations of several risk factors across different lymphoma subtypes.23 regarding fl , in 2013 a large pooled analysis carried on by the interlymph consortium made an attempt to assess associations between medical , hormonal , family history , lifestyle and occupational factors with the risk of developing fl . moreover , fl risk was increased in subjects with a first - degree relative with non - hodgkin lymphoma in spray painters among women with sjgren and among cigarette smokers and obese subjects . no specific observation mentioned a link between infection and risk of fl.24 another large retrospective case control study using seer and medicare database investigated the role of infection - related conditions and different nhl subtypes.25 cases were defined as individuals with a seer diagnosis of primary lymphoid malignancy between 1992 and 2005 . claims for sinusitis , laryngitis and herpes zoster were present in the history of fl patients , sinusitis , laryngitis and herpes zoster were significant at longer latencies . most fl cases carried the t(14;18 ) , which was hypothesized to be transformed by exogenous antigen stimulation , such as from a viral infection.26 antigenic stimulation and/or subclinical immune deficiency , predisposing patients to both infections and lymphoma , were claimed as possible association between infection - related conditions and lymphoma . the gene sequence for the immunoglobulin ( ig ) heavy - chain ( h ) and light - chain ( l ) variable regions are assembled in the early stages of b - cell development in the bone marrow from distinct variable ( v ) , diversity ( d ) and joining ( j ) segments through a process of somatic dna rearrangement known as v(d)j recombination . in later stages , which takes place in the germinal center ( gc ) of the secondary lymphoid tissues , naive b - cells with low - affinity functional surface ig ( sig ) are induced to proliferate . in fl cells , b - cell receptor ( bcr ) expression is retained , despite the characteristic chromosomal translocation t(14;18 ) , because bcr is fundamental for the transduction of the signals that maintain the survival and growth of fl clones . bcr variable - region mannoses in fl are recognized by lectins of common opportunistic bacteria , such as burkholderia cenocepacia and pseudomonas aeruginosa , that are usually found in soil and water ; these lectins represent a potent stimulus for the proliferation of b cells expressing this kind of glycan - terminated glycan.27,28,29 therefore , these studies directly support the potential importance of microbial exposure in the proliferation and survival of fl clones , and they might be a key to a better understanding of the pathogenesis of fl . although an expanding literature has examined several risk factors potentially correlated with the occurrence of fl , the etiology of the proliferative stimulus is generally unknown , and the few relationships observed suggest a complex multifactorial etiology . interestingly , regarding fl subtype , a trend toward statistical risk was observed in countries with a high prevalence of hbv infection while no statistical risk was seen in countries with a low prevalence of hbv infection.30 the conclusion of the study was that was difficult to determine if the increased risk of fl in areas of high prevalence of hbv infections is due simply to a larger number of hbv infections or a true causal relationship . in the latter case , hbv might be responsible for lymphomagenesis through a chronic stimulation of b - cells which may predispose to dna damage and transformation into neoplastic cells , or through an immunologic response to chronic local antigenic stimulation . as opposed to hbv , a case - control study carried on by the interlymph did not show an increased association between hcv infection and fl , that was restricted to other specific b - nhl subtypes like diffuse large b - cell lymphoma ( dlbcl ) , marginal zone lymphoma , and lymphoplasmacytic lymphoma.19 an interesting clinicopathological finding came up from a spanish study that analyzed a retrospective series of 58 patients with a diagnosis of hcv - positive b - cell lymphoproliferative disorder ; eight of them were affected by fl , and at least half of them expressed bcl2 and p53 . interestingly , the authors reported a cohort of 11 patients in which a clonal b cell expansion in the peripheral blood and bone marrow could be revealed , in the absence of conclusive histological evidence of neoplastic infiltration . these expanded clones make up a definite group of hcv - associated monoclonal b - cell lymphocytosis that should be monitored because a 10% risk of evolution to overt lymphoma has been demonstrated.31 currently , the association between ebv and follicular lymphoma is reported only in the form of isolated case reports in patients with various form of immunodeficiency or in the context of transformation to diffuse large cell lymphoma or classical hodgkin lymphoma . they identified 10 cases of ebv - positive fl ( prevalence=2.6% ) with a significant prevalence of grades 3a3b fl ( 9 out of 10 ) and frequent strong coexpression of cd30 ; all cases demonstrated progression of the disease to a higher grade fl or diffuse large b - cell lymphoma . given the increased incidence of ebv in high - grade fl and the fact that the cases are clinically and morphologically indistinguishable from ebv - negative fl patients , the authors suggested the screening for eber in all high - grade cases.32 recently an intriguing association between coxiella burnetii / q fever and the incidence of b - cell lymphomas was proposed by melenotte et al . in a large scale study.33 starting from the observation of the occurrence of lymphoma in a patient with q fever , they screened over 1000 consecutive patients of the french national referral center for q fever database and examined if there was an association between the two diseases . an excess risk of dlbcl and fl was found in individuals who had q fever compared with the general population and above all patients with a persistent localized infection were found to have a greater risk of lymphoma . these results support the evidence that a novel factor should be added to the list of bacteria that promote human b - cell lymphomas , in particular , fl . the most relevant studies reporting a link between follicular lymphoma and infectious agents are summarized in table 1 . taken in mind the gastric malt as a well - accepted example of antigen - driven neoplastic cell proliferation , portlock et al . explored the association between infectious agents and nhl in a cohort of 56 patients with an untreated advanced non - bulky indolent lymphoma.34 all patients were tested for hp , hcv , borrelia burgdorferi , chlamydia psittaci and small bowel bacterial overgrowth ; in this series , a documented infection was found in 37% of the patients , with a prevalence of hp . therefore in 2007 , they launched a prospective clinical trial testing the role of prolonged clarithromycin antibiotic therapy as a first treatment in the same category of indolent advanced - stage lymphoma patients . although the small sample size , they reported lymphoma objective responses in 9 of 32 patients ( 28.1% ) with a long treatment - free survival for patients responding to antibiotics.35 the association between infectious agents and fl added new important information about the role played by the antigen stimulation in fl ; moreover , the possibility to treat the neoplastic disease in a simple and efficient way could be considered a step toward developing a lymphoma preventive strategy by reducing the antigen drive . in the last years , several infectious agents , like hepatitis c , human immunodeficiency , and epstein - barr viruses , and helicobacter pylori , chlamydia psittaci , and coxiella burnetii bacteria have been reported as involved in the malignant transformation of b or t lymphocytes , and therefore associated with the pathogenesis of lymphoproliferative disorders . regarding fl , based on available data , evidence linking this lymphoma subtype and exogenous infectious agents are weak , and currently , fl can not be considered as an infection - driven disease . however , some clinical , epidemiologic studies and case reports indicate that it is still somehow premature to conclude that exogenous agents have a negligible role in the genesis of fl . it has been estimated that chronic infections caused by viruses , bacteria , and parasites are the causative agents of nearly 1015% of global cancers burden.36,37 these infectious agents promote a cascade of events culminating in chronic inflammatory responses . in particular , in antigen - driven hematologic malignancies , like hp - associated with malt , the chronic stimulation of the innate immune system causes a clonal expansion of b - lymphocytes , which leads to the production of oxidative reactions ; these events result in genetic alterations , which eventually result in the development of a neoplastic monoclonal lymphoproliferation . as well as for malt lymphomas , also for fl could be postulated an inflammatory response secondary to an infectious - driven chronic antigenic stimulation , inducing t(14:18 ) translocation , leading to the transformation of a germinal center - derived b - cell . modern next generation sequencing tools for microbiome analysis are becoming widely available and intriguing correlations between the type of bacterial colonization in multiple districts , and some diseases have been established . intestinal microbiota either directly or indirectly through the immune system can lead to aberrant dna replication , particularly in some b lymphocytes which are vulnerable to genetic instability and activation , eventually affecting several pathways associated with lymphomagenesis.38 finally , a chronic antigenic infectious stimulation was shown to be fundamental also in the cell perturbation of the microenvironment in sustaining the neoplastic cell growth . indeed , also this pathway could play a role in the oncogenic cascade leading to fl,39,40 and the encouraging results obtained by a novel panel of inhibitors of the signal transduction of the bcr , has led to further investigate the crosstalk between the downstream bcr signaling cascade and the microenvironment . the btk inhibitor ibrutinib and the pi3k inhibitor idelalisib have demonstrated good safety profile and promising clinical efficacy , affecting the survival of neoplastic b cells by preventing lymphocyte adhesion and homing , and inhibiting the microenvironment signals that commonly sustain the malignant clone.41,42 the pathogenesis of fl is a multistep process in which the t(14;18 ) translocation in a b lymphocyte appears to be fundamental for the initiation of the neoplastic cascade . even if still unclear , infectious agents could play a role as a first hit responsible for the b - cell malignant transformation and growth . precise elucidation of the mechanisms underlying lympho - proliferations may provide important clues for understanding how immune disturbance contributes to the development of this subtype of lymphoma . moreover , the responses shown by bcr inhibitors and by antibacterial treatments , which can have cytoprotection properties like rifaximin , considered a gut microenvironment modulator , provide an intriguing argument for a causative link between infectious agents and b - cell lymphoproliferation .

in october 2001 , the nordic cochrane centre published a cochrane review of mammography screening , which questioned whether screening reduces breast cancer mortality.1 within the same month , the centre published a more comprehensive review in lancet that also reported on the harms of screening and found considerable overdiagnosis and overtreatment ( a 30% increase in the number of mastectomies and tumourectomies).2 this resulted in a heated debate , which is still ongoing.3 the cochrane review was updated in 2006,4 to include overdiagnosis , and again in 2009.5 recently , several studies have questioned whether screening is as beneficial as originally claimed,68 and confirmed that overdiagnosis is a major harm of breast cancer screening.911 the us preventive services task force published updated screening recommendations in november 2009 and asserted that the benefit is smaller than previously thought and that the harms include overdiagnosis and overtreatment , but it did not quantify these harms.12 the task force changed its previous recommendations and now recommends that women aged 4049 years discuss with their physician whether breast screening is right for them , and it further recommends biennial screening instead of annual screening for all age groups.12 these recommendations were repeated in the 2011 canadian guidelines for breast screening.13 screening is likely to miss aggressive cancers because they grow fast , leaving little time to detect them in their preclinical phases.6 further , the basic assumption that finding and treating early - stage disease will prevent late stage or metastatic disease may not be correct , as breast cancer screening has not reduced the occurrence of large breast cancers14 or late - stage breast cancers,11 despite the large and sustained increases in early invasive cancers and ductal carcinoma in situ with screening . a systematic review from 2009 showed that the rate of overdiagnosis in organised breast screening programmes was 52% , which means that one in three cancers diagnosed in a screened population is overdiagnosed.9 it is quite likely that many screen - detected cancers would have regressed spontaneously in the absence of screening.15 16 we explored how the first comprehensive systematic review on mammography screening ever performed , the one from 2001 published in lancet,2 and the subsequent systematic cochrane reviews from 20064 and 20095 have been cited from 2001 to april 2012 . we investigated whether there were differences between general medical journals and specialty journals regarding which results were mentioned and how overdiagnosis , overtreatment , breast cancer mortality , total mortality , and the methods of the reviews were described . vested interests on behalf of both journals and contributing authors may be more pronounced in specialty journals , and this may influence views on specific interventions , such as mammography screening . we searched for articles quoting one of the three versions of the review2 4 5 ( date of last search 20 april 2012 ) . we used the source titles function in the institute for scientific information ( isi ) web of knowledge to count the number of times each review had been cited in individual journals . we only included journals in which four or more articles had cited one of the three versions of the review . this criterion led to the exclusion of specialty journals of little relevance for our study , for example , nephrology and research in gerontological nursing . we could not include the 2001 cochrane review1 because it was not indexed by the isi web of knowledge . this version of the review1 is not comparable to the other three versions,25 as the editors of the cochrane breast cancer group had refused to publish these data on overdiagnosis and overtreatment . a journal was classified as a general medical journal if it did not preferentially publish papers from a particular medical specialty . a journal was classified as a specialty journal if it preferentially published articles from a particular medical specialty or topic . when we rated how the papers cited the review , we looked for statements applicable to the following categories : breast cancer mortality methods used in the review we rated the quoting articles general opinions about the results and methods of the review using the labels accept , neutral , reject , unclear , or not applicable , using the following definitions : accept : the authors explicitly agreed with the results or methods , or quoted the numerical results without comments . neutral : the results or methods were mentioned and the author presented arguments both for and against them . reject : the authors explicitly stated that the results or methods were flawed , wrong , or false , or only presented arguments against them . only reporting a result from a favourable subgroup analysis unclear : the results or methods were mentioned , but it was not possible to tell if the authors agreed with them or not , or the results were only mentioned qualitatively . not applicable : the review was quoted for something else than its results or methods . the articles quoting the review were assessed in relation to the five categories ( overdiagnosis , overtreatment , breast cancer mortality , total mortality and methods ) separately , and no overall assessment of the articles general opinion about the review was made . texts classified as not applicable regarding any of the five categories were reread to determine and note which topics were discussed . two researchers ( kr , andreas brnden petersen , see acknowledgements ) assessed the text independently . disagreements were settled by discussion . in order to ensure blinded data extraction , an assistant ( mads clausen , see acknowledgements ) not involved with data extraction identified the text sections citing one of the three review versions and copied them into a microsoft word document . only this text was copied , and the two data extractors were therefore unaware of the author and journal names , time of publication and the title of the article . the fonts of the copied text were converted into times new roman , saved in a new document and the text labelled with a random number using the the key to matching the text with the articles was not available to data extractors until the assessments had been completed . the person responsible for copying the text made sure it did not contain any information that might reveal which of the three versions of the review had been cited . when there was more than one reference within the copied text , the reference to the review was highlighted to make it clear which statements referred to the review . all article types , as well as letters to the editor , were included and were classified as research papers , systematic reviews , editorials , letters , guidelines and narratives . p values were calculated using fisher 's exact test ( two - tailed p values ( http://www.swogstat.org/stat/public/fisher.htm ) ) . in total , 523 articles cited one of the three versions of the review : 360 cited the 2001 lancet review,2 123 the 2006 cochrane review4 and 40 the 2009 cochrane review.5 three articles cited both the 2001 and the 2006 versions of the review ; for these , we only used information related to the 2001 citation . including only journals that had published at least four articles , which cited one or more of the three versions of the review , the search identified 151 , 27 and 15 articles , respectively ( 193 in total , or 37% of the total of 523 articles ) . we excluded 22 additional articles , two because there was no reference to the review in the text , even though the review was listed as a reference,17 18 and 20 ( 10 , 5 and 5 citing the 2001 , 2006 and 2009 versions , respectively ) because they had one or more authors affiliated with the nordic cochrane centre . thus , 171 articles were included for assessment . in total , 63 articles ( 37% ) were from general medical journals and 108 ( 63% ) from specialty journals . a total of 80 ( 47% ) were from european journals and 91 ( 53% ) from north american journals . the general medical journals included were lancet ( 21 articles ) , bmj ( 13 articles ) , annals of internal medicine ( 13 articles ) , journal of the american medical association ( 7 articles ) , new england journal of medicine ( 5 articles ) and international journal of epidemiology ( 4 articles ) . the specialty journals included journal of the national cancer institute ( 13 articles ) , cancer ( 13 articles ) , american journal of roentgenology ( 7 articles ) and 15 others ( see box 1 ) . most of the included articles were either research papers ( n=63 , 37% ) or narrative articles ( n=44 , 26% ; table 1 ) . box 1the specialty journals included in this studyspecialty journals includedjournal of the national cancer institute ( 13)cancer ( 13)european journal of cancer ( 7)british journal of cancer ( 7)american journal of roentgenology ( 7)cancer causes and control ( 6)annals of oncology ( 6)european journal of surgical oncology ( 6)journal of medical screening ( 5)cancer epidemiology , biomarkers and prevention ( 5)ca : a cancer journal for clinicians ( 5)journal of clinical oncology ( 5)radiologic clinics of north america ( 5)oncologist ( 4)breast cancer research and treatment ( 4)breast ( 4)radiology ( 3)journal of surgical oncology ( 3 ) specialty journals included journal of the national cancer institute ( 13 ) european journal of cancer ( 7 ) british journal of cancer ( 7 ) american journal of roentgenology ( 7 ) cancer causes and control ( 6 ) annals of oncology ( 6 ) european journal of surgical oncology ( 6 ) journal of medical screening ( 5 ) cancer epidemiology , biomarkers and prevention ( 5 ) ca : a cancer journal for clinicians ( 5 ) journal of clinical oncology ( 5 ) radiologic clinics of north america ( 5 ) breast cancer research and treatment ( 4 ) journal of surgical oncology ( 3 ) the article types included in this study eu , european ; na , north american . the text of 32 of the 171 included articles ( 19% ) was rated as not applicable for all the five categories ( overdiagnosis , overtreatment , breast cancer mortality , total mortality and methods ) . in total , 15 of these 32 articles discussed the controversy when the first review was published , without specifically mentioning any of the categories . other subjects discussed were screening of women under the age of 50 ( two articles ) , and benefits of breast cancer screening other than those in our categories ( two articles ; see online supplementary appendix 1 for a full list of topics ) . the review s conclusions regarding overdiagnosis were not quoted in 87% ( 149/171 ) of the included articles and the results for breast cancer mortality were not quoted in 53% ( 91/171 ) of the included articles . general medical journals were more likely to accept the results or methods of systematic reviews than specialty journals , for example , overdiagnosis was classified as accepted in 11% ( 7/63 ) of articles in general medical journals , but in only 3% ( 3/108 ) of the articles in specialty journals ( p=0.05 ) , and the methods were accepted in 14% ( 9/63 ) of articles in general medical journals , but only in 1% ( 1/108 ) of articles in specialty journals ( p=0.001 ) . specialty journals were also more likely to reject the results for breast cancer mortality , namely for 26% ( 28/108 ) of articles compared with 8% ( 5/63 ; p=0.02 ) in general medical journals . the differences between general medical and specialty journals in relation to rejecting the categories overdiagnosis , overtreatment , total mortality and methods were small ( table 2 ) . the european and north american journals were equally likely to reject or accept the review 's methods or results ( data not shown ) . the number of citations of the three versions of the review differed a lot over time ( see table 3 ) . some years had very few citations , the lowest being 2012 and 2006 where the review was cited only 1 and 6 times , respectively . the highest number of citations was in 2002 ( 42 citations ) . there were no clear trends over time regarding the number of articles accepting or rejecting the methods and conclusions of the reviews , although the breast cancer mortality results may have received greater acceptance in recent years , for example , in 2002 , there was no acceptance of the breast cancer mortality results ( 0 of 42 ) , whereas 19% ( 3/16 ) explicitly accepted them in 2010 ( p=0.02 ; data not shown ) . number of citations of one of the three reviews per year the 2001 version of the review had more categories rejected and fewer categories accepted than the 2006 and 2009 versions , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version ( p=0.0002 ; see table 4 ) . comparison of the three versions of the review in terms of accepting or rejecting the five categories percentages in brackets are calculated from the total number of articles cited in that version of the review . the total number of articles cited in the 2006 version : 22 . the total number of articles cited in the 2009 version : 10 . although we deliberately reduced the sample size by requiring at least four citations for each included journal , we had enough articles that quoted the review for our comparisons . specialty journals were more likely to reject the estimate of the effect of screening on breast cancer mortality than the six general medical journals we included . articles in general medical journals were also more approving of four of the five individual categories we assessed ( overdiagnosis , overtreatment , total mortality and methods ) than the specialty journals were and the difference was statistically significant for all the categories , except for breast cancer mortality . we have previously found that scientific articles on breast screening tend to emphasise the major benefits of mammography screening over its major harms and that overdiagnosis was more often downplayed or rejected in articles written by authors affiliated with screening by specialty or funding , compared with authors unrelated with screening.19 recommendations in guidelines for breast screening are also influenced by the authors medical specialty.20 the difference we found between the general medical and specialty journals could be explained by conflicts of interest , which are likely to be more prevalent in specialty journals owned by political interest groups such as the american cancer society or by medical societies with members whose income may depend on the intervention . all the six general medical journals , but only 22% ( 4/18 ) of the specialty journals follow the international committee of medical journal editors ( icmje ) uniform requirements for manuscripts submitted to biomedical journals.21 even though journals have conflict of interest reporting policies , the conflicts of interest reported are not always reliable.22 all the general medical journals included are members of the world association of medical editors ( wame ) ; however , this is only the case for 22% ( 4/18 ) of the specialty journals included . wame aims to improve the editorial standards and , among other things , to ensure a balanced debate on controversial issues.23 being a member of wame helps with transparency in terms of their guidelines for conflicts of interest , but it also reminds editors to ensure that their journals are covering both sides of a debate . the results and conclusions on breast cancer mortality and overdiagnosis were more often accepted in 2010 than in any other year ( data not shown ) . the ongoing independent review of the national health service ( nhs ) breast screening programme announced by mike richards , the uk national clinical director for cancer and end of life care , department of health , in october 2011 is a further indication of this development.24 also , the us preventive services task force changed its recommendations for breast screening in 2009.12 though our data did not show strong time trends , we believe that these developments demonstrate a growing acceptance of the results and conclusions of our systematic review . in support of this , the 2009 version of the cochrane review has received more approval than disapproval , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version.2531 the us preventive services task force was heavily criticised after the publication of its new recommendations in 2009,29 32 but the criticism came from people with vested interests , and the independent canadian task force supported the conclusions of the us preventive services task force and the 2009 cochrane review5 in 2011.13 the 2001 review published in lancet was by far the most cited of the three reviews . it was 5 years older than the cochrane review from 2006 , but the vast majority of the citations came within the first year of publication . it was unique at the time , as it questioned whether mammography screening was effective , based on a thorough quality assessment of all the randomised controlled trials , and also was the first systematic review to quantify overdiagnosis . a minor part of the included articles ( 19% , 32/171 ) did not refer to any of our five specified outcomes . in nearly half of the cases ( 47% ) , this was due to the article referring only to the debate that followed the first review,33 and not its results or methods . the texts also dealt with topics such as false positives or screening women under the age of 50 years . this was the case for articles in both the general medical and specialty journals , and for articles in the european and north american journals . the text typically dealt with only one or two of our categories , for example , overdiagnosis , and did not mention overtreatment or any other categories . none of the articles rejected overdiagnosis ( 0 of 171 articles ) , which could be because they did not mention the issue at all . this was the case in 76% of scientific articles on breast screening in a previous study by jrgensen et al.19 our definition of rejection was that the author should explicitly state that the review 's estimate was flawed , wrong or false , or that they should in some way argue against it . with this strict definition , we did not capture authors who have consistently stated over the years in other articles than those we included that they do not believe that overdiagnosis is a problem , and we also did not present their views on the subject . numerous articles were classified as unclear for one or more of our categories . the texts in question did not allow an interpretation in any direction and we did not rate the articles as accepting or rejecting the review s results and methods unless it was perfectly clear what the authors meant . this reflects that authors often do not present clear opinions of the intervention which they discuss . an additional explanation for the many articles found to be unclear could be that we did not assess the entire article , and arguments could have been presented elsewhere in the text . letters were included in this study , which could explain why some of the articles were classified as not applicable in all the five categories . the specialists who read and respond to letters in their own journals might be more likely to react negatively towards the review because of conflicts of interest.19 specialists with a connection to mammography screening also reply to articles in general medical journals when they concern mammography screening . therefore , it is quite likely that there is a greater difference between the specialists involved with the screening programmes and the doctors not involved in breast cancer screening , in terms of accepting and rejecting the results and methods , than we have found in this study . the results and conclusions on breast cancer mortality and overdiagnosis were more often accepted in 2010 than in any other year ( data not shown ) . the ongoing independent review of the national health service ( nhs ) breast screening programme announced by mike richards , the uk national clinical director for cancer and end of life care , department of health , in october 2011 is a further indication of this development.24 also , the us preventive services task force changed its recommendations for breast screening in 2009.12 though our data did not show strong time trends , we believe that these developments demonstrate a growing acceptance of the results and conclusions of our systematic review . in support of this , the 2009 version of the cochrane review has received more approval than disapproval , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version.2531 the us preventive services task force was heavily criticised after the publication of its new recommendations in 2009,29 32 but the criticism came from people with vested interests , and the independent canadian task force supported the conclusions of the us preventive services task force and the 2009 cochrane review5 in 2011.13 the 2001 review published in lancet was by far the most cited of the three reviews . it was 5 years older than the cochrane review from 2006 , but the vast majority of the citations came within the first year of publication . it was unique at the time , as it questioned whether mammography screening was effective , based on a thorough quality assessment of all the randomised controlled trials , and also was the first systematic review to quantify overdiagnosis . a minor part of the included articles ( 19% , 32/171 ) did not refer to any of our five specified outcomes . in nearly half of the cases ( 47% ) , this was due to the article referring only to the debate that followed the first review,33 and not its results or methods . the texts also dealt with topics such as false positives or screening women under the age of 50 years . this was the case for articles in both the general medical and specialty journals , and for articles in the european and north american journals . the text typically dealt with only one or two of our categories , for example , overdiagnosis , and did not mention overtreatment or any other categories . none of the articles rejected overdiagnosis ( 0 of 171 articles ) , which could be because they did not mention the issue at all . this was the case in 76% of scientific articles on breast screening in a previous study by jrgensen et al.19 our definition of rejection was that the author should explicitly state that the review 's estimate was flawed , wrong or false , or that they should in some way argue against it . with this strict definition , we did not capture authors who have consistently stated over the years in other articles than those we included that they do not believe that overdiagnosis is a problem , and we also did not present their views on the subject . the texts in question did not allow an interpretation in any direction and we did not rate the articles as accepting or rejecting the review s results and methods unless it was perfectly clear what the authors meant . this reflects that authors often do not present clear opinions of the intervention which they discuss . an additional explanation for the many articles found to be unclear could be that we did not assess the entire article , and arguments could have been presented elsewhere in the text . letters were included in this study , which could explain why some of the articles were classified as not applicable in all the five categories . the specialists who read and respond to letters in their own journals might be more likely to react negatively towards the review because of conflicts of interest.19 specialists with a connection to mammography screening also reply to articles in general medical journals when they concern mammography screening . therefore , it is quite likely that there is a greater difference between the specialists involved with the screening programmes and the doctors not involved in breast cancer screening , in terms of accepting and rejecting the results and methods , than we have found in this study . articles in specialty journals were less approving of the results and methods of the systematic review of breast screening than those in general medical journals . this may be explained by conflicts of interest , as several specialty journals were published by groups with vested interests in breast screening , and several articles had authors with vested interests .

introductionin 2001 , a cochrane review of mammography screening questioned whether screening reduces breast cancer mortality , and a more comprehensive review in lancet , also in 2001 , reported considerable overdiagnosis and overtreatment . this led to a heated debate and a recent review of the evidence by uk experts intended to be independent.objectiveto explore if general medical and specialty journals differed in accepting the results and methods of three cochrane reviews on mammography screening.methodswe identified articles citing the lancet review from 2001 or updated versions of the cochrane review ( last search 20 april 2012 ) . we explored which results were quoted , whether the methods and results were accepted ( explicit agreement or quoted without caveats ) , differences between general and specialty journals , and change over time.resultswe included 171 articles . the results for overdiagnosis were not quoted in 87% ( 148/171 ) of included articles and the results for breast cancer mortality were not quoted in 53% ( 91/171 ) of articles . 11% ( 7/63 ) of articles in general medical journals accepted the results for overdiagnosis compared with 3% ( 3/108 ) in specialty journals ( p=0.05 ) . 14% ( 9/63 ) of articles in general medical journals accepted the methods of the review compared with 1% ( 1/108 ) in specialty journals ( p=0.001 ) . specialty journals were more likely to explicitly reject the estimated effect on breast cancer mortality 26% ( 28/108 ) , compared with 8% ( 5/63 ) in general medical journals , p=0.02.conclusionsarticles in specialty journals were more likely to explicitly reject results from the cochrane reviews , and less likely to accept the results and methods , than articles in general medical journals . several specialty journals are published by interest groups and some authors have vested interests in mammography screening .

Introduction Methods Results Discussion Development over time Limitations Conclusion Supplementary Material Supplementary Material

in october 2001 , the nordic cochrane centre published a cochrane review of mammography screening , which questioned whether screening reduces breast cancer mortality.1 within the same month , the centre published a more comprehensive review in lancet that also reported on the harms of screening and found considerable overdiagnosis and overtreatment ( a 30% increase in the number of mastectomies and tumourectomies).2 this resulted in a heated debate , which is still ongoing.3 the cochrane review was updated in 2006,4 to include overdiagnosis , and again in 2009.5 recently , several studies have questioned whether screening is as beneficial as originally claimed,68 and confirmed that overdiagnosis is a major harm of breast cancer screening.911 the us preventive services task force published updated screening recommendations in november 2009 and asserted that the benefit is smaller than previously thought and that the harms include overdiagnosis and overtreatment , but it did not quantify these harms.12 the task force changed its previous recommendations and now recommends that women aged 4049 years discuss with their physician whether breast screening is right for them , and it further recommends biennial screening instead of annual screening for all age groups.12 these recommendations were repeated in the 2011 canadian guidelines for breast screening.13 screening is likely to miss aggressive cancers because they grow fast , leaving little time to detect them in their preclinical phases.6 further , the basic assumption that finding and treating early - stage disease will prevent late stage or metastatic disease may not be correct , as breast cancer screening has not reduced the occurrence of large breast cancers14 or late - stage breast cancers,11 despite the large and sustained increases in early invasive cancers and ductal carcinoma in situ with screening . a systematic review from 2009 showed that the rate of overdiagnosis in organised breast screening programmes was 52% , which means that one in three cancers diagnosed in a screened population is overdiagnosed.9 it is quite likely that many screen - detected cancers would have regressed spontaneously in the absence of screening.15 16 we explored how the first comprehensive systematic review on mammography screening ever performed , the one from 2001 published in lancet,2 and the subsequent systematic cochrane reviews from 20064 and 20095 have been cited from 2001 to april 2012 . we investigated whether there were differences between general medical journals and specialty journals regarding which results were mentioned and how overdiagnosis , overtreatment , breast cancer mortality , total mortality , and the methods of the reviews were described . vested interests on behalf of both journals and contributing authors may be more pronounced in specialty journals , and this may influence views on specific interventions , such as mammography screening . we searched for articles quoting one of the three versions of the review2 4 5 ( date of last search 20 april 2012 ) . we only included journals in which four or more articles had cited one of the three versions of the review . this criterion led to the exclusion of specialty journals of little relevance for our study , for example , nephrology and research in gerontological nursing . this version of the review1 is not comparable to the other three versions,25 as the editors of the cochrane breast cancer group had refused to publish these data on overdiagnosis and overtreatment . when we rated how the papers cited the review , we looked for statements applicable to the following categories : breast cancer mortality methods used in the review we rated the quoting articles general opinions about the results and methods of the review using the labels accept , neutral , reject , unclear , or not applicable , using the following definitions : accept : the authors explicitly agreed with the results or methods , or quoted the numerical results without comments . neutral : the results or methods were mentioned and the author presented arguments both for and against them . only reporting a result from a favourable subgroup analysis unclear : the results or methods were mentioned , but it was not possible to tell if the authors agreed with them or not , or the results were only mentioned qualitatively . the articles quoting the review were assessed in relation to the five categories ( overdiagnosis , overtreatment , breast cancer mortality , total mortality and methods ) separately , and no overall assessment of the articles general opinion about the review was made . texts classified as not applicable regarding any of the five categories were reread to determine and note which topics were discussed . only this text was copied , and the two data extractors were therefore unaware of the author and journal names , time of publication and the title of the article . the fonts of the copied text were converted into times new roman , saved in a new document and the text labelled with a random number using the the key to matching the text with the articles was not available to data extractors until the assessments had been completed . the person responsible for copying the text made sure it did not contain any information that might reveal which of the three versions of the review had been cited . in total , 523 articles cited one of the three versions of the review : 360 cited the 2001 lancet review,2 123 the 2006 cochrane review4 and 40 the 2009 cochrane review.5 three articles cited both the 2001 and the 2006 versions of the review ; for these , we only used information related to the 2001 citation . including only journals that had published at least four articles , which cited one or more of the three versions of the review , the search identified 151 , 27 and 15 articles , respectively ( 193 in total , or 37% of the total of 523 articles ) . we excluded 22 additional articles , two because there was no reference to the review in the text , even though the review was listed as a reference,17 18 and 20 ( 10 , 5 and 5 citing the 2001 , 2006 and 2009 versions , respectively ) because they had one or more authors affiliated with the nordic cochrane centre . in total , 63 articles ( 37% ) were from general medical journals and 108 ( 63% ) from specialty journals . the general medical journals included were lancet ( 21 articles ) , bmj ( 13 articles ) , annals of internal medicine ( 13 articles ) , journal of the american medical association ( 7 articles ) , new england journal of medicine ( 5 articles ) and international journal of epidemiology ( 4 articles ) . the specialty journals included journal of the national cancer institute ( 13 articles ) , cancer ( 13 articles ) , american journal of roentgenology ( 7 articles ) and 15 others ( see box 1 ) . most of the included articles were either research papers ( n=63 , 37% ) or narrative articles ( n=44 , 26% ; table 1 ) . box 1the specialty journals included in this studyspecialty journals includedjournal of the national cancer institute ( 13)cancer ( 13)european journal of cancer ( 7)british journal of cancer ( 7)american journal of roentgenology ( 7)cancer causes and control ( 6)annals of oncology ( 6)european journal of surgical oncology ( 6)journal of medical screening ( 5)cancer epidemiology , biomarkers and prevention ( 5)ca : a cancer journal for clinicians ( 5)journal of clinical oncology ( 5)radiologic clinics of north america ( 5)oncologist ( 4)breast cancer research and treatment ( 4)breast ( 4)radiology ( 3)journal of surgical oncology ( 3 ) specialty journals included journal of the national cancer institute ( 13 ) european journal of cancer ( 7 ) british journal of cancer ( 7 ) american journal of roentgenology ( 7 ) cancer causes and control ( 6 ) annals of oncology ( 6 ) european journal of surgical oncology ( 6 ) journal of medical screening ( 5 ) cancer epidemiology , biomarkers and prevention ( 5 ) ca : a cancer journal for clinicians ( 5 ) journal of clinical oncology ( 5 ) radiologic clinics of north america ( 5 ) breast cancer research and treatment ( 4 ) journal of surgical oncology ( 3 ) the article types included in this study eu , european ; na , north american . the text of 32 of the 171 included articles ( 19% ) was rated as not applicable for all the five categories ( overdiagnosis , overtreatment , breast cancer mortality , total mortality and methods ) . in total , 15 of these 32 articles discussed the controversy when the first review was published , without specifically mentioning any of the categories . other subjects discussed were screening of women under the age of 50 ( two articles ) , and benefits of breast cancer screening other than those in our categories ( two articles ; see online supplementary appendix 1 for a full list of topics ) . the review s conclusions regarding overdiagnosis were not quoted in 87% ( 149/171 ) of the included articles and the results for breast cancer mortality were not quoted in 53% ( 91/171 ) of the included articles . general medical journals were more likely to accept the results or methods of systematic reviews than specialty journals , for example , overdiagnosis was classified as accepted in 11% ( 7/63 ) of articles in general medical journals , but in only 3% ( 3/108 ) of the articles in specialty journals ( p=0.05 ) , and the methods were accepted in 14% ( 9/63 ) of articles in general medical journals , but only in 1% ( 1/108 ) of articles in specialty journals ( p=0.001 ) . specialty journals were also more likely to reject the results for breast cancer mortality , namely for 26% ( 28/108 ) of articles compared with 8% ( 5/63 ; p=0.02 ) in general medical journals . the differences between general medical and specialty journals in relation to rejecting the categories overdiagnosis , overtreatment , total mortality and methods were small ( table 2 ) . the european and north american journals were equally likely to reject or accept the review 's methods or results ( data not shown ) . the number of citations of the three versions of the review differed a lot over time ( see table 3 ) . some years had very few citations , the lowest being 2012 and 2006 where the review was cited only 1 and 6 times , respectively . there were no clear trends over time regarding the number of articles accepting or rejecting the methods and conclusions of the reviews , although the breast cancer mortality results may have received greater acceptance in recent years , for example , in 2002 , there was no acceptance of the breast cancer mortality results ( 0 of 42 ) , whereas 19% ( 3/16 ) explicitly accepted them in 2010 ( p=0.02 ; data not shown ) . number of citations of one of the three reviews per year the 2001 version of the review had more categories rejected and fewer categories accepted than the 2006 and 2009 versions , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version ( p=0.0002 ; see table 4 ) . comparison of the three versions of the review in terms of accepting or rejecting the five categories percentages in brackets are calculated from the total number of articles cited in that version of the review . the total number of articles cited in the 2009 version : 10 . specialty journals were more likely to reject the estimate of the effect of screening on breast cancer mortality than the six general medical journals we included . articles in general medical journals were also more approving of four of the five individual categories we assessed ( overdiagnosis , overtreatment , total mortality and methods ) than the specialty journals were and the difference was statistically significant for all the categories , except for breast cancer mortality . we have previously found that scientific articles on breast screening tend to emphasise the major benefits of mammography screening over its major harms and that overdiagnosis was more often downplayed or rejected in articles written by authors affiliated with screening by specialty or funding , compared with authors unrelated with screening.19 recommendations in guidelines for breast screening are also influenced by the authors medical specialty.20 the difference we found between the general medical and specialty journals could be explained by conflicts of interest , which are likely to be more prevalent in specialty journals owned by political interest groups such as the american cancer society or by medical societies with members whose income may depend on the intervention . all the six general medical journals , but only 22% ( 4/18 ) of the specialty journals follow the international committee of medical journal editors ( icmje ) uniform requirements for manuscripts submitted to biomedical journals.21 even though journals have conflict of interest reporting policies , the conflicts of interest reported are not always reliable.22 all the general medical journals included are members of the world association of medical editors ( wame ) ; however , this is only the case for 22% ( 4/18 ) of the specialty journals included . the results and conclusions on breast cancer mortality and overdiagnosis were more often accepted in 2010 than in any other year ( data not shown ) . the ongoing independent review of the national health service ( nhs ) breast screening programme announced by mike richards , the uk national clinical director for cancer and end of life care , department of health , in october 2011 is a further indication of this development.24 also , the us preventive services task force changed its recommendations for breast screening in 2009.12 though our data did not show strong time trends , we believe that these developments demonstrate a growing acceptance of the results and conclusions of our systematic review . in support of this , the 2009 version of the cochrane review has received more approval than disapproval , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version.2531 the us preventive services task force was heavily criticised after the publication of its new recommendations in 2009,29 32 but the criticism came from people with vested interests , and the independent canadian task force supported the conclusions of the us preventive services task force and the 2009 cochrane review5 in 2011.13 the 2001 review published in lancet was by far the most cited of the three reviews . it was 5 years older than the cochrane review from 2006 , but the vast majority of the citations came within the first year of publication . it was unique at the time , as it questioned whether mammography screening was effective , based on a thorough quality assessment of all the randomised controlled trials , and also was the first systematic review to quantify overdiagnosis . a minor part of the included articles ( 19% , 32/171 ) did not refer to any of our five specified outcomes . in nearly half of the cases ( 47% ) , this was due to the article referring only to the debate that followed the first review,33 and not its results or methods . this was the case for articles in both the general medical and specialty journals , and for articles in the european and north american journals . none of the articles rejected overdiagnosis ( 0 of 171 articles ) , which could be because they did not mention the issue at all . this was the case in 76% of scientific articles on breast screening in a previous study by jrgensen et al.19 our definition of rejection was that the author should explicitly state that the review 's estimate was flawed , wrong or false , or that they should in some way argue against it . the texts in question did not allow an interpretation in any direction and we did not rate the articles as accepting or rejecting the review s results and methods unless it was perfectly clear what the authors meant . an additional explanation for the many articles found to be unclear could be that we did not assess the entire article , and arguments could have been presented elsewhere in the text . the specialists who read and respond to letters in their own journals might be more likely to react negatively towards the review because of conflicts of interest.19 specialists with a connection to mammography screening also reply to articles in general medical journals when they concern mammography screening . therefore , it is quite likely that there is a greater difference between the specialists involved with the screening programmes and the doctors not involved in breast cancer screening , in terms of accepting and rejecting the results and methods , than we have found in this study . the results and conclusions on breast cancer mortality and overdiagnosis were more often accepted in 2010 than in any other year ( data not shown ) . the ongoing independent review of the national health service ( nhs ) breast screening programme announced by mike richards , the uk national clinical director for cancer and end of life care , department of health , in october 2011 is a further indication of this development.24 also , the us preventive services task force changed its recommendations for breast screening in 2009.12 though our data did not show strong time trends , we believe that these developments demonstrate a growing acceptance of the results and conclusions of our systematic review . in support of this , the 2009 version of the cochrane review has received more approval than disapproval , for example , 30% ( 3/10 ) accepted the results for breast cancer mortality presented in the 2009 version of the review , compared with 0 ( 0/140 ) in the 2001 version.2531 the us preventive services task force was heavily criticised after the publication of its new recommendations in 2009,29 32 but the criticism came from people with vested interests , and the independent canadian task force supported the conclusions of the us preventive services task force and the 2009 cochrane review5 in 2011.13 the 2001 review published in lancet was by far the most cited of the three reviews . it was 5 years older than the cochrane review from 2006 , but the vast majority of the citations came within the first year of publication . it was unique at the time , as it questioned whether mammography screening was effective , based on a thorough quality assessment of all the randomised controlled trials , and also was the first systematic review to quantify overdiagnosis . a minor part of the included articles ( 19% , 32/171 ) did not refer to any of our five specified outcomes . in nearly half of the cases ( 47% ) , this was due to the article referring only to the debate that followed the first review,33 and not its results or methods . this was the case for articles in both the general medical and specialty journals , and for articles in the european and north american journals . none of the articles rejected overdiagnosis ( 0 of 171 articles ) , which could be because they did not mention the issue at all . this was the case in 76% of scientific articles on breast screening in a previous study by jrgensen et al.19 our definition of rejection was that the author should explicitly state that the review 's estimate was flawed , wrong or false , or that they should in some way argue against it . the texts in question did not allow an interpretation in any direction and we did not rate the articles as accepting or rejecting the review s results and methods unless it was perfectly clear what the authors meant . an additional explanation for the many articles found to be unclear could be that we did not assess the entire article , and arguments could have been presented elsewhere in the text . the specialists who read and respond to letters in their own journals might be more likely to react negatively towards the review because of conflicts of interest.19 specialists with a connection to mammography screening also reply to articles in general medical journals when they concern mammography screening . therefore , it is quite likely that there is a greater difference between the specialists involved with the screening programmes and the doctors not involved in breast cancer screening , in terms of accepting and rejecting the results and methods , than we have found in this study . articles in specialty journals were less approving of the results and methods of the systematic review of breast screening than those in general medical journals . this may be explained by conflicts of interest , as several specialty journals were published by groups with vested interests in breast screening , and several articles had authors with vested interests .

several studies had compared the difference between surgical and nonsurgical treatment for patients with herniated disc , but the baseline differences between their groups or small sample sizes or lack of validated outcome measures in these studies limits to get an evidence - based conclusions that is regarded as to the optimal treatment . fortunately , only 3% to 6% of lumbosacral disc herniations become symptomatic9,12,19,24 ) and management of lumbar disc herniation has a wide range of variable modality . patients are commonly treated in primary care , but a small proportion is referred to secondary care and may eventually undergo surgery when a related symptom continues for at least 6 weeks . conservative treatment is primarily aimed at pain reduction , by either analgesics or decreasing pressure on the nerve root without a conventional surgery . there seems to be a consensus that surgery is indicated in carefully selected patients with severe sciatica or serious or progressive neurologic deficits and image demonstrations correlating with the patient 's examination findings15 ) . there are many previous articles that announced several modalities for treating motor weakness caused by lumbar disc herniation , but few randomized controlled trials that had compared the efficacy of one treatment over another treatment except cross - over treatment15 ) . in this study the objective of this study is to analyze the effectiveness of surgery over conservative treatment for patients who present motor weakness caused by lumbar disc herniation . fifty - five patients with motor weakness caused by a lumbar disc herniation from 2006 to 2010 were participated in this study . we confirmed that all herniated disc lesions led to the patient 's symptoms by an image study using magnetic resonance image ( mri ) . in this study , we included patients who underwent surgery or conservative management for motor deficit resulted from lumbar disc herniation . however , the patients with the following history were excluded : prior lumbar spine surgery , cauda equina syndrome , developmental spine deformities , vertebral fractures , spine infection or tumor , inflammatory spondylopathy , pregnancy , or severe comorbid conditions . in addition , patients who were treated with both surgical and conservative treatments and disappeared were excluded . one group included 26 patients who underwent surgical treatment and another group consisted of 20 patients who did n't get a surgical treatment but only conservative treatment . the herniation types in the patients on this study were divided into subligamentous , transligamentous and sequestrated herniation . subligamentous herniation implies that the displaced nuclear material is still confined by the outermost fibers of the annulus . the disc herniation has traveled up behind the vertebral body above , or down behind the vertebral body below . transligamentous herniation is that part of the displaced nuclear material has burst through the posterior fivers of the annulus and the pll to lie in the spinal canal . however , there is still a connection between the extruded discal material and the disc space cavity . sequestrated herniation notes nuclear material has not only ruptured through the annular - pll complex , it has completely separated from the nuclear cavity , and the discal fragment lies free in the spinal canal . the herniation zones on an axial view in the patients on this study were divided into the central , subarticular and foraminal herniation . central herniation implies that it has its thickest part of displaced nuclear material at the center of spinal canal from disc space . in this region , foraminal zone is defined as the space through neural foramen from medical to lateral margin of the pedicle structure , and lastly , subarticular zone is located between central and foraminal zone . we performed only standard often microdiscectomy , which involved a surgical removal of the herniated disc materials by an operation . the conservative treatment group from other department in our center was received epidural steroid injection , selective nerve root block , active physical therapy and other non - operative methods . we monitored a visual analogue rating scale ( vas ) of back and leg pain , oswestry disability index ( odi ) and degree of motor weakness on 1 , 3 , 6 months and 1-year . vas is a measurement instrument that tries to measure a characteristic or attitude that is believed to range across a continuum of values and can not easily be directly measured . operationally , a vas is usually a horizontal line , 100 mm in length , anchored by word descriptors at each end . the patient marks on the line the point that they feel represents their perception of their current state ( degree of pain ) . the vas score is determined by measuring in millimeters from the end of the line on the left hand to the point that the patient marks22 ) . patient - completed questionnaire that gives a subjective percentage score of level of function ( disability ) in activities of daily living in those rehabilitating from low back pain . there are 10 items ( pain intensity , personal care , lifting , sitting , standing , sleeping , social life , traveling , changing degree of pain ) . the questions are designed in a way that to realize how the back or leg pain is affecting the patient 's ability to manage in everyday life3,10 ) . the degree of weakness was estimated by medical research council scale for muscle strength6 ) . data analysis was performed using spss version 20.0 ( spss inc . , chicago , il , usa ) . descriptive statistics were obtained to determine the influences of each group 's clinical feature to results and test , exact test and t - test were conducted to assess the overall differences between groups . we used mann - whitney u test to analyze the difference by variables between groups at each designated follow - up times and repeated measure anova ( rm - anova ) was used to found an interaction between time and group by variables . fifty - five patients with motor weakness caused by a lumbar disc herniation from 2006 to 2010 were participated in this study . we confirmed that all herniated disc lesions led to the patient 's symptoms by an image study using magnetic resonance image ( mri ) . in this study , we included patients who underwent surgery or conservative management for motor deficit resulted from lumbar disc herniation . however , the patients with the following history were excluded : prior lumbar spine surgery , cauda equina syndrome , developmental spine deformities , vertebral fractures , spine infection or tumor , inflammatory spondylopathy , pregnancy , or severe comorbid conditions . in addition , patients who were treated with both surgical and conservative treatments and disappeared were excluded . one group included 26 patients who underwent surgical treatment and another group consisted of 20 patients who did n't get a surgical treatment but only conservative treatment . the herniation types in the patients on this study were divided into subligamentous , transligamentous and sequestrated herniation . subligamentous herniation implies that the displaced nuclear material is still confined by the outermost fibers of the annulus . the disc herniation has traveled up behind the vertebral body above , or down behind the vertebral body below . transligamentous herniation is that part of the displaced nuclear material has burst through the posterior fivers of the annulus and the pll to lie in the spinal canal . however , there is still a connection between the extruded discal material and the disc space cavity . sequestrated herniation notes nuclear material has not only ruptured through the annular - pll complex , it has completely separated from the nuclear cavity , and the discal fragment lies free in the spinal canal . the herniation zones on an axial view in the patients on this study were divided into the central , subarticular and foraminal herniation . central herniation implies that it has its thickest part of displaced nuclear material at the center of spinal canal from disc space . in this region , foraminal zone is defined as the space through neural foramen from medical to lateral margin of the pedicle structure , and lastly , subarticular zone is located between central and foraminal zone . we performed only standard often microdiscectomy , which involved a surgical removal of the herniated disc materials by an operation . the conservative treatment group from other department in our center was received epidural steroid injection , selective nerve root block , active physical therapy and other non - operative methods . we monitored a visual analogue rating scale ( vas ) of back and leg pain , oswestry disability index ( odi ) and degree of motor weakness on 1 , 3 , 6 months and 1-year . vas is a measurement instrument that tries to measure a characteristic or attitude that is believed to range across a continuum of values and can not easily be directly measured . operationally , a vas is usually a horizontal line , 100 mm in length , anchored by word descriptors at each end . the patient marks on the line the point that they feel represents their perception of their current state ( degree of pain ) . the vas score is determined by measuring in millimeters from the end of the line on the left hand to the point that the patient marks22 ) . patient - completed questionnaire that gives a subjective percentage score of level of function ( disability ) in activities of daily living in those rehabilitating from low back pain . there are 10 items ( pain intensity , personal care , lifting , sitting , standing , sleeping , social life , traveling , changing degree of pain ) . the questions are designed in a way that to realize how the back or leg pain is affecting the patient 's ability to manage in everyday life3,10 ) . the degree of weakness was estimated by medical research council scale for muscle strength6 ) . descriptive statistics were obtained to determine the influences of each group 's clinical feature to results and test , exact test and t - test were conducted to assess the overall differences between groups . we used mann - whitney u test to analyze the difference by variables between groups at each designated follow - up times and repeated measure anova ( rm - anova ) was used to found an interaction between time and group by variables . the mean age of all participants were 48.6 ( surgery : 50.8 , non - surgery : 43.9 ) years old and the ratio of male and female was 2.83 to 1 ( male : 34 , female : 12 ) . the most common disc herniation level , disc herniation type and disc location were l4 - 5 , transligamentous type and subarticular zone . there were no statistically significant differences on the distribution in disc herniation level , type and location between two groups ( p>0.05 ) . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . 3 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . 4 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . the mean age of all participants were 48.6 ( surgery : 50.8 , non - surgery : 43.9 ) years old and the ratio of male and female was 2.83 to 1 ( male : 34 , female : 12 ) . the most common disc herniation level , disc herniation type and disc location were l4 - 5 , transligamentous type and subarticular zone . there were no statistically significant differences on the distribution in disc herniation level , type and location between two groups ( p>0.05 ) . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . 2 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . 3 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . 4 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . 2 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . 3 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . 4 . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . for decades , the different recommendations and comparisons among surgical and conservative treatment for patients with lumbar disc herniation were published . for those with moderate or severe symptoms , surgery may facilitate recovery and result in better outcomes compared with nonsurgical treatment1 ) . also , some patients need surgical intervention due to the neurological deficit and prolonged or uncontrolled pain . weber5,28 ) estimated that one year after disease onset , about 25% of patients would need a surgery . the larger maine lumbar spine study showed that 15% of patients who initially received conservative treatment would undergo surgical intervention within 3 months1,5 ) . postacchini21 ) recommends considering surgery after 6 months of unsuccessful conservative treatment , dvorak et al.8 ) after 4 months , dauch et al.7 ) after 6 weeks , hurme and alaranta14 ) after 3 weeks , and jnsson17 ) after " a certain amount of time " . peul et al.20,27 ) randomly assigned 141 patients with sciatica to an early surgery ( at a mean of 2.2 weeks ) and 142 patients with same symptom to conservative management . of those managed conservatively , 55 patients ( 38.7% ) were converted to surgery after a mean of 18.7 weeks . they found that those patients who undertaken an early operation were relieved in their leg pain more quickly than another one with delayed operation . this is relevant because a faster recovery rate could , besides the clinical benefit , have an economic advantage in a relatively young patient population15,20,27 ) . a cost effectiveness study which was performed alongside the trial of peul et al.15,20,27 ) showed that surgical group was cost - effective with a willingness as per quality - adjusted life years . moreover , many studies have analyzed long - term outcomes of patients with sciatica caused by herniated lumbar disc1,13,28 ) . in the weber study1,28 ) , good results were reported in 70.2% of patients who got a surgical modality as a first treatment after 4 years and 51.5% of those initially received conservative treatment . however , unfortunately this difference did not reveal a statistical significance , and there were similar improvement in the predominant pain symptom and functional status in long - term period . there are various reasons that long - term outcomes of patients with a herniated lumbar disc are similar to surgical and conservative treatment . recently , a large study was reported in 2006 by autio et al.2,4,5 ) , in which 68 of 160 enrolled patients ( 42.5% ) documented by lumbar mri revealed a diminished volume of herniated lumbar disc 2 months later since the occurrence of the disease . in the other studies , the occurrence rate of spontaneous regression of herniated lumbar disc was around 35 - 63% on average , during a period of 6 months to 1 year2,5,18,26 ) . this phenomenon may be related to dehydration and/or shrinkage , retraction of herniated discs , and inflammation - related resorption of the herniated disc5,11,25 ) . in addition , radicular pain can often affect the patient 's description of weakness and the findings on clinical examination , and thus , mimic muscle weakness , and the improvement of motor weakness could be shown in conservative treatment false - positively . from the reasons described as above , the newly accepted indication of non - operative management for patients with herniated disc is the absence of progressive neurological signs or cauda equine syndrome . nevertheless , most patients who present with mild weakness will be tried with non - operative treatment . there are wide variety of non - operative treatment modalities : bed rest , lumbar support , oral analgesics , muscle relaxants , spinal manipulation , physiotherapy , behavioral therapy and epidural steroid injections16,20,23,24 ) . among these modality , bed rest is thought to reduce the pressure of the intervertebral disc over the nerve root , even though there is no definite evidence that this affects the natural history of radicular weakness23 ) . this study analyzed patients with motor weakness caused by a herniated lumbar disc that underwent either surgical or nonsurgical treatment modality during 1 year retrospectively . the patients were treated surgically in this study had better outcomes than nonsurgical group at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . in addition , the improvement of pain symptom and functional status had similar outcomes between two groups . in this result on first 1 month , in terms of motor recovery , there is a more steep recovery in a surgical group compare to non - surgical group . 1 shows similar slope on both group after 1 month and the same result in the end . in addition , there were also the alleviation for pain on back and leg from surgical modality , even if we ca n't find out statistical significant differences . nevertheless , surgical method could be a best choice in an early stage , there could be always unpredictable complications . in surgical complications , surgeon and patient need to understand better about a risk versus a benefit of surgical treatment . for example , sometimes conservative treatment can be a optional treatment in a patient with a tremendous risk for general anesthesia , bleeding tendency , when a patient are reluctant for surgery and so on . because there is no difference between surgical and non - surgical group in a view of the long - term outcome despite there is still no absolute conclusion . lastly , there are several limitations originated from relatively small sample size on a statistical aspect , not enough follow - up period limited in 1 year and a risk from lack of consistency for measuring the symptom grade between the different departments . surgical treatment showed more benefits on recovery of motor function in the short - term period , especially 1 month , compare to conservative one statistically . although there was no statistically significance , we can also expect to obtain a rapid recovery on pain symptom with surgical modality within first one month postoperatively . we believe that early and proper surgical treatment in a case of motor weakness from disc herniation could be a good way for providing a chance for rapid alleviation . however , there are several limitations in this study and further studies are required to get a more proper and reasonable consensus .

objectivethe aim of this study is to assess outcomes during first one year for patients with severe motor weakness caused by lumbar disc herniation that underwent surgical or nonsurgical treatment.methodsthe 46 patients with motor weakness because of lumbar disc herniation who were treated at neurosurgical department and rehabilitation in our hospital from 2006 to 2010 , retrospectively . each group had 26 surgical treatments and 20 conservative treatments . we followed up 1 , 3 , 6 months and 12 month and monitored a visual analogue rating scale ( vas ) of back and leg pain , oswestry disability index ( odi ) and degree of motor weakness . we analyzed the differences between surgical and nonsurgical groups using mann - whitney u test and repeat measure anova in each follow - up periods.resultsin the recovery of motor weakness , surgical treatment uncovered a rapid functional recovery in the early periods ( p=0.003 ) and no difference between groups at the end of follow - up period was found ( p>0.05 ) . in vas of back and leg , the interaction between time and group was not found ( p>0.05 ) and there was no difference between groups ( p>0.05 ) . in odi , the interaction between time and group was not found ( p>0.05 ) and there was no difference between groups ( p>0.05).conclusionsurgical treatment for motor weakness caused by herniated intervertebral disc resulted in a rapid recovery in the short - term period , especially 1 month . we think early and proper surgical treatment in a case of motor weakness from disc herniation could be a good way for providing a chance for rapid alleviation .

INTRODUCTION MATERIALS AND METHODS Patient population Radiological classification Treatment modality Outcome measures Statistical analysis RESULTS Patient characteristics Outcomes Motor recovery VAS score on back VAS score on leg ODI score DISCUSSION CONCLUSION

several studies had compared the difference between surgical and nonsurgical treatment for patients with herniated disc , but the baseline differences between their groups or small sample sizes or lack of validated outcome measures in these studies limits to get an evidence - based conclusions that is regarded as to the optimal treatment . fortunately , only 3% to 6% of lumbosacral disc herniations become symptomatic9,12,19,24 ) and management of lumbar disc herniation has a wide range of variable modality . there seems to be a consensus that surgery is indicated in carefully selected patients with severe sciatica or serious or progressive neurologic deficits and image demonstrations correlating with the patient 's examination findings15 ) . there are many previous articles that announced several modalities for treating motor weakness caused by lumbar disc herniation , but few randomized controlled trials that had compared the efficacy of one treatment over another treatment except cross - over treatment15 ) . in this study the objective of this study is to analyze the effectiveness of surgery over conservative treatment for patients who present motor weakness caused by lumbar disc herniation . fifty - five patients with motor weakness caused by a lumbar disc herniation from 2006 to 2010 were participated in this study . in this study , we included patients who underwent surgery or conservative management for motor deficit resulted from lumbar disc herniation . however , the patients with the following history were excluded : prior lumbar spine surgery , cauda equina syndrome , developmental spine deformities , vertebral fractures , spine infection or tumor , inflammatory spondylopathy , pregnancy , or severe comorbid conditions . in addition , patients who were treated with both surgical and conservative treatments and disappeared were excluded . one group included 26 patients who underwent surgical treatment and another group consisted of 20 patients who did n't get a surgical treatment but only conservative treatment . the herniation types in the patients on this study were divided into subligamentous , transligamentous and sequestrated herniation . transligamentous herniation is that part of the displaced nuclear material has burst through the posterior fivers of the annulus and the pll to lie in the spinal canal . sequestrated herniation notes nuclear material has not only ruptured through the annular - pll complex , it has completely separated from the nuclear cavity , and the discal fragment lies free in the spinal canal . the herniation zones on an axial view in the patients on this study were divided into the central , subarticular and foraminal herniation . central herniation implies that it has its thickest part of displaced nuclear material at the center of spinal canal from disc space . the conservative treatment group from other department in our center was received epidural steroid injection , selective nerve root block , active physical therapy and other non - operative methods . we monitored a visual analogue rating scale ( vas ) of back and leg pain , oswestry disability index ( odi ) and degree of motor weakness on 1 , 3 , 6 months and 1-year . the patient marks on the line the point that they feel represents their perception of their current state ( degree of pain ) . the vas score is determined by measuring in millimeters from the end of the line on the left hand to the point that the patient marks22 ) . patient - completed questionnaire that gives a subjective percentage score of level of function ( disability ) in activities of daily living in those rehabilitating from low back pain . there are 10 items ( pain intensity , personal care , lifting , sitting , standing , sleeping , social life , traveling , changing degree of pain ) . the questions are designed in a way that to realize how the back or leg pain is affecting the patient 's ability to manage in everyday life3,10 ) . descriptive statistics were obtained to determine the influences of each group 's clinical feature to results and test , exact test and t - test were conducted to assess the overall differences between groups . we used mann - whitney u test to analyze the difference by variables between groups at each designated follow - up times and repeated measure anova ( rm - anova ) was used to found an interaction between time and group by variables . fifty - five patients with motor weakness caused by a lumbar disc herniation from 2006 to 2010 were participated in this study . in this study , we included patients who underwent surgery or conservative management for motor deficit resulted from lumbar disc herniation . however , the patients with the following history were excluded : prior lumbar spine surgery , cauda equina syndrome , developmental spine deformities , vertebral fractures , spine infection or tumor , inflammatory spondylopathy , pregnancy , or severe comorbid conditions . in addition , patients who were treated with both surgical and conservative treatments and disappeared were excluded . one group included 26 patients who underwent surgical treatment and another group consisted of 20 patients who did n't get a surgical treatment but only conservative treatment . the herniation types in the patients on this study were divided into subligamentous , transligamentous and sequestrated herniation . the disc herniation has traveled up behind the vertebral body above , or down behind the vertebral body below . transligamentous herniation is that part of the displaced nuclear material has burst through the posterior fivers of the annulus and the pll to lie in the spinal canal . sequestrated herniation notes nuclear material has not only ruptured through the annular - pll complex , it has completely separated from the nuclear cavity , and the discal fragment lies free in the spinal canal . the herniation zones on an axial view in the patients on this study were divided into the central , subarticular and foraminal herniation . central herniation implies that it has its thickest part of displaced nuclear material at the center of spinal canal from disc space . the conservative treatment group from other department in our center was received epidural steroid injection , selective nerve root block , active physical therapy and other non - operative methods . we monitored a visual analogue rating scale ( vas ) of back and leg pain , oswestry disability index ( odi ) and degree of motor weakness on 1 , 3 , 6 months and 1-year . the patient marks on the line the point that they feel represents their perception of their current state ( degree of pain ) . the vas score is determined by measuring in millimeters from the end of the line on the left hand to the point that the patient marks22 ) . there are 10 items ( pain intensity , personal care , lifting , sitting , standing , sleeping , social life , traveling , changing degree of pain ) . the questions are designed in a way that to realize how the back or leg pain is affecting the patient 's ability to manage in everyday life3,10 ) . descriptive statistics were obtained to determine the influences of each group 's clinical feature to results and test , exact test and t - test were conducted to assess the overall differences between groups . we used mann - whitney u test to analyze the difference by variables between groups at each designated follow - up times and repeated measure anova ( rm - anova ) was used to found an interaction between time and group by variables . the most common disc herniation level , disc herniation type and disc location were l4 - 5 , transligamentous type and subarticular zone . there were no statistically significant differences on the distribution in disc herniation level , type and location between two groups ( p>0.05 ) . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . the most common disc herniation level , disc herniation type and disc location were l4 - 5 , transligamentous type and subarticular zone . there were no statistically significant differences on the distribution in disc herniation level , type and location between two groups ( p>0.05 ) . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . the assessment of differences in motor recovery between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 2 , and the interaction between time course and group are pre - sented in fig . there were statistically significant differences at the 1 ( p=0.031 ) , 3 ( p=0.014 ) and 6 ( p=0.002 ) month monitoring point of follow - up period between two groups and significant interaction between time and group ( p=0.009 ) . the results reveal that a rapid motor recovery could be obtained with surgical treatment at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . the assessment of differences in vas score on back between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 3 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we could not find out whether there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in vas score on leg between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 4 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . however , we can find out there was a tendency of rapid decrease of pain score in a surgical group within first 1 month postoperatively . the assessment of differences in odi score between two groups was performed using a mann - whitney u test and rm - anova . the effect of treatment type at each monitoring point of follow - up period are presented in table 5 , and the interaction between time and group are presented in fig . there were no statistically significant differences at any monitoring point of follow - up period between two groups and interaction between time and groups . the results reveal that odi score was not affected by a sort of treatment during the follow - up period . for decades , the different recommendations and comparisons among surgical and conservative treatment for patients with lumbar disc herniation were published . postacchini21 ) recommends considering surgery after 6 months of unsuccessful conservative treatment , dvorak et al.8 ) after 4 months , dauch et al.7 ) after 6 weeks , hurme and alaranta14 ) after 3 weeks , and jnsson17 ) after " a certain amount of time " . peul et al.20,27 ) randomly assigned 141 patients with sciatica to an early surgery ( at a mean of 2.2 weeks ) and 142 patients with same symptom to conservative management . they found that those patients who undertaken an early operation were relieved in their leg pain more quickly than another one with delayed operation . a cost effectiveness study which was performed alongside the trial of peul et al.15,20,27 ) showed that surgical group was cost - effective with a willingness as per quality - adjusted life years . moreover , many studies have analyzed long - term outcomes of patients with sciatica caused by herniated lumbar disc1,13,28 ) . in the weber study1,28 ) , good results were reported in 70.2% of patients who got a surgical modality as a first treatment after 4 years and 51.5% of those initially received conservative treatment . however , unfortunately this difference did not reveal a statistical significance , and there were similar improvement in the predominant pain symptom and functional status in long - term period . there are various reasons that long - term outcomes of patients with a herniated lumbar disc are similar to surgical and conservative treatment . recently , a large study was reported in 2006 by autio et al.2,4,5 ) , in which 68 of 160 enrolled patients ( 42.5% ) documented by lumbar mri revealed a diminished volume of herniated lumbar disc 2 months later since the occurrence of the disease . in the other studies , the occurrence rate of spontaneous regression of herniated lumbar disc was around 35 - 63% on average , during a period of 6 months to 1 year2,5,18,26 ) . in addition , radicular pain can often affect the patient 's description of weakness and the findings on clinical examination , and thus , mimic muscle weakness , and the improvement of motor weakness could be shown in conservative treatment false - positively . from the reasons described as above , the newly accepted indication of non - operative management for patients with herniated disc is the absence of progressive neurological signs or cauda equine syndrome . among these modality , bed rest is thought to reduce the pressure of the intervertebral disc over the nerve root , even though there is no definite evidence that this affects the natural history of radicular weakness23 ) . this study analyzed patients with motor weakness caused by a herniated lumbar disc that underwent either surgical or nonsurgical treatment modality during 1 year retrospectively . the patients were treated surgically in this study had better outcomes than nonsurgical group at the early stage of follow - up period , even though the degree of motor recovery at 12th month has no significant difference . in this result on first 1 month , in terms of motor recovery , there is a more steep recovery in a surgical group compare to non - surgical group . 1 shows similar slope on both group after 1 month and the same result in the end . in addition , there were also the alleviation for pain on back and leg from surgical modality , even if we ca n't find out statistical significant differences . nevertheless , surgical method could be a best choice in an early stage , there could be always unpredictable complications . for example , sometimes conservative treatment can be a optional treatment in a patient with a tremendous risk for general anesthesia , bleeding tendency , when a patient are reluctant for surgery and so on . because there is no difference between surgical and non - surgical group in a view of the long - term outcome despite there is still no absolute conclusion . lastly , there are several limitations originated from relatively small sample size on a statistical aspect , not enough follow - up period limited in 1 year and a risk from lack of consistency for measuring the symptom grade between the different departments . surgical treatment showed more benefits on recovery of motor function in the short - term period , especially 1 month , compare to conservative one statistically . although there was no statistically significance , we can also expect to obtain a rapid recovery on pain symptom with surgical modality within first one month postoperatively . we believe that early and proper surgical treatment in a case of motor weakness from disc herniation could be a good way for providing a chance for rapid alleviation . however , there are several limitations in this study and further studies are required to get a more proper and reasonable consensus .

to stimulate the advancement of computer - aided diagnostic research for medical image and signals , there is need to have an online depository to share clinical ground truth with the public and provide open access for researchers to evaluate their computer - aided algorithms . new methods and systems on medical image and signals are mostly based on datasets by each developer ; however , such a strategy makes the performance comparison imperfect and unreliable . the perfect performance testing demands a solid evaluation using case data from different modalities and after approval by medical experts . to clear up the word case data , an example on mammography could be helpful ; in such a case , at least two views of each breast with digital radiography should be provided along with magnetic resonance imaging ( mri ) or ultrasound . furthermore , the data classification ( to normal and disease ) and possibly regions of abnormality labeled by the expert as ground truth or gold standard or interpretation are needed for a perfect case data . the main idea behind designing this website was to provide a collection concentrated on medical datasets rather than only a general data collection . there were only a few attempts made to establish appropriate reference datasets for medical imaging and signal processing until the past 10 years . the available datasets did not include perfect case data because most of them had no ground truth and almost all of them were not free of charge . however , undeniable need for such datasets led to numerous online medical image and signal databases in recent years . furthermore , we provide a full list of databases ( in mentioned websites or other individual sources ) as a supplementary . some websites that list and/or host multiple collections of medical datasets in this study , we introduce isfahan misp dataset website and furthermore , we introduce a collection of available datasets ( sorted according to anatomical / physiological information and imaging modality ) , which are freely available on the internet . there are also some websites that list and/or host multiple collections of data , and we shortly discuss about them . there were only a few attempts made to establish appropriate reference datasets for medical imaging and signal processing until the past 10 years . the available datasets did not include perfect case data because most of them had no ground truth and almost all of them were not free of charge . however , undeniable need for such datasets led to numerous online medical image and signal databases in recent years . furthermore , we provide a full list of databases ( in mentioned websites or other individual sources ) as a supplementary . some websites that list and/or host multiple collections of medical datasets in this study , we introduce isfahan misp dataset website and furthermore , we introduce a collection of available datasets ( sorted according to anatomical / physiological information and imaging modality ) , which are freely available on the internet . there are also some websites that list and/or host multiple collections of data , and we shortly discuss about them . the working group in misp research center had set one of its goals to develop a reference medical image and signal database for image and signal processing research and development . the main aim of this collection was to provide reliable datasets for researchers to compare their algorithms on common framework and to be able to interpret and compare the performance of their newly developed methods . the collected databases were devised to be in accordance with patient privacy and copyright issues . they all have written and signed forms by the institutional review board , also known as an independent ethics committee . the ground truth was provided for datasets based on evaluation of one , two , or three experts . some datasets were collected from different modalities or from diverse anatomical structures of one patient . most of the mentioned datasets were available with a published paper in famous journals to demonstrate possible application of the dataset . the features of mysql are listed below : relational database management system . a relational database stores information in different tables , rather than in one giant table . these tables can be referenced to each other , to access and maintain data easily.open source database system . the database software can be used and modified by anyone according to their needs.fast , reliable , and easy - to - use . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . relational database management system . a relational database stores information in different tables , rather than in one giant table . these tables can be referenced to each other , to access and maintain data easily . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . a separate thread ( which is always running ) handles each incoming connection and manages the connections . multiple clients can perform read operations simultaneously , but while writing , only one client , who needs access to updated data , is held up . even though the threads share the same process space , they execute individually , and because of this separation , multiprocessor machines can spread the thread across many central processing units ( cpus ) as long as the host operating system ( os ) supports multiple cpus . multithreading is the key feature to support mysql s performance design goals , and it can be counted as the core feature around which mysql is built . php is server side scripting language compatible with all known oss like windows , linux , etc . nowadays , php frameworks are being extensively used by programmers to address the performance tuning issues faster and with ease . they offer extensible architecture and features that make source code programming easier by providing standard templates and plug - ins . the main features of php are listed below : powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . there are even many database managers written in php.faster web applications : because php does not use a lot of a system s resources to run , it operates much faster than other scripting languages . even when used with other softwares , php still retains speed without slowing down other processes . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years.object oriented : php actually has the ability to call java and windows com objects . other classes can actually borrow from those custom classes and this specification extends the capabilities of php even further.high level freedom : when comparing php to a language such as aspx , the level of freedom is far superior . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . in cases like development of applications with aspx php also is not os specific and can be run on all well - known oss.free of charge : there are no costs associated with using php , including updates . therefore , the fact that one can code programs with php for free is a huge benefit that will not be provided with jps , asp , or other scripting languages that require paid hosting . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . faster web applications : because php does not use a lot of a system s resources to run , it operates much faster than other scripting languages . even when used with other softwares , php still retains speed without slowing down other processes . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years . object oriented : php actually has the ability to call java and windows com objects . other classes can actually borrow from those custom classes and this specification extends the capabilities of php even further . high level freedom : when comparing php to a language such as aspx , the level of freedom is far superior . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . in cases like development of applications with aspx php also is not os specific and can be run on all well - known os s . free of charge : there are no costs associated with using php , including updates . keeping costs down is a goal of any business and developers as well . therefore , the fact that one can code programs with php for free is a huge benefit that will not be provided with jps , asp , or other scripting languages that require paid hosting . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . regarding above features , we used php for web programming and mysql for database managing . was a fast , secure , and easy - to - use online database for medical signals and images . among these features , provided registered users the ability to download medical signals and images selectively and freely . it was also an appropriate place for researchers around the world to share their data and supplementary materials with each others , because users could freely , easily , securely , and unlimitedly upload their datasets while managing their privacies ( citation and fee ) . uploaded datasets would be categorized and published after verification . commenting and replying for the comments were available for all datasets ; therefore , researchers could share their experiences . to improve functionality in search engines ( e.g. , google ) , automatic sitemap and semi - automatic seo indexing to provide more description on contents of misp dataset , two examples of the available sets are described below . as a long - term goal of misp research center , the datasets would be added to this website , and it was open to accept databases from other researchers by convincing them to the reality that making online free datasets can improve the research quality of the owner and the users . the first sample dataset was on ocular images and included the following image sets : ( 1)misp isfahan optical coherence tomography ( oct ) dataset for segmentation.this dataset contained 13 three - dimensional ( 3d ) macular sd - oct images obtained from eyes without pathologies using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1].the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named totalmanual_13.(2)misp isfahan oct dataset for denoising.this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 6 . the positions of the mentioned slices were stored in another mat file named random_topcon.(3)misp isfahan oct dataset for vessel segmentation of oct and scanning laser ophthalmology ( slo).this dataset contained 24 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies using heidelberg hra oct scanner imaging system in ophthalmology dept . , each dataset consisted of a slo image and limited number of oct scans with size of 496 512 ( namely , for a data with 19 selected oct slices , the whole data size was 496 512 19 ) . non - vessel manually to make a standard in evaluation of the proposed method . 24.(4)misp isfahan oct dataset from normal population.this dataset contained 112 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies from 76 people using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . 112.(5)misp isfahan enhance depth imaging oct dataset from normal population.this dataset contained 19 3d macular sd - oct images ( along with slo images ) obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . 19.(6)misp isfahan corneal oct dataset from normal population.this dataset contained 15 3d corneal sd - oct images obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 15 . misp isfahan optical coherence tomography ( oct ) dataset for segmentation . this dataset contained 13 three - dimensional ( 3d ) macular sd - oct images obtained from eyes without pathologies using topcon 3d oct-1000 imaging system in ophthalmology dept . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1 ] . the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 6 . misp isfahan oct dataset for vessel segmentation of oct and scanning laser ophthalmology ( slo ) . this dataset contained 24 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 ( namely , for a data with 19 selected oct slices , the whole data size was 496 512 19 ) . non - vessel manually to make a standard in evaluation of the proposed method . the datasets were in mat format and were named 1 to 24 . this dataset contained 112 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies from 76 people using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 112 . this dataset contained 19 3d macular sd - oct images ( along with slo images ) obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 19 . this dataset contained 15 3d corneal sd - oct images obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 15 . the x , y , z size of the obtained volumes was 512 650 128 voxels the second sample dataset was on audio and acoustic signals and included the following datasets : ( 1)voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease).data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . all participants or their legal guardian signed a consent form to participate in the experiments . the study had been approved by the biomedical ethics board of isfahan university of medical sciences . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally.acoustic recording study : the acoustic recording was conducted in the test room with indoor lighting , which was at 24c . there was ambient noise from outside of the room but it was minimized during the recordings . swallowing sounds were recorded via microphone ( c417 omnidirectional condenser lavalier microphone , akg acoustics , austria ) connected to a portable digital sound recorder ( edirol r-44 , japan ) . this rubber piece was attached to the surface of the skin using double - sided adhesive tape . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . the microphone was positioned over the laryngopharynx of each patient for 15 min when the participant was in a seated position watching a nature movie . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . the number of swallows that each participant had within the 15-min time period was varied . all data recordings were carried out by a speech language pathologist and a biomedical engineer.(2)voice samples of patients with internal nasal valve collapse before and after functional rhinoplasty.this dataset contained voice recordings of participants who had internal nasal valve collapse . these voice samples were corresponding to the following two groups : before and after functional rhinoplasty . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . to minimize the environmental noise , recordings were done in an acoustic room ( with noise of 28 db based on sound level meter [ tes-1351 ] ) . the participants were in a seated position and a microphone ( somic senic st-818 3.5 mm on - ear stereo headphones with adjustable microphone and 2.5 m cable ) was located 5 cm from the center of their lips on the right corner of mouth.the participants were guided to generate the vowels / a / and / i / in three tests for a period of 5 s. the reason for choosing these vowels was their different location of articulation in the vocal tract . /i / is the highest front vowel , and / a / is the lowest back vowel.vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . voice signals were obtained again 3 months after the surgery under the same conditions . prior to recording the second voice samples , the participants underwent examinations by an ent specialist and also were examined again in terms of inclusion and exclusion criteria of the study . the collected voice samples were encoded by a person who was not involved in the analysis of voice signals . voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease ) . data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . all participants or their legal guardian signed a consent form to participate in the experiments . the study had been approved by the biomedical ethics board of isfahan university of medical sciences . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally . acoustic recording study : the acoustic recording was conducted in the test room with indoor lighting , which was at 24c . there was ambient noise from outside of the room but it was minimized during the recordings . swallowing sounds were recorded via microphone ( c417 omnidirectional condenser lavalier microphone , akg acoustics , austria ) connected to a portable digital sound recorder ( edirol r-44 , japan ) . this rubber piece was attached to the surface of the skin using double - sided adhesive tape . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . the microphone was positioned over the laryngopharynx of each patient for 15 min when the participant was in a seated position watching a nature movie . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . the number of swallows that each participant had within the 15-min time period was varied . all data recordings were carried out by a speech language pathologist and a biomedical engineer . these voice samples were corresponding to the following two groups : before and after functional rhinoplasty . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . to minimize the environmental noise , recordings were done in an acoustic room ( with noise of 28 db based on sound level meter [ tes-1351 ] ) . the participants were in a seated position and a microphone ( somic senic st-818 3.5 mm on - ear stereo headphones with adjustable microphone and 2.5 m cable ) was located 5 cm from the center of their lips on the right corner of mouth . the participants were guided to generate the vowels / a / and / i / in three tests for a period of 5 s. the reason for choosing these vowels was their different location of articulation in the vocal tract . /i / is the highest front vowel , and / a / is the lowest back vowel . vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . voice signals were obtained again 3 months after the surgery under the same conditions . prior to recording the second voice samples , the participants underwent examinations by an ent specialist and also were examined again in terms of inclusion and exclusion criteria of the study . the collected voice samples were encoded by a person who was not involved in the analysis of voice signals . the features of mysql are listed below : relational database management system . a relational database stores information in different tables , rather than in one giant table . these tables can be referenced to each other , to access and maintain data easily.open source database system . the database software can be used and modified by anyone according to their needs.fast , reliable , and easy - to - use . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . relational database management system . a relational database stores information in different tables , rather than in one giant table . these tables can be referenced to each other , to access and maintain data easily . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . a separate thread ( which is always running ) handles each incoming connection and manages the connections . multiple clients can perform read operations simultaneously , but while writing , only one client , who needs access to updated data , is held up . even though the threads share the same process space , they execute individually , and because of this separation , multiprocessor machines can spread the thread across many central processing units ( cpus ) as long as the host operating system ( os ) supports multiple cpus . multithreading is the key feature to support mysql s performance design goals , and it can be counted as the core feature around which mysql is built . php is server side scripting language compatible with all known oss like windows , linux , etc . nowadays , php frameworks are being extensively used by programmers to address the performance tuning issues faster and with ease . they offer extensible architecture and features that make source code programming easier by providing standard templates and plug - ins . the main features of php are listed below : powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . there are even many database managers written in php.faster web applications : because php does not use a lot of a system s resources to run , it operates much faster than other scripting languages . even when used with other softwares , php still retains speed without slowing down other processes . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years.object oriented : php actually has the ability to call java and windows com objects . other classes can actually borrow from those custom classes and this specification extends the capabilities of php even further.high level freedom : when comparing php to a language such as aspx , the level of freedom is far superior . as mentioned before , php is open - source . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . in cases like development of applications with aspx php also is not os specific and can be run on all well - known oss.free of charge : there are no costs associated with using php , including updates . therefore , the fact that one can code programs with php for free is a huge benefit that will not be provided with jps , asp , or other scripting languages that require paid hosting . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . faster web applications : because php does not use a lot of a system s resources to run , it operates much faster than other scripting languages . even when used with other softwares , php still retains speed without slowing down other processes . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years . object oriented : php actually has the ability to call java and windows com objects . other classes can actually borrow from those custom classes and this specification extends the capabilities of php even further . high level freedom : when comparing php to a language such as aspx , the level of freedom is far superior . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . in cases like development of applications with aspx php also is not os specific and can be run on all well - known os s . free of charge : there are no costs associated with using php , including updates . keeping costs down is a goal of any business and developers as well . therefore , the fact that one can code programs with php for free is a huge benefit that will not be provided with jps , asp , or other scripting languages that require paid hosting . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . regarding above features , we used php for web programming and mysql for database managing . was a fast , secure , and easy - to - use online database for medical signals and images . among these features , it was also an appropriate place for researchers around the world to share their data and supplementary materials with each others , because users could freely , easily , securely , and unlimitedly upload their datasets while managing their privacies ( citation and fee ) . uploaded datasets would be categorized and published after verification . commenting and replying for the comments were available for all datasets ; therefore , researchers could share their experiences . to improve functionality in search engines ( e.g. , google ) , automatic sitemap and semi - automatic seo indexing to provide more description on contents of misp dataset , two examples of the available sets are described below . as a long - term goal of misp research center , the datasets would be added to this website , and it was open to accept databases from other researchers by convincing them to the reality that making online free datasets can improve the research quality of the owner and the users . the first sample dataset was on ocular images and included the following image sets : ( 1)misp isfahan optical coherence tomography ( oct ) dataset for segmentation.this dataset contained 13 three - dimensional ( 3d ) macular sd - oct images obtained from eyes without pathologies using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1].the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named totalmanual_13.(2)misp isfahan oct dataset for denoising.this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . the datasets were in mat format and were named 1 to 6 . participants in the dataset the positions of the mentioned slices were stored in another mat file named random_topcon.(3)misp isfahan oct dataset for vessel segmentation of oct and scanning laser ophthalmology ( slo).this dataset contained 24 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies using heidelberg hra oct scanner imaging system in ophthalmology dept . , each dataset consisted of a slo image and limited number of oct scans with size of 496 512 ( namely , for a data with 19 selected oct slices , the whole data size was 496 512 19 ) . non - vessel manually to make a standard in evaluation of the proposed method . 24.(4)misp isfahan oct dataset from normal population.this dataset contained 112 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies from 76 people using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . 112.(5)misp isfahan enhance depth imaging oct dataset from normal population.this dataset contained 19 3d macular sd - oct images ( along with slo images ) obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . 19.(6)misp isfahan corneal oct dataset from normal population.this dataset contained 15 3d corneal sd - oct images obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 15 . this dataset contained 13 three - dimensional ( 3d ) macular sd - oct images obtained from eyes without pathologies using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1 ] . the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 6 . the positions of the mentioned slices were stored in another mat file named random_topcon . misp isfahan oct dataset for vessel segmentation of oct and scanning laser ophthalmology ( slo ) . this dataset contained 24 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 ( namely , for a data with 19 selected oct slices , the whole data size was 496 512 19 ) . non - vessel manually to make a standard in evaluation of the proposed method . the datasets were in mat format and were named 1 to 24 . this dataset contained 112 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies from 76 people using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 112 . this dataset contained 19 3d macular sd - oct images ( along with slo images ) obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 19 . this dataset contained 15 3d corneal sd - oct images obtained using heidelberg hra oct scanner imaging system in ophthalmology dept . each dataset consisted of a limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 15 . the x , y , z size of the obtained volumes was 512 650 128 voxels the second sample dataset was on audio and acoustic signals and included the following datasets : ( 1)voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease).data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . all participants or their legal guardian signed a consent form to participate in the experiments . the study had been approved by the biomedical ethics board of isfahan university of medical sciences . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally.acoustic recording study : the acoustic recording was conducted in the test room with indoor lighting , which was at 24c . there was ambient noise from outside of the room but it was minimized during the recordings . swallowing sounds were recorded via microphone ( c417 omnidirectional condenser lavalier microphone , akg acoustics , austria ) connected to a portable digital sound recorder ( edirol r-44 , japan ) . this rubber piece was attached to the surface of the skin using double - sided adhesive tape . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . the microphone was positioned over the laryngopharynx of each patient for 15 min when the participant was in a seated position watching a nature movie . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . the number of swallows that each participant had within the 15-min time period was varied . all data recordings were carried out by a speech language pathologist and a biomedical engineer.(2)voice samples of patients with internal nasal valve collapse before and after functional rhinoplasty.this dataset contained voice recordings of participants who had internal nasal valve collapse . these voice samples were corresponding to the following two groups : before and after functional rhinoplasty . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . to minimize the environmental noise , recordings were done in an acoustic room ( with noise of 28 db based on sound level meter [ tes-1351 ] ) . the participants were in a seated position and a microphone ( somic senic st-818 3.5 mm on - ear stereo headphones with adjustable microphone and 2.5 m cable ) was located 5 cm from the center of their lips on the right corner of mouth.the participants were guided to generate the vowels / a / and / i / in three tests for a period of 5 s. the reason for choosing these vowels was their different location of articulation in the vocal tract . / is the highest front vowel , and / a / is the lowest back vowel.vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . voice signals were obtained again 3 months after the surgery under the same conditions . prior to recording the second voice samples , the participants underwent examinations by an ent specialist and also were examined again in terms of inclusion and exclusion criteria of the study . the collected voice samples were encoded by a person who was not involved in the analysis of voice signals . voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease ) . data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . all participants or their legal guardian signed a consent form to participate in the experiments . the study had been approved by the biomedical ethics board of isfahan university of medical sciences . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally . acoustic recording study : the acoustic recording was conducted in the test room with indoor lighting , which was at 24c . there was ambient noise from outside of the room but it was minimized during the recordings . swallowing sounds were recorded via microphone ( c417 omnidirectional condenser lavalier microphone , akg acoustics , austria ) connected to a portable digital sound recorder ( edirol r-44 , japan ) . this rubber piece was attached to the surface of the skin using double - sided adhesive tape . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . the microphone was positioned over the laryngopharynx of each patient for 15 min when the participant was in a seated position watching a nature movie . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . the number of swallows that each participant had within the 15-min time period was varied . all data recordings were carried out by a speech language pathologist and a biomedical engineer . these voice samples were corresponding to the following two groups : before and after functional rhinoplasty . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . to minimize the environmental noise , recordings were done in an acoustic room ( with noise of 28 db based on sound level meter [ tes-1351 ] ) . the participants were in a seated position and a microphone ( somic senic st-818 3.5 mm on - ear stereo headphones with adjustable microphone and 2.5 m cable ) was located 5 cm from the center of their lips on the right corner of mouth . the participants were guided to generate the vowels / a / and / i / in three tests for a period of 5 s. the reason for choosing these vowels was their different location of articulation in the vocal tract . /i / is the highest front vowel , and / a / is the lowest back vowel . vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . voice signals were obtained again 3 months after the surgery under the same conditions . prior to recording the second voice samples , the participants underwent examinations by an ent specialist and also were examined again in terms of inclusion and exclusion criteria of the study . the collected voice samples were encoded by a person who was not involved in the analysis of voice signals . the proposed online dataset aims at filling the gap in medical image and signal depository , which can provide the sets to be downloaded and uploaded with different privacies set by the owners . the biosigdata.com is a free service , and the structure makes it a fast , secure , and easy - to - use online database .

an online depository was introduced to share clinical ground truth with the public and provide open access for researchers to evaluate their computer - aided algorithms . php was used for web programming and mysql for database managing . the website was entitled biosigdata.com . it was a fast , secure , and easy - to - use online database for medical signals and images . freely registered users could download the datasets and could also share their own supplementary materials while maintaining their privacies ( citation and fee ) . commenting was also available for all datasets , and automatic sitemap and semi - automatic seo indexing have been set for the site . a comprehensive list of available websites for medical datasets is also presented as a supplementary ( http://journalonweb.com/tempaccess/4800.584.jmss_55_16i3253.pdf ) .

Introduction Comprehensive collection of available online datasets for medical image and signal processing (MISP) Construction and Content Technical background Sample description for available sets in MISP dataset website Conclusion Financial support and sponsorship Conflicts of interest

to stimulate the advancement of computer - aided diagnostic research for medical image and signals , there is need to have an online depository to share clinical ground truth with the public and provide open access for researchers to evaluate their computer - aided algorithms . furthermore , the data classification ( to normal and disease ) and possibly regions of abnormality labeled by the expert as ground truth or gold standard or interpretation are needed for a perfect case data . the main idea behind designing this website was to provide a collection concentrated on medical datasets rather than only a general data collection . there were only a few attempts made to establish appropriate reference datasets for medical imaging and signal processing until the past 10 years . the available datasets did not include perfect case data because most of them had no ground truth and almost all of them were not free of charge . however , undeniable need for such datasets led to numerous online medical image and signal databases in recent years . furthermore , we provide a full list of databases ( in mentioned websites or other individual sources ) as a supplementary . some websites that list and/or host multiple collections of medical datasets in this study , we introduce isfahan misp dataset website and furthermore , we introduce a collection of available datasets ( sorted according to anatomical / physiological information and imaging modality ) , which are freely available on the internet . there are also some websites that list and/or host multiple collections of data , and we shortly discuss about them . there were only a few attempts made to establish appropriate reference datasets for medical imaging and signal processing until the past 10 years . the available datasets did not include perfect case data because most of them had no ground truth and almost all of them were not free of charge . furthermore , we provide a full list of databases ( in mentioned websites or other individual sources ) as a supplementary . some websites that list and/or host multiple collections of medical datasets in this study , we introduce isfahan misp dataset website and furthermore , we introduce a collection of available datasets ( sorted according to anatomical / physiological information and imaging modality ) , which are freely available on the internet . there are also some websites that list and/or host multiple collections of data , and we shortly discuss about them . the working group in misp research center had set one of its goals to develop a reference medical image and signal database for image and signal processing research and development . the main aim of this collection was to provide reliable datasets for researchers to compare their algorithms on common framework and to be able to interpret and compare the performance of their newly developed methods . the ground truth was provided for datasets based on evaluation of one , two , or three experts . most of the mentioned datasets were available with a published paper in famous journals to demonstrate possible application of the dataset . the features of mysql are listed below : relational database management system . a relational database stores information in different tables , rather than in one giant table . the database software can be used and modified by anyone according to their needs.fast , reliable , and easy - to - use . relational database management system . a separate thread ( which is always running ) handles each incoming connection and manages the connections . even though the threads share the same process space , they execute individually , and because of this separation , multiprocessor machines can spread the thread across many central processing units ( cpus ) as long as the host operating system ( os ) supports multiple cpus . multithreading is the key feature to support mysql s performance design goals , and it can be counted as the core feature around which mysql is built . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years.object oriented : php actually has the ability to call java and windows com objects . therefore , the fact that one can code programs with php for free is a huge benefit that will not be provided with jps , asp , or other scripting languages that require paid hosting . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years . other classes can actually borrow from those custom classes and this specification extends the capabilities of php even further . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . regarding above features , we used php for web programming and mysql for database managing . was a fast , secure , and easy - to - use online database for medical signals and images . among these features , provided registered users the ability to download medical signals and images selectively and freely . it was also an appropriate place for researchers around the world to share their data and supplementary materials with each others , because users could freely , easily , securely , and unlimitedly upload their datasets while managing their privacies ( citation and fee ) . commenting and replying for the comments were available for all datasets ; therefore , researchers could share their experiences . , google ) , automatic sitemap and semi - automatic seo indexing to provide more description on contents of misp dataset , two examples of the available sets are described below . as a long - term goal of misp research center , the datasets would be added to this website , and it was open to accept databases from other researchers by convincing them to the reality that making online free datasets can improve the research quality of the owner and the users . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1].the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named totalmanual_13. the datasets were in mat format and were named 1 to 6 . 24. each dataset consisted of a slo image and limited number of oct scans with size of 496 512 . each dataset consisted of a limited number of oct scans with size of 496 512 . the datasets were in mat format and were named 1 to 15 . misp isfahan optical coherence tomography ( oct ) dataset for segmentation . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1 ] . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 6 . the datasets were in mat format and were named 1 to 24 . the datasets were in mat format and were named 1 to 112 . the datasets were in mat format and were named 1 to 19 . the datasets were in mat format and were named 1 to 15 . the x , y , z size of the obtained volumes was 512 650 128 voxels the second sample dataset was on audio and acoustic signals and included the following datasets : ( 1)voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease).data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . there was ambient noise from outside of the room but it was minimized during the recordings . this rubber piece was attached to the surface of the skin using double - sided adhesive tape . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . all data recordings were carried out by a speech language pathologist and a biomedical engineer. inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . /i / is the highest front vowel , and / a / is the lowest back vowel.vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . there was ambient noise from outside of the room but it was minimized during the recordings . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . these voice samples were corresponding to the following two groups : before and after functional rhinoplasty . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . /i / is the highest front vowel , and / a / is the lowest back vowel . the database software can be used and modified by anyone according to their needs.fast , reliable , and easy - to - use . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . relational database management system . a multithreaded application performs many tasks at the same time as if multiple instances of that application were running simultaneously . even though the threads share the same process space , they execute individually , and because of this separation , multiprocessor machines can spread the thread across many central processing units ( cpus ) as long as the host operating system ( os ) supports multiple cpus . multithreading is the key feature to support mysql s performance design goals , and it can be counted as the core feature around which mysql is built . the main features of php are listed below : powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . there are even many database managers written in php.faster web applications : because php does not use a lot of a system s resources to run , it operates much faster than other scripting languages . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years.object oriented : php actually has the ability to call java and windows com objects . one can use any text editor to code php such as notebook++ , jedit , emacs , bluefish , or even just notepad if felt inclined . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . powerful database - driven functionality : php integrates well with mysql and contains a library full of useful functions to help the usage of mysql . it can be concluded that php is a mature language , and it is also fairly stable because all the kinks have been worked out over the years . there are no licenses , restrictions , or royalty fees involved at all , and php is 100% free for anyone to use . regarding above features , we used php for web programming and mysql for database managing . was a fast , secure , and easy - to - use online database for medical signals and images . among these features , it was also an appropriate place for researchers around the world to share their data and supplementary materials with each others , because users could freely , easily , securely , and unlimitedly upload their datasets while managing their privacies ( citation and fee ) . commenting and replying for the comments were available for all datasets ; therefore , researchers could share their experiences . , google ) , automatic sitemap and semi - automatic seo indexing to provide more description on contents of misp dataset , two examples of the available sets are described below . as a long - term goal of misp research center , the datasets would be added to this website , and it was open to accept databases from other researchers by convincing them to the reality that making online free datasets can improve the research quality of the owner and the users . the first sample dataset was on ocular images and included the following image sets : ( 1)misp isfahan optical coherence tomography ( oct ) dataset for segmentation.this dataset contained 13 three - dimensional ( 3d ) macular sd - oct images obtained from eyes without pathologies using topcon 3d oct-1000 imaging system in ophthalmology dept . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1].the values of the proposed layer localization were provided for 12 boundaries and were stored in another mat file named randomimages ( the rows corresponded to each dataset and the columns contained the traced slices ) . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named totalmanual_13. the datasets were in mat format and were named 1 to 6 . the datasets were in mat format and were named 1 to 15 . , feiz hospital , isfahan , iran . the datasets were in mat format and were named 1 to 13 . the x , y , z size of the obtained volumes was 512 650 128 voxels , 7 3.125 3.125 mm , and voxel size 13.67 4.81 24.41 m [ as shown in figure 1 ] . furthermore , the validation was based on manual tracing by two observers , and mean value of the mentioned tracings were provided in mat files named this dataset contained six 3d oct data using topcon 3d oct-1000 imaging system in ophthalmology dept . the datasets were in mat format and were named 1 to 6 . each dataset consisted of a slo image and limited number of oct scans with size of 496 512 ( namely , for a data with 19 selected oct slices , the whole data size was 496 512 19 ) . the datasets were in mat format and were named 1 to 24 . this dataset contained 112 3d macular sd - oct images ( along with slo images ) obtained from eyes without pathologies from 76 people using heidelberg hra oct scanner imaging system in ophthalmology dept . the datasets were in mat format and were named 1 to 112 . the datasets were in mat format and were named 1 to 19 . the datasets were in mat format and were named 1 to 15 . the x , y , z size of the obtained volumes was 512 650 128 voxels the second sample dataset was on audio and acoustic signals and included the following datasets : ( 1)voice samples of patients with parkinson s disease ( spontaneous swallows in parkinson s disease).data were collected from 34 patients ( 19 males ) who suffered from parkinson s disease ( pd ) ( age = 59.85 11.46 years ) . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally.acoustic recording study : the acoustic recording was conducted in the test room with indoor lighting , which was at 24c . there was ambient noise from outside of the room but it was minimized during the recordings . the recorder amplified and digitized the received signal at a sampling rate of 44.1 khz . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . the number of swallows that each participant had within the 15-min time period was varied . (2)voice samples of patients with internal nasal valve collapse before and after functional rhinoplasty.this dataset contained voice recordings of participants who had internal nasal valve collapse . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . to minimize the environmental noise , recordings were done in an acoustic room ( with noise of 28 db based on sound level meter [ tes-1351 ] ) . / is the highest front vowel , and / a / is the lowest back vowel.vocal intensity during vowel prolongation was held at 75 2 db ( measured with sound level meter ) by asking each participant in person . patients demographic data , including age , sex , time from commencement of disease , and the hoehen and yahr scale score , which shows the stage of pd , were collected via an informed interview . the two tests were performed independently , whereas videofluoroscopy study was done to validate which participant suffered from aspiration generally . there was ambient noise from outside of the room but it was minimized during the recordings . the microphone was positioned over the laryngopharynx of each patient for 15 min when the participant was in a seated position watching a nature movie . because no food was used in this test , spontaneous swallows that were registered were innate saliva swallows . inclusion criteria were the lack of cold , hoarseness in all participants , and menstruation in females on the voice recording day . the participants were familiarized with the method of the test before recordings . the participants were in a seated position and a microphone ( somic senic st-818 3.5 mm on - ear stereo headphones with adjustable microphone and 2.5 m cable ) was located 5 cm from the center of their lips on the right corner of mouth . /i / is the highest front vowel , and / a / is the lowest back vowel . the patients voice sample was recorded at the same time using praat ( version 5.0.23 ) software at a sampling frequency of 44.1 khz using a laptop equipped with a sound card . the biosigdata.com is a free service , and the structure makes it a fast , secure , and easy - to - use online database .

there is a complex interplay between wider determinants of health , population movement , and shifting patterns of health and disease . health professionals are required to deliver high quality care to patients with diverse needs and backgrounds . postgraduate education must evolve to prepare health professionals to address the health challenges that globalisation brings . the potential benefits of health systems adopting a global health perspective in healthcare practice and management are well recognised . global health education aims to awaken health professionals to the interplay between local and global health , health systems and globalisation . reduction of health inequalities and improvement of health and well - being can only be realised if health professionals understand the global arena in which they are working . the need for appropriate global health training for doctors has been repeatedly raised , and uk medical royal colleges have responded to this call with conferences , position statements and strategies . despite this , the commission on medical education for the twenty - first century noted a mismatch between present professional competencies and the requirements of an increasingly interdependent world. a review of eleven uk postgraduate medical and surgical curricula found that only six contained any specific global health competencies , but all curricula contained generic competencies for which a global perspective could be advantageous . in undergraduate medical education , global health learning outcomes have been proposed . the general medical council includes the learning outcome : discuss from a global perspective the determinants of health and disease and variations in health care delivery and medical practice for uk medical undergraduates . global health competencies have also been explored for uk paediatricians and north american postgraduate health professionals . however , there is no current consensus on the minimum global health competencies required of uk postgraduate doctors , and current curricula vary significantly in terms of global health coverage . this study aimed to develop core global health competencies relevant to all uk postgraduate health professionals . as the study progressed , it was recognised that the learning needs and training pathways of doctors and other health professionals are sufficiently diverse that this could not be achieved to a good standard within the constraints of the timeframe and given the lack of representation of other health professionals among the author group . hence , the scope of the study was narrowed after round one to the development of core competencies for postgraduate doctors in the uk and provision of a framework for global health education that may inform curricula in other countries and for other health professionals . we carried out a modified policy delphi consultation to gather and incorporate wide - ranging views of stakeholders . consultation took place between march and june 2015 and allowed broad consultation ( round one ) , followed by indepth discussions with experts ( round two ) , and then further consultation with all participants ( round three ) . we reviewed published literature and existing postgraduate medical curricula and proposed seven key global health competencies . a draft competency document was developed as a basis for consultation in round one ( supplementary file 1 ) , which represents the main modification from the standard policy delphi . an online questionnaire was circulated to patient , health professional , educator and academic groups who were asked to cascade the questionnaire through their networks and on social media ( supplementary file 2 ) . it invited multiple choice and free text responses about the relevance and feasibility of the competencies for uk doctors as well as for other health professionals . participants were invited to offer ideas of how each competency may link to training or work of health professionals in the uk . consent to participation was deemed implicit in taking the survey . to incentivise participation , we offered participants the chance to win a book token . the survey remained open for 2 weeks . to inform revision of the competency document for round two , one author compiled descriptive statistics from quantitative results and two authors independently identified themes arising from the qualitative data . not all suggestions could be accommodated , with the most common reasons for exclusions being conflicting opinions from participants and suggested additions that were beyond the scope of the document . where there were conflicting opinions , we reached consensus through discussion and reference to published literature in round two , we interviewed key stakeholders , including patient representatives , global health educators , clinical leaders and trainee representatives . telephone or face - to - face interviews were each carried out by one researcher , who took notes during the interview . interview notes were compiled and used to explore themes , including areas of disagreement , drawing on advice from experts ( e.g. in economics and ethics ) and reference to published literature . three , we invited all first - round participants who had provided a contact address and all second - round participants to comment on the competency document and verify whether their comments had been adequately addressed . comments were solicited via an online questionnaire , which was emailed to participants with the updated document . the survey remained open for 1 week , with a reminder sent after 4 days . an online questionnaire was circulated to patient , health professional , educator and academic groups who were asked to cascade the questionnaire through their networks and on social media ( supplementary file 2 ) . it invited multiple choice and free text responses about the relevance and feasibility of the competencies for uk doctors as well as for other health professionals . participants were invited to offer ideas of how each competency may link to training or work of health professionals in the uk . consent to participation was deemed implicit in taking the survey . to incentivise participation , we offered participants the chance to win a book token . the survey remained open for 2 weeks . to inform revision of the competency document for round two , one author compiled descriptive statistics from quantitative results and two authors independently identified themes arising from the qualitative data . not all suggestions could be accommodated , with the most common reasons for exclusions being conflicting opinions from participants and suggested additions that were beyond the scope of the document . where there were conflicting opinions , we reached consensus through discussion and reference to published literature in round two , we interviewed key stakeholders , including patient representatives , global health educators , clinical leaders and trainee representatives . telephone or face - to - face interviews were each carried out by one researcher , who took notes during the interview . interview notes were compiled and used to explore themes , including areas of disagreement , drawing on advice from experts ( e.g. in economics and ethics ) and reference to published literature . in round three , we invited all first - round participants who had provided a contact address and all second - round participants to comment on the competency document and verify whether their comments had been adequately addressed . comments were solicited via an online questionnaire , which was emailed to participants with the updated document . the survey remained open for 1 week , with a reminder sent after 4 days . five inter - related competencies were defined ( figure 2 and supplementary file 3 ) after contribution from over 250 individuals ( figure 1 ) . * participants were able to select more than one continent of work for those that work across more than one location , therefore the total number of responses for continent of work exceeds 255 . * participants were able to select more than one continent of work for those that work across more than one location , therefore the total number of responses for continent of work exceeds 255 . over 60% of participants indicated that all of the proposed competencies were relevant to doctors . participants felt that the level of detail to which a trainee would need to address each competency would vary depending on their profession and speciality . in round one , participants deemed that the competencies were less relevant to and demonstrable by non - medical health professionals . we addressed this feedback by narrowing the aim of the research to the development of competencies for postgraduate doctors . participants in all rounds suggested that the competencies may yet have relevance for other health professionals , but may require tailoring . we expanded the range of knowledge areas and practice examples provided within each competency to represent the diversity of focuses that may be needed . we also added an acknowledgement in the introduction of the competencies document that educators will need to tailor these competencies to trainees ' learning needs and the setting in which the competency is being assessed . in all rounds , concerns were expressed by participants about overburdening curricula . in response , we amalgamated inter - related competencies and refined competencies such that global health topics can be incorporated into curricula by expanding ( rather than adding to the number of ) existing competencies . for example to demonstrate an awareness of equity in healthcare access and delivery is a competency frequently encountered in training curricula , which can be enhanced by including a global health perspective , such as consider barriers faced by asylum seekers , undocumented migrants , and survivors of torture. participants called for clarity of language , terms and intended audience ( for example , doctors versus all health professional ) , which we addressed by adding definitions and revising the document for clarity . alignment of the competencies with an established learning taxonomy was suggested and we did this using bloom 's taxonomy . participants felt that the competencies should reflect a person - centred approach to healthcare , focusing on the patient experience . it is recommended that a person - centred approach is taken to reflect that global health education ultimately aims to improve patient care . whether global health should be taught through a global health framework , or structured according to existing health professional competencies , was discussed . some participants felt that an ecological model ( from population - level down to individual - level topics ) should structure learning in global health ; others felt that the competencies would appear more relevant if they began with competencies focused on interaction with individuals . further conflicting perspectives emerged regarding the relative importance and relevance of each competency . in response , a statement and diagram to clarify that all competencies are inter - related and equally important has been included in the final document ( figure 2 ) . integration of the competencies into curricula and approach to learning should be tailored as appropriate within each professional field . for round one , competencies included human rights and ethics and cultural diversity and health , which , respectively , 92% and 93% of participants thought were appropriate and feasible competencies for doctors . after round one , we amalgamated these competencies into diversity , human rights and ethics. competencies before round one included socioeconomic determinants of health and environmental determinants of health , which were deemed appropriate and feasible for doctors by 88% and 72% of participants , respectively . in all rounds , comments about environmental determinants of health were at two extremes : some participants stated that understanding environmental issues and their transnational nature is essential for doctors ; others felt that addressing environmental issues is beyond their remit . attempting to respect both views , we included environmental determinants of health within a competency on socioeconomic determinants , and developed tangible practice examples to highlight how environmental issues may fall within the role of health professionals . in round one , 85% of participants thought that global burden of disease was an appropriate and feasible competency for doctors . some participants felt that the examples given were too specific to be relevant to all doctors regardless of specialty ; therefore , we replaced specific disease examples with broader examples and concepts . some participants suggested that there should be more focus on certain disease areas ( mainly non - communicable diseases and mental health ) , and certain patient groups ( older people , refugees , asylum seekers and undocumented migrants ) . participants commented that there should be a shift of focus from disease and its treatment to health and its promotion . we made changes throughout the competency ( including referencing demographic transition rather than problems of ageing populations ) , and changed the title of the competency to global epidemiology. this competency was deemed appropriate and feasible for doctors by 83% of participants . we revised the competency to ensure clarity and focus on the relationship of the roles and duties of doctors . participants suggested many additions , such as health impact assessment , transnational health threats and international resources for health ( e.g. transplant organs ) . to avoid being directive and overburdening many participants felt that doctors lack an understanding of their own health system ; therefore , understanding other health systems is not feasible ; others felt that understanding the components of a health system with examples from other countries could aid comprehension of the local health system . in round two , participants highlighted the importance of understanding how health system configuration and healthcare workers ' roles affect population health ; therefore , we added further reference to health professionals ' roles , migration and work abroad . in round one , for each of the seven proposed competencies , the majority of participants indicated that it was relevant and feasible for all health professionals , with a proportion of participants selecting agree or strongly agree that it is relevant and feasible as follows : human rights and ethics 86% , cultural diversity and health 89% , socioeconomic determinants of health 68% , environmental determinants of health 54% , global burden of disease 56% , global health governance 58% and health systems 60% . these findings suggest that global health education is relevant and feasible to all health professionals , with particular relevance seen for competencies relating to diversity , ethics and human rights . where fewer respondents agreed that the competency was relevant to all health professionals , comments suggested that this may be due to the competency being too specific or written using language and concepts that are findings from rounds two and three , including comments from non - medical health professionals and health educators , also suggested that the global health competencies arising from this document have some relevance to , and may benefit , wider health professional education . for round one , competencies included human rights and ethics and cultural diversity and health , which , respectively , 92% and 93% of participants thought were appropriate and feasible competencies for doctors . after round one , we amalgamated these competencies into diversity , human rights and ethics. competencies before round one included socioeconomic determinants of health and environmental determinants of health , which were deemed appropriate and feasible for doctors by 88% and 72% of participants , respectively . in all rounds , comments about environmental determinants of health were at two extremes : some participants stated that understanding environmental issues and their transnational nature is essential for doctors ; others felt that addressing environmental issues is beyond their remit . attempting to respect both views , we included environmental determinants of health within a competency on socioeconomic determinants , and developed tangible practice examples to highlight how environmental issues may fall within the role of health professionals . in round one , 85% of participants thought that global burden of disease was an appropriate and feasible competency for doctors . some participants felt that the examples given were too specific to be relevant to all doctors regardless of specialty ; therefore , we replaced specific disease examples with broader examples and concepts . some participants suggested that there should be more focus on certain disease areas ( mainly non - communicable diseases and mental health ) , and certain patient groups ( older people , refugees , asylum seekers and undocumented migrants ) . participants commented that there should be a shift of focus from disease and its treatment to health and its promotion . we made changes throughout the competency ( including referencing demographic transition rather than problems of ageing populations ) , and changed the title of the competency to we revised the competency to ensure clarity and focus on the relationship of the roles and duties of doctors . participants suggested many additions , such as health impact assessment , transnational health threats and international resources for health ( e.g. transplant organs ) . to avoid in round one , the competency health systems was rated appropriate and feasible for doctors by 82% of participants . many participants felt that doctors lack an understanding of their own health system ; therefore , understanding other health systems is not feasible ; others felt that understanding the components of a health system with examples from other countries could aid comprehension of the local health system . in round two , participants highlighted the importance of understanding how health system configuration and healthcare workers ' roles affect population health ; therefore , we added further reference to health professionals ' roles , migration and work abroad . in round one , for each of the seven proposed competencies , the majority of participants indicated that it was relevant and feasible for all health professionals , with a proportion of participants selecting agree or strongly agree that it is relevant and feasible as follows : human rights and ethics 86% , cultural diversity and health 89% , socioeconomic determinants of health 68% , environmental determinants of health 54% , global burden of disease 56% , global health governance 58% and health systems 60% . these findings suggest that global health education is relevant and feasible to all health professionals , with particular relevance seen for competencies relating to diversity , ethics and human rights . where fewer respondents agreed that the competency was relevant to all health professionals , comments suggested that this may be due to the competency being too specific or written using language and concepts that are findings from rounds two and three , including comments from non - medical health professionals and health educators , also suggested that the global health competencies arising from this document have some relevance to , and may benefit , wider health professional education . this is the first large scale consultation on global health competencies for uk doctors , consulting over 250 diverse stakeholders with discussion and reflection on global health competencies for postgraduate medical training . the resulting five core competencies provide an achievable minimum level of core global health competence , required by all postgraduate doctors . the findings of the consultation demonstrate a perceived difference in the topics and approach to learning of global health required by doctors versus other health professionals . the breadth of the five competencies and the accompanying examples , as well as the input from and endorsement by a range of health professionals , suggest that the competencies may also inform curriculum development for other postgraduate health professionals . attainment of these competencies by a medical workforce would help to ensure that health services are equipped to care for diverse populations , deal with global influences on health and meet health challenges of the future . analysis of participants ' responses confirmed that learning needs are diverse and views of which topics are relevant and what is essential learning vary amongst stakeholders . for example , participants had varying views on whether doctors need to learn about the structure and function of different health systems , environmental issues or laws applying to migration ; all of which are determinants of health and bear some relationship to healthcare provision . an individual 's views on the relevance of global health competencies may be subject to the individual 's type of work , location , level of responsibility , previous exposure to this subject area or conceptualisation of professionalism , social accountability and the roles of health professionals . as more doctors opt to spend time working in different and diverse healthcare settings , postgraduate education leads may wish to support the design of educational tools to aid doctors intending to work overseas with the clinical knowledge , skills and attitudes required to work effectively in unfamiliar settings . although the global health competencies proposed here may serve as a building block for this , they are designed with a focus on what a doctor working in the uk needs to know . the five main learning areas that need to be addressed according to this study are supported by previous work , such as that developing global health learning outcomes for medical undergraduates , competencies for uk postgraduate paediatricians and competencies for usa health professionals , and by forthcoming competencies from the uk department for international development ; all of which identify similar competency areas . the attempt to incorporate global health within core curricula the findings of this study diverge from previous studies in a number of ways , highlighting the evolving nature of global health and medical education dialogues . examples of these areas of divergence include the incorporation of ethics within a competency addressing diversity and human rights ; the equal attention to environmental determinants of health alongside social and economic determinants ; the more indepth exploration of global health governance and health systems as they impact on the design and delivery of services locally ; and a step away from global burden of disease towards a focus on health promotion by using the term global epidemiology. this reinforces the importance of ongoing review and update of health professional curricula to reflect the changing nature and understanding of health and healthcare in our ever more globalised world . strengths of this study include the number of participants and diversity of their backgrounds , which allowed the combination of perspectives from a variety of health professionals , key health leaders and lay people . although the majority of respondents worked in the uk , we also gleaned the opinion of those working in other parts of the world , including low and middle income countries . we encouraged participants to cascade the survey via their networks and social media , and the response rate for round one can not be calculated . although the study involved a large number of participants and multiple interactions with study coordinators , the addition of face - to - face group discussions could have generated further ideas and indepth discussion of contentious issues . furthermore , resource limitations prevented us from recording and transcribing interviews ; therefore , there was risk of loss of depth of findings in round two . the identification of participants was dependent on health groups , networks and experts identified by , known to or recommended to the authors ; therefore , the population sampled may not represent the full diversity of stakeholders . in the uk , the need for improved global health training of health care professionals and creation of healthcare environments that support global health initiatives has been identified . based on the findings of this study , we recommend that : all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , andregular review of global health competencies is undertaken . all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , and regular review of global health competencies is undertaken . postgraduate medical education can better prepare doctors for work in our increasingly globalised world through the inclusion of a global health perspective in training . in order to incorporate these competencies into existing speciality curricula without overburdening trainees , it will be important for educators in each speciality to tailor the competencies to the educational needs of their trainees . incorporation of core global health competencies into existing postgraduate health professional education may ensure that health systems are equipped to care for diverse populations , deal with global influences on health and meet the health challenges of the future . as healthcare and global health are ever changing , these competencies will require regular review and updates , and novel approaches to their integration and delivery . in the uk , the need for improved global health training of health care professionals and creation of healthcare environments that support global health initiatives has been identified . based on the findings of this study , we recommend that : all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , andregular review of global health competencies is undertaken . all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , and regular review of global health competencies is undertaken . postgraduate medical education can better prepare doctors for work in our increasingly globalised world through the inclusion of a global health perspective in training . in order to incorporate these competencies into existing speciality curricula without overburdening trainees , it will be important for educators in each speciality to tailor the competencies to the educational needs of their trainees . incorporation of core global health competencies into existing postgraduate health professional education may ensure that health systems are equipped to care for diverse populations , deal with global influences on health and meet the health challenges of the future . as healthcare and global health are ever changing , these competencies will require regular review and updates , and novel approaches to their integration and delivery .

backgroundglobalisation is having profound impacts on health and healthcare . we solicited the views of a wide range of stakeholders in order to develop core global health competencies for postgraduate doctors.methodspublished literature and existing curricula informed writing of seven global health competencies for consultation . a modified policy delphi involved an online survey and face - to - face and telephone interviews over three rounds.resultsover 250 stakeholders participated , including doctors , other health professionals , policymakers and members of the public from all continents of the world . participants indicated that global health competence is essential for postgraduate doctors and other health professionals . concerns were expressed about overburdening curricula and identifying what is essential for whom . conflicting perspectives emerged about the importance and relevance of different global health topics . five core competencies were developed : ( 1 ) diversity , human rights and ethics ; ( 2 ) environmental , social and economic determinants of health ; ( 3 ) global epidemiology ; ( 4 ) global health governance ; and ( 5 ) health systems and health professionals.conclusionsglobal health can bring important perspectives to postgraduate curricula , enhancing the ability of doctors to provide quality care . these global health competencies require tailoring to meet different trainees ' needs and facilitate their incorporation into curricula . healthcare and global health are ever - changing ; therefore , the competencies will need to be regularly reviewed and updated .

Introduction Methods Round one Round two Round three Results Competency one: diversity, human rights and ethics Competency two: environmental, social and economic determinants of health Competency three: global epidemiology Competency four: global health governance Competency five: health systems and health professionals Relevance of the competencies to other health professionals Discussion Recommendations Conclusions Supplementary data

there is a complex interplay between wider determinants of health , population movement , and shifting patterns of health and disease . health professionals are required to deliver high quality care to patients with diverse needs and backgrounds . the potential benefits of health systems adopting a global health perspective in healthcare practice and management are well recognised . global health education aims to awaken health professionals to the interplay between local and global health , health systems and globalisation . reduction of health inequalities and improvement of health and well - being can only be realised if health professionals understand the global arena in which they are working . a review of eleven uk postgraduate medical and surgical curricula found that only six contained any specific global health competencies , but all curricula contained generic competencies for which a global perspective could be advantageous . the general medical council includes the learning outcome : discuss from a global perspective the determinants of health and disease and variations in health care delivery and medical practice for uk medical undergraduates . global health competencies have also been explored for uk paediatricians and north american postgraduate health professionals . however , there is no current consensus on the minimum global health competencies required of uk postgraduate doctors , and current curricula vary significantly in terms of global health coverage . this study aimed to develop core global health competencies relevant to all uk postgraduate health professionals . as the study progressed , it was recognised that the learning needs and training pathways of doctors and other health professionals are sufficiently diverse that this could not be achieved to a good standard within the constraints of the timeframe and given the lack of representation of other health professionals among the author group . hence , the scope of the study was narrowed after round one to the development of core competencies for postgraduate doctors in the uk and provision of a framework for global health education that may inform curricula in other countries and for other health professionals . we carried out a modified policy delphi consultation to gather and incorporate wide - ranging views of stakeholders . we reviewed published literature and existing postgraduate medical curricula and proposed seven key global health competencies . a draft competency document was developed as a basis for consultation in round one ( supplementary file 1 ) , which represents the main modification from the standard policy delphi . an online questionnaire was circulated to patient , health professional , educator and academic groups who were asked to cascade the questionnaire through their networks and on social media ( supplementary file 2 ) . it invited multiple choice and free text responses about the relevance and feasibility of the competencies for uk doctors as well as for other health professionals . participants were invited to offer ideas of how each competency may link to training or work of health professionals in the uk . not all suggestions could be accommodated , with the most common reasons for exclusions being conflicting opinions from participants and suggested additions that were beyond the scope of the document . where there were conflicting opinions , we reached consensus through discussion and reference to published literature in round two , we interviewed key stakeholders , including patient representatives , global health educators , clinical leaders and trainee representatives . telephone or face - to - face interviews were each carried out by one researcher , who took notes during the interview . an online questionnaire was circulated to patient , health professional , educator and academic groups who were asked to cascade the questionnaire through their networks and on social media ( supplementary file 2 ) . it invited multiple choice and free text responses about the relevance and feasibility of the competencies for uk doctors as well as for other health professionals . participants were invited to offer ideas of how each competency may link to training or work of health professionals in the uk . not all suggestions could be accommodated , with the most common reasons for exclusions being conflicting opinions from participants and suggested additions that were beyond the scope of the document . where there were conflicting opinions , we reached consensus through discussion and reference to published literature in round two , we interviewed key stakeholders , including patient representatives , global health educators , clinical leaders and trainee representatives . telephone or face - to - face interviews were each carried out by one researcher , who took notes during the interview . interview notes were compiled and used to explore themes , including areas of disagreement , drawing on advice from experts ( e.g. five inter - related competencies were defined ( figure 2 and supplementary file 3 ) after contribution from over 250 individuals ( figure 1 ) . over 60% of participants indicated that all of the proposed competencies were relevant to doctors . participants felt that the level of detail to which a trainee would need to address each competency would vary depending on their profession and speciality . in round one , participants deemed that the competencies were less relevant to and demonstrable by non - medical health professionals . we addressed this feedback by narrowing the aim of the research to the development of competencies for postgraduate doctors . participants in all rounds suggested that the competencies may yet have relevance for other health professionals , but may require tailoring . we expanded the range of knowledge areas and practice examples provided within each competency to represent the diversity of focuses that may be needed . we also added an acknowledgement in the introduction of the competencies document that educators will need to tailor these competencies to trainees ' learning needs and the setting in which the competency is being assessed . in all rounds , concerns were expressed by participants about overburdening curricula . in response , we amalgamated inter - related competencies and refined competencies such that global health topics can be incorporated into curricula by expanding ( rather than adding to the number of ) existing competencies . for example to demonstrate an awareness of equity in healthcare access and delivery is a competency frequently encountered in training curricula , which can be enhanced by including a global health perspective , such as consider barriers faced by asylum seekers , undocumented migrants , and survivors of torture. alignment of the competencies with an established learning taxonomy was suggested and we did this using bloom 's taxonomy . it is recommended that a person - centred approach is taken to reflect that global health education ultimately aims to improve patient care . whether global health should be taught through a global health framework , or structured according to existing health professional competencies , was discussed . some participants felt that an ecological model ( from population - level down to individual - level topics ) should structure learning in global health ; others felt that the competencies would appear more relevant if they began with competencies focused on interaction with individuals . further conflicting perspectives emerged regarding the relative importance and relevance of each competency . integration of the competencies into curricula and approach to learning should be tailored as appropriate within each professional field . for round one , competencies included human rights and ethics and cultural diversity and health , which , respectively , 92% and 93% of participants thought were appropriate and feasible competencies for doctors . after round one , we amalgamated these competencies into diversity , human rights and ethics. competencies before round one included socioeconomic determinants of health and environmental determinants of health , which were deemed appropriate and feasible for doctors by 88% and 72% of participants , respectively . in all rounds , comments about environmental determinants of health were at two extremes : some participants stated that understanding environmental issues and their transnational nature is essential for doctors ; others felt that addressing environmental issues is beyond their remit . attempting to respect both views , we included environmental determinants of health within a competency on socioeconomic determinants , and developed tangible practice examples to highlight how environmental issues may fall within the role of health professionals . in round one , 85% of participants thought that global burden of disease was an appropriate and feasible competency for doctors . some participants felt that the examples given were too specific to be relevant to all doctors regardless of specialty ; therefore , we replaced specific disease examples with broader examples and concepts . we made changes throughout the competency ( including referencing demographic transition rather than problems of ageing populations ) , and changed the title of the competency to global epidemiology. we revised the competency to ensure clarity and focus on the relationship of the roles and duties of doctors . to avoid being directive and overburdening many participants felt that doctors lack an understanding of their own health system ; therefore , understanding other health systems is not feasible ; others felt that understanding the components of a health system with examples from other countries could aid comprehension of the local health system . in round two , participants highlighted the importance of understanding how health system configuration and healthcare workers ' roles affect population health ; therefore , we added further reference to health professionals ' roles , migration and work abroad . in round one , for each of the seven proposed competencies , the majority of participants indicated that it was relevant and feasible for all health professionals , with a proportion of participants selecting agree or strongly agree that it is relevant and feasible as follows : human rights and ethics 86% , cultural diversity and health 89% , socioeconomic determinants of health 68% , environmental determinants of health 54% , global burden of disease 56% , global health governance 58% and health systems 60% . these findings suggest that global health education is relevant and feasible to all health professionals , with particular relevance seen for competencies relating to diversity , ethics and human rights . where fewer respondents agreed that the competency was relevant to all health professionals , comments suggested that this may be due to the competency being too specific or written using language and concepts that are findings from rounds two and three , including comments from non - medical health professionals and health educators , also suggested that the global health competencies arising from this document have some relevance to , and may benefit , wider health professional education . for round one , competencies included human rights and ethics and cultural diversity and health , which , respectively , 92% and 93% of participants thought were appropriate and feasible competencies for doctors . after round one , we amalgamated these competencies into diversity , human rights and ethics. competencies before round one included socioeconomic determinants of health and environmental determinants of health , which were deemed appropriate and feasible for doctors by 88% and 72% of participants , respectively . in all rounds , comments about environmental determinants of health were at two extremes : some participants stated that understanding environmental issues and their transnational nature is essential for doctors ; others felt that addressing environmental issues is beyond their remit . attempting to respect both views , we included environmental determinants of health within a competency on socioeconomic determinants , and developed tangible practice examples to highlight how environmental issues may fall within the role of health professionals . some participants felt that the examples given were too specific to be relevant to all doctors regardless of specialty ; therefore , we replaced specific disease examples with broader examples and concepts . we made changes throughout the competency ( including referencing demographic transition rather than problems of ageing populations ) , and changed the title of the competency to we revised the competency to ensure clarity and focus on the relationship of the roles and duties of doctors . to avoid in round one , the competency health systems was rated appropriate and feasible for doctors by 82% of participants . many participants felt that doctors lack an understanding of their own health system ; therefore , understanding other health systems is not feasible ; others felt that understanding the components of a health system with examples from other countries could aid comprehension of the local health system . in round two , participants highlighted the importance of understanding how health system configuration and healthcare workers ' roles affect population health ; therefore , we added further reference to health professionals ' roles , migration and work abroad . in round one , for each of the seven proposed competencies , the majority of participants indicated that it was relevant and feasible for all health professionals , with a proportion of participants selecting agree or strongly agree that it is relevant and feasible as follows : human rights and ethics 86% , cultural diversity and health 89% , socioeconomic determinants of health 68% , environmental determinants of health 54% , global burden of disease 56% , global health governance 58% and health systems 60% . these findings suggest that global health education is relevant and feasible to all health professionals , with particular relevance seen for competencies relating to diversity , ethics and human rights . where fewer respondents agreed that the competency was relevant to all health professionals , comments suggested that this may be due to the competency being too specific or written using language and concepts that are findings from rounds two and three , including comments from non - medical health professionals and health educators , also suggested that the global health competencies arising from this document have some relevance to , and may benefit , wider health professional education . this is the first large scale consultation on global health competencies for uk doctors , consulting over 250 diverse stakeholders with discussion and reflection on global health competencies for postgraduate medical training . the resulting five core competencies provide an achievable minimum level of core global health competence , required by all postgraduate doctors . the findings of the consultation demonstrate a perceived difference in the topics and approach to learning of global health required by doctors versus other health professionals . the breadth of the five competencies and the accompanying examples , as well as the input from and endorsement by a range of health professionals , suggest that the competencies may also inform curriculum development for other postgraduate health professionals . attainment of these competencies by a medical workforce would help to ensure that health services are equipped to care for diverse populations , deal with global influences on health and meet health challenges of the future . analysis of participants ' responses confirmed that learning needs are diverse and views of which topics are relevant and what is essential learning vary amongst stakeholders . for example , participants had varying views on whether doctors need to learn about the structure and function of different health systems , environmental issues or laws applying to migration ; all of which are determinants of health and bear some relationship to healthcare provision . an individual 's views on the relevance of global health competencies may be subject to the individual 's type of work , location , level of responsibility , previous exposure to this subject area or conceptualisation of professionalism , social accountability and the roles of health professionals . although the global health competencies proposed here may serve as a building block for this , they are designed with a focus on what a doctor working in the uk needs to know . the five main learning areas that need to be addressed according to this study are supported by previous work , such as that developing global health learning outcomes for medical undergraduates , competencies for uk postgraduate paediatricians and competencies for usa health professionals , and by forthcoming competencies from the uk department for international development ; all of which identify similar competency areas . the attempt to incorporate global health within core curricula the findings of this study diverge from previous studies in a number of ways , highlighting the evolving nature of global health and medical education dialogues . examples of these areas of divergence include the incorporation of ethics within a competency addressing diversity and human rights ; the equal attention to environmental determinants of health alongside social and economic determinants ; the more indepth exploration of global health governance and health systems as they impact on the design and delivery of services locally ; and a step away from global burden of disease towards a focus on health promotion by using the term global epidemiology. this reinforces the importance of ongoing review and update of health professional curricula to reflect the changing nature and understanding of health and healthcare in our ever more globalised world . strengths of this study include the number of participants and diversity of their backgrounds , which allowed the combination of perspectives from a variety of health professionals , key health leaders and lay people . although the majority of respondents worked in the uk , we also gleaned the opinion of those working in other parts of the world , including low and middle income countries . although the study involved a large number of participants and multiple interactions with study coordinators , the addition of face - to - face group discussions could have generated further ideas and indepth discussion of contentious issues . furthermore , resource limitations prevented us from recording and transcribing interviews ; therefore , there was risk of loss of depth of findings in round two . the identification of participants was dependent on health groups , networks and experts identified by , known to or recommended to the authors ; therefore , the population sampled may not represent the full diversity of stakeholders . in the uk , the need for improved global health training of health care professionals and creation of healthcare environments that support global health initiatives has been identified . based on the findings of this study , we recommend that : all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , andregular review of global health competencies is undertaken . all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , and regular review of global health competencies is undertaken . postgraduate medical education can better prepare doctors for work in our increasingly globalised world through the inclusion of a global health perspective in training . in order to incorporate these competencies into existing speciality curricula without overburdening trainees , it will be important for educators in each speciality to tailor the competencies to the educational needs of their trainees . incorporation of core global health competencies into existing postgraduate health professional education may ensure that health systems are equipped to care for diverse populations , deal with global influences on health and meet the health challenges of the future . as healthcare and global health are ever changing , these competencies will require regular review and updates , and novel approaches to their integration and delivery . in the uk , the need for improved global health training of health care professionals and creation of healthcare environments that support global health initiatives has been identified . based on the findings of this study , we recommend that : all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , andregular review of global health competencies is undertaken . all postgraduate medical education bodies identify how these competencies relate to their trainees ' learning needs and incorporate global health into their existing curricula , non - medical health professional educators explore how these competencies can be adapted and incorporated into curricula for their trainees and postgraduates , guided by consultation with trainees , health professionals and other stakeholders , new learning , teaching and assessment mechanisms to address these competencies are developed , delivered and evaluated , and regular review of global health competencies is undertaken . postgraduate medical education can better prepare doctors for work in our increasingly globalised world through the inclusion of a global health perspective in training . in order to incorporate these competencies into existing speciality curricula without overburdening trainees , it will be important for educators in each speciality to tailor the competencies to the educational needs of their trainees . incorporation of core global health competencies into existing postgraduate health professional education may ensure that health systems are equipped to care for diverse populations , deal with global influences on health and meet the health challenges of the future . as healthcare and global health are ever changing , these competencies will require regular review and updates , and novel approaches to their integration and delivery .

duchenne muscular dystrophy ( dmd ) is a fatal neuromuscular disease , affecting 1 : 3500 live male births . it occurs as a result of mutations , mainly deletions , in the dystrophin gene . mutations lead to absence of or defect in the protein dystrophin from the muscle fibers causing cycles of muscle fiber degeneration and regeneration with replacement by fat and connective tissue . diagnosis is based on clinical examination observing the child run , jump , climb stairs , and get up from the floor ; blood test : serum creatine kinase ( ck ) levels up to 50100-fold above normal ; genetic testing : approximately 65% of patients with dmd have intragenic out - of - frame ( gross rearrangements ) deletions and approximately 10% have duplications of one or more exons of the dystrophin gene [ 2 , 3 ] ; and muscle biopsy : dystrophin analysis will always be abnormal and offers a further route to confirm the diagnosis . at the moment , there is no curative treatment for this devastating disease , and the main goal of interventions is to maintain ambulation as long as possible and to minimize the impact of the predictable complications of the disease , such as joint contractures , scoliosis , cardiomyopathy , and respiratory insufficiency . the objective of this review is to trace the natural history of the disease , in particular , with regard to the development of spinal deformity and how this complication has been modified by surgical interventions and overall by corticosteroid treatment . clinical evolution of muscular weakness in patients with duchenne muscular dystrophy is peculiarly marked by its progressive nature . as dmd boys appear healthy at birth , the natural history of untreated dmd leads to the development of an abnormal gait , calf hypertrophy , and difficulty rising from the floor when at 25 years of age . if not correctly diagnosed and treated , the boys become progressively unsteady in their walking , have a propensity to fall , use gower 's manoeuvre to stand up again , and acquire a waddling gait . gower 's manoeuvre is always present , with boys needing to turn onto their front and rise to standing from the floor using a broad - based stance , usually with the support of their hands on their thighs . common features of the disease are calves muscle hypertrophy and , frequently , developmental delay with delayed speech . respiratory failure is the major cause of death and occurs in the second or third decade of life ; it is caused by progressive respiratory muscle weakness and includes progressive restrictive ventilatory defects , chronic hypoventilation , and pulmonary infections . the remaining 10% of deaths occur due to myocardial disease and its sequelae including heart failure and dysrhythmia . interventions designed to lessen the predictable complications of the disease have successfully changed its course that is now compatible with survival into adult life . the provision of noninvasive mechanical ventilation , assisted coughing , and cardioprotective medication allows survival into the late twenties and thirties . the natural history of the disease has also been significantly changed by the use of corticosteroids ( cs ) . efficacy has been established in improving muscle strength and timed functional tests over period of 618 months [ 9 , 10 ] . follow - up studies show long - term benefit with marked reduction in spinal deformity and prolonged ambulation . more recently , it was shown that the early use of cs has significant advantages : boys starting treatment between ages 2 and 4 maintain ambulation beyond age 16 [ 13 , 14 ] . the clinical and laboratory diagnosis of dmd is now feasible much earlier than in the past and cs treatment can begin earlier in the course of the disease hopefully providing greater benefit than if treatment is delayed . it should be noted in fact that the marked elevation of ck , a recognized marker of muscle fiber necrosis , is already present at birth [ 16 , 17 ] . a florid dystrophic process is already evident in the muscle biopsy of newborns with dmd [ 16 , 18 ] . dmd infants and young boys in the first 3 years of age have already measurable deficits in gross and fine motor function . in addition , motor function declines within the first 3 years of life compared to age - matched peers . a progressive scoliosis develops in over 90% of patients as a combined result of wheel chair dependence , paralysis of the extensor muscles , contractures , and growth spurt ( figure 1 ) . a severe collapsing scoliosis can interfere with breathing by reducing the lung function and can obstacle seating position , greatly decreasing quality of life of dmd patients . the spinal deformity differs from that seen in patients with idiopathic scoliosis and is described as a c - type curve ( a gentle , sweeping curve , with apex at the thoracolumbar junction ) . it can cover up to 15 segments and , as a consequence , on clinical observation , be missed by the untrained eye . the spinal deformity results from a varying combination of curves in the coronal ( scoliosis ) and sagittal ( kyphosis , lordosis ) planes and excessive flexibility or stiffness of the spine due to the paralysis ( collapse ) or fibrous replacement ( rigid spine ) of the axial musculature . onset of scoliosis and loss of autonomous ambulation occur together in boys with dmd , generally between the ages of 10 and 14 years . there are different patterns of progression of scoliosis [ 22 , 23 ] . in the coronal plane , scoliosis can evolve in a linear and constant way when the child is wheelchair bound or alternate a period of slow progression to a rapid increase of the curve or have a rapid evolution over a few months too . in most of the cases , the final result is a kyphoscoliosis with collapsing spine or , less frequently , a hyperlordosis or a lordoscoliosis with rigid spine [ 22 , 24 ] . in addition to skeletal deformities on the sagittal and anterior - posterior plane , almost 100% of patients have a more or less distinct pelvic tilt ( 015 ) . the majority of patients complain about pain when sitting in a wheelchair because of the one - sided load on one buttock . when scoliosis is suspected , an x - ray of the entire spine ( ap and lateral view ) is mandatory . the various modalities of physiotherapy , including pool exercises , daily mobilization of contractures , and orthoses , like night splints or supportive sitting devices , have not shown a significant effect on progression of scoliosis . rehabilitation in lightweight knee - ankle - foot orthoses at the point of loss of ambulation , with or without tendon release , has been proven effective in preventing / reducing progression of scoliosis during the pubertal growth spurt [ 2528 ] . nonsurgical methods of spinal correction include the use of body jackets , custom - made seating inserts , and wheelchair modifications [ 29 , 30 ] . bracing is known to be ineffective to stop the progression of scoliosis in these children , as progression occurred in 94% despite bracing , and can be predicted to be 10 per year . use of bracing should be therefore reserved for patients who refuse surgery or patients who are inoperable . spine surgery with posterior spinal fusion is the gold standard treatment for severe progressive scoliosis in dmd patients with the subsequent indications : documented curve progression , loss of seating balance , pain and/or discomfort . surgical treatment is mainly performed to restore the balance of the spinal column in both coronal and sagittal planes , to improve life quality of patients , facilitate nursing , and improve sitting balance and comfort . instrumentation techniques have evolved over the years to achieve these goals by decreasing surgical time and blood loss with minimum neurovascular complications . several instrumentation techniques have been applied for scoliosis correction in dmd patients ranging from halo casts with traction wires and buttons , harrington rods , and luque 's segmental spinal fixation to more recent techniques using pedicle screws and hooks . use of harrington instrumentation technique showed significant improvement in curve correction ( 60% on average ) , delay of curve progression , and shortening of constrained postoperative recumbence period . long - term studies have shown that this is a successful procedure with limited complications [ 24 , 31 , 37 ] . the most commonly described method for fusion to the pelvis is luque rod instrumentation with use of the galveston technique . the use of the galveston fixation with the placement of the pelvic portion of the rods between the tables of the ilia above the sciatic notch allows for correction of pelvic obliquity . long - term studies have shown that the luque - galveston system with spinal fusion is an efficient , safe , well - designed , easily adaptable , and reproducible technical method for the dmd patient with a moderate spinal curve needing correction . success rates are highest if surgery is performed early , when the spine is still mobile at a cobb angle of 2040 and the cardiac and respiratory function is , in part , preserved . the rate of complications is so reduced and if there is a pelvic obliquity , the fixation to the pelvis is always indicated . the search for a safe and resistant surgical technique has led to the advent of pedicle screws and hook system . pedicle screws are penetrating anchors , which are superior to gripping fixation obtained by laminar wires and cables . the pedicle screw system was still found to be superior in achieving a better major curve correction and lesser neurological complications . overall , the described instrumentations appear to provide and maintain an optimal degree of correction at medium to long - term follow - up but the advantages of lowest blood loss and least surgical time without the need for pelvic fixation seem to swing the verdict in favor of the pedicle screw system . patients with dmd have increased blood loss during spinal surgery compared to non - dmd patients . in duchenne patients , labarque et al . found that platelets have a disorganized cytoskeleton due to dysfunctional dystrophin that may result in increased bleeding during surgery . independently of the surgical technique , patients operated with a scoliotic curve less than 40 had better results , even in time , than patients operated with a more severe scoliotic curve ( 40 or more ) . patients with dmd develop a restrictive respiratory pattern with reduction of maximal respiratory pressures and forced vital capacity ( fvc ) that eventually causes respiratory insufficiency and death ( figure 2 ) . in these patients , lung function increases up to the age of 1012 years hitting its plateau ; then lung function decreases with an estimated loss of 10% per year of fvc . no correlation has been found between grade and progression of respiratory dysfunction and severity of scoliosis , because intrinsic respiratory muscle 's weakness is actually the main determinant of decline in respiratory function in dmd . kennedy et al . , in a study on spinal surgery and lung function , reported no differences between surgical group and nonsurgical group in the rate of deterioration of % fvc which was 35% per year concluding that spinal stabilization in dmd does not alter the decline in pulmonary function nor does it improve survival . in addition , no difference was found in the rate of vital capacity decrease between operated or nonoperated patients and , in operated patients ' subgroup , between patients with scoliotic curve less than 40 and patients with more severe scoliotic patterns ( > 40 ) . in spite of these progresses , spinal stabilization in patients with dmd remains a demanding surgery because of dystrophic involvement of the paraspinal muscles and vascular smooth fibers , abnormal platelet function with a higher risk of intraoperatory bleeding requiring an adequate surgical technique , and a proper availability of blood units . intraoperative bleeding is the main risk factor ; however , several other complications are common in spinal surgery in dmd patients : intraoperative infection , implant failure , vertebral fracture , pullout of the screws , sacral decubitus , and death [ 46 , 47 ] . this surgery should be performed only by experienced surgeons and highly specialized centers ; moreover , it does not influence the natural course of respiratory decline . the first scientific evidence on the beneficial effect of steroids in duchenne muscular dystrophy was documented over 40 years ago by drachman et al . ; since then , several other studies have demonstrated the efficacy of corticosteroid therapy in delaying the loss of independence and autonomous ambulation and in maintaining an adequate pulmonary function . prednisone or deflazacort has been demonstrated to have a beneficial effect on muscle strength and function in boys with dmd and should be offered as treatment [ 15 , 48 ] . currently , corticosteroids are the gold standard treatment for muscle weakness in ambulant children with dmd . the most common daily dosage regimes are 0.75 mg / kg / day prednisone / prednisolone and 0.9 mg / kg / day deflazacort . other studies , using steroids with various combinations of daily , alternate - day , or cyclical prednisone treatment [ 13 , 4951 ] , have also demonstrated benefit in functional parameters . however , despite the fact that corticosteroids are routinely prescribed to dmd patients in most countries , there is no consensus on the optimal age to initiate treatment , optimal dose , and optimal dose schedule [ 6 , 15 ] . although most of the different schedules / dosages claim to be effective at improving muscle strength and function , none has been shown to be able to maintain this result with time . all long - term studies , independently of schedule / dosage , have shown that after a variable period of improvement , patients invariably lose muscle strength and function , although at a lower rate compared to patients that had not taken corticosteroids . the effect of corticosteroid treatment , at its best , is only able to slow the progressive course of the disease . this has been documented in a long - term study of alternate - day corticosteroids in five 2- to 4-year - old dmd patients [ 13 , 14 , 52 ] . four patients , aged 16 to 18 were fully ambulant , and 3 of them could still climb stairs . short stature and delayed puberty this and other studies [ 15 , 48 ] suggest that long - term corticosteroid treatment is effective in prolonging function but not in recovering lost function and , therefore , its early use seems appropriate . the exact mechanisms by which steroids slow the dystrophic process are still under investigation . various possibilities have been proposed based mainly on observations in mouse models of muscular dystrophy and a limited number of studies in patients . the effects of steroids in animal models include attenuating muscle fiber necrosis ; decreasing the entry of calcium into cells [ 55 , 56 ] ; regulating gene expression ; reducing cytotoxic t lymphocytes ; increasing laminin expression and myogenic repair ; decreasing muscle apoptosis and cellular infiltration ; protecting against mechanically induced fiber damage ( possibly by stabilizing the muscle fiber membranes ) ; increasing muscle levels of taurine and creatine ; reducing muscle degeneration and increasing survival ; alleviating myofiber pathology by activation of the calcineurin / nf - at pathway ; increasing the number of myoblasts ; enhancing the myogenesis of satellite cells ; and increasing dystrophin - related protein expression . in dmd muscle / cell , the effects of steroids include postrascriptionally mediated utrophin accumulation ; increasing muscle mass by inhibition of muscle proteolysis [ 68 , 69 ] ; enhancing dystrophin expression ; inhibiting myotube death during myogenesis ; and reducing the number of mononuclear inflammatory cells and dendritic cells . it is unlikely that the effect of prednisone results from its immunosuppressive action given that azathioprine decreases mononuclear subsets infiltrating muscle to a similar degree as does prednisone , although azathioprine - treated patients do not show a clinical improvement . it is important to support parents with adequate dietary counseling to limit weight gain , to cut down on high calorie foods , and to maintain a healthy diet . long - term daily use of steroids has an effect on a loss of final height at the end of growth . in several comparative studies , children who had used steroids were shorter by > 10 cm compared to children who had not undertaken this therapy . bone mineral density is one of the most important side effects related to the steroids ; however , it was also demonstrated that the bone density reduction occurs in dmd even before steroid use . cataract is another complication suffered by children with dmd , and , for this reason , ophthalmological examination is necessary to monitor for the development of cataracts and increased intraocular pressure . other common side effects include cushingoid appearance , hirsutism , acne , behavioral changes consisting of irritability and hyperactivity , osteonecrosis , and hypertension . the positive effect of corticosteroid treatment in the prevention / delay of the development of scoliosis deformity has been recognized by all long - term studies [ 11 , 7578 ] . of particular interest is the canadian deflazacort study involving 54 dmd boys who were followed for 15 years [ 12 , 75 , 7981 ] . fifty - four dmd boys aged 7 to 10 and able to walk were enrolled in a nonrandomized comparative study ; thirty patients were treated with deflazacort ( treatment group ) , and twenty - four were not ( control group ) . patients in the treatment group had a better pulmonary function ; they were able to walk longer and to climb stairs for a mean of 1.5 years longer compared to patients in the control group . at the last follow - up ( fifteen years ) , six ( 20% ) in the deflazacort group and twenty - two ( 92% ) in the control group developed scoliosis and underwent spinal surgery . reported a lower prevalence and an average milder scoliotic curve in patients treated with steroids . houde et al . , in another study of deflazacort use , report that scoliosis was much less severe in treated ( 14 2.5 ) than in untreated boys ( 46 24 ) . examining the orthopedic outcomes of long - term daily corticosteroid treatment in dmd , showed that treated boys had a significantly lower prevalence of scoliosis than the untreated group ( 31 versus 91% ) . the average scoliotic curve was also significantly milder in the treated group ( 11.6 ) compared with the untreated group ( 33.2 ) . moreover , in another recent cohort study that analyzed the effect of prednisone or deflazacort , boys who had received steroid therapy were significantly less likely to undergo spinal surgery . steroid therapy seems to be effective in preserving respiratory muscle strength in dmd , even if it remains uncertain how long this effect can be sustained over time . respiratory outcome studies of dmd patients treated with steroids showed improved values of % fvc . in a retrospective study including forty - nine dmd patients treated with corticosteroids for 7 years , balaban et al . showed that both deflazacort and prednisolone had a beneficial long - term effect on pulmonary function . long - term steroid therapy is also associated with improved peak cough flow and respiratory muscle strength in patients with dmd . a recent similar study also showed that cs can stabilize or delay the loss of lung function even in nonambulant patients or patients older than 10 years and in those treated after 7 years of age . the most promising therapies for dmd are gene therapy , exon skipping , and stop codon read - through , all aiming at restoring the expression of dystrophin . a drisapersen phase iii clinical trial ( nct01254019 ) , with 186 patients , aiming to induce skipping of exon 51 and de novo dystrophin production in patient muscle , failed to show significant improvement of the primary outcome measure , the 6-minute - walk test . eteplirsen , targeting skipping of exon 51 , showed variable levels of dystrophin restoration and stabilization of clinical outcome in a subset of patients in an open - label extension study . however , it remains to be seen whether eteplirsen can maintain a significant clinical benefit with time . the ataluren trial ( stop codon read - through ) with 174 patients showed a marginally significant improvement in the 6-minute - walk test compared to placebo . however , this drug has shown very little evidence of dystrophin restoration and the trial utilized a very subjective scoring method [ 87 , 88 ] . the failure of the only phase iii study of antisense oligonucleotide ( drisapersen ) performed so far has raised much discussion about the validity of dystrophin as a biomarker and the 6-minute - walk test as an outcome measure [ 8688 ] . it has also been suggested to optimize aspects of the clinical trial design , including younger age of treatment , because older boys have fewer myofibers left to rescue . first , the inclusion of patients on cs , both in the treatment and in the control groups , may be problematic . this setting supposes that if the active treatment has a positive effect , it will be added to the already known positive effect of cs . if this is not the case , we can miss the opportunity to use an effective treatment possibly with fewer side effects compared to cs . second , the choice of the clinical outcome measures should match the type of improvement that is expected . in a rapidly progressive disease like dmd muscle strength ( maximal isometric muscle force ) and muscle function ( 6-minute - walk test ) are not appropriate endpoints for any intervention that has an impact limited to slow progression . muscle strength and muscle function , in this situation , may have a transient improvement at some point in time ; however , they are expected to deteriorate with time , although at a lower rate compared to no treatment . some endpoints suitable to demonstrate slowing progression of the disease are ( 1 ) survival to death ; ( 2 ) survival to death or any respiratory intervention ; and ( 3 ) prolongation of independent walking . it is evident that all these endpoints require a long period of treatment particularly if treatment is started early in the course of the disease . in dmd patients , the development of spinal deformity has been dramatically changed by the progressive diffusion of cs treatment . there is now a general consensus that long - term cs therapy ( 1 ) prolongs ambulation , ( 2 ) reduces the need for spinal surgery , ( 3 ) reduces cardiopulmonary dysfunction , ( 4 ) delays the need for mechanical ventilation , and ( 5 ) increases survival and quality of life of dmd patients . . the goal of future dmd treatments should be to find a product at least as good as glucocorticoids with a lower side effect profile or with a significant glucocorticoid sparing effect . along this road , the new emerging and promising treatments are nonsense suppression therapies for boys with premature stop codon mutations and exon skipping by means of antisense oligonucleotides . there are plenty of lessons to be learnt from the recent failure of a phase iii exon skipping clinical trial , which should help to overcome the roadblock . first , there is the urgent need to standardize methods for dystrophin quantification and optimize several aspects of the clinical trial design . second , approvals of exon skipping and splice modulation as therapies for dmd require that a correlation be shown between dystrophin expression and clinical outcomes . but while restoration of dystrophin can be verified quickly , prolongation of walking , that is , the desirable clinical outcome , will take 10 or more years to be shown if treatments are started early . because of this unavoidable misalignment in time , accelerated approval should be based on surrogate biochemical evidence only ( de novo dystrophin demonstration in muscle ) . in addition placebo controlled trials will be unfeasible if a decade or more of blindness is needed to show slowing of disease progression in the treated group compared to placebo .

duchenne muscular dystrophy is a progressive disease with loss of ambulation at around 9 - 10 years of age , followed , if untreated , by development of scoliosis , respiratory insufficiency , and death in the second decade of life . this review highlights the natural history of the disease , in particular , with regard to the development of the spinal deformity and how this complication has been modified by surgical interventions and overall by corticosteroid treatment . the beneficial effect of corticosteroids may have also an impact on the clinical trial design of the new emerging causative therapies .

1. Introduction 2. Natural History 3. Spinal Deformity 4. Corticosteroids in DMD 5. New Emerging Therapies 6. Conclusions and Future Direction

duchenne muscular dystrophy ( dmd ) is a fatal neuromuscular disease , affecting 1 : 3500 live male births . it occurs as a result of mutations , mainly deletions , in the dystrophin gene . mutations lead to absence of or defect in the protein dystrophin from the muscle fibers causing cycles of muscle fiber degeneration and regeneration with replacement by fat and connective tissue . diagnosis is based on clinical examination observing the child run , jump , climb stairs , and get up from the floor ; blood test : serum creatine kinase ( ck ) levels up to 50100-fold above normal ; genetic testing : approximately 65% of patients with dmd have intragenic out - of - frame ( gross rearrangements ) deletions and approximately 10% have duplications of one or more exons of the dystrophin gene [ 2 , 3 ] ; and muscle biopsy : dystrophin analysis will always be abnormal and offers a further route to confirm the diagnosis . at the moment , there is no curative treatment for this devastating disease , and the main goal of interventions is to maintain ambulation as long as possible and to minimize the impact of the predictable complications of the disease , such as joint contractures , scoliosis , cardiomyopathy , and respiratory insufficiency . the objective of this review is to trace the natural history of the disease , in particular , with regard to the development of spinal deformity and how this complication has been modified by surgical interventions and overall by corticosteroid treatment . clinical evolution of muscular weakness in patients with duchenne muscular dystrophy is peculiarly marked by its progressive nature . as dmd boys appear healthy at birth , the natural history of untreated dmd leads to the development of an abnormal gait , calf hypertrophy , and difficulty rising from the floor when at 25 years of age . if not correctly diagnosed and treated , the boys become progressively unsteady in their walking , have a propensity to fall , use gower 's manoeuvre to stand up again , and acquire a waddling gait . gower 's manoeuvre is always present , with boys needing to turn onto their front and rise to standing from the floor using a broad - based stance , usually with the support of their hands on their thighs . common features of the disease are calves muscle hypertrophy and , frequently , developmental delay with delayed speech . respiratory failure is the major cause of death and occurs in the second or third decade of life ; it is caused by progressive respiratory muscle weakness and includes progressive restrictive ventilatory defects , chronic hypoventilation , and pulmonary infections . interventions designed to lessen the predictable complications of the disease have successfully changed its course that is now compatible with survival into adult life . the provision of noninvasive mechanical ventilation , assisted coughing , and cardioprotective medication allows survival into the late twenties and thirties . the natural history of the disease has also been significantly changed by the use of corticosteroids ( cs ) . efficacy has been established in improving muscle strength and timed functional tests over period of 618 months [ 9 , 10 ] . follow - up studies show long - term benefit with marked reduction in spinal deformity and prolonged ambulation . the clinical and laboratory diagnosis of dmd is now feasible much earlier than in the past and cs treatment can begin earlier in the course of the disease hopefully providing greater benefit than if treatment is delayed . a florid dystrophic process is already evident in the muscle biopsy of newborns with dmd [ 16 , 18 ] . dmd infants and young boys in the first 3 years of age have already measurable deficits in gross and fine motor function . in addition , motor function declines within the first 3 years of life compared to age - matched peers . a progressive scoliosis develops in over 90% of patients as a combined result of wheel chair dependence , paralysis of the extensor muscles , contractures , and growth spurt ( figure 1 ) . a severe collapsing scoliosis can interfere with breathing by reducing the lung function and can obstacle seating position , greatly decreasing quality of life of dmd patients . the spinal deformity differs from that seen in patients with idiopathic scoliosis and is described as a c - type curve ( a gentle , sweeping curve , with apex at the thoracolumbar junction ) . the spinal deformity results from a varying combination of curves in the coronal ( scoliosis ) and sagittal ( kyphosis , lordosis ) planes and excessive flexibility or stiffness of the spine due to the paralysis ( collapse ) or fibrous replacement ( rigid spine ) of the axial musculature . onset of scoliosis and loss of autonomous ambulation occur together in boys with dmd , generally between the ages of 10 and 14 years . there are different patterns of progression of scoliosis [ 22 , 23 ] . in the coronal plane , scoliosis can evolve in a linear and constant way when the child is wheelchair bound or alternate a period of slow progression to a rapid increase of the curve or have a rapid evolution over a few months too . in most of the cases , the final result is a kyphoscoliosis with collapsing spine or , less frequently , a hyperlordosis or a lordoscoliosis with rigid spine [ 22 , 24 ] . in addition to skeletal deformities on the sagittal and anterior - posterior plane , almost 100% of patients have a more or less distinct pelvic tilt ( 015 ) . the majority of patients complain about pain when sitting in a wheelchair because of the one - sided load on one buttock . when scoliosis is suspected , an x - ray of the entire spine ( ap and lateral view ) is mandatory . the various modalities of physiotherapy , including pool exercises , daily mobilization of contractures , and orthoses , like night splints or supportive sitting devices , have not shown a significant effect on progression of scoliosis . rehabilitation in lightweight knee - ankle - foot orthoses at the point of loss of ambulation , with or without tendon release , has been proven effective in preventing / reducing progression of scoliosis during the pubertal growth spurt [ 2528 ] . nonsurgical methods of spinal correction include the use of body jackets , custom - made seating inserts , and wheelchair modifications [ 29 , 30 ] . bracing is known to be ineffective to stop the progression of scoliosis in these children , as progression occurred in 94% despite bracing , and can be predicted to be 10 per year . spine surgery with posterior spinal fusion is the gold standard treatment for severe progressive scoliosis in dmd patients with the subsequent indications : documented curve progression , loss of seating balance , pain and/or discomfort . surgical treatment is mainly performed to restore the balance of the spinal column in both coronal and sagittal planes , to improve life quality of patients , facilitate nursing , and improve sitting balance and comfort . several instrumentation techniques have been applied for scoliosis correction in dmd patients ranging from halo casts with traction wires and buttons , harrington rods , and luque 's segmental spinal fixation to more recent techniques using pedicle screws and hooks . use of harrington instrumentation technique showed significant improvement in curve correction ( 60% on average ) , delay of curve progression , and shortening of constrained postoperative recumbence period . long - term studies have shown that this is a successful procedure with limited complications [ 24 , 31 , 37 ] . the most commonly described method for fusion to the pelvis is luque rod instrumentation with use of the galveston technique . the use of the galveston fixation with the placement of the pelvic portion of the rods between the tables of the ilia above the sciatic notch allows for correction of pelvic obliquity . long - term studies have shown that the luque - galveston system with spinal fusion is an efficient , safe , well - designed , easily adaptable , and reproducible technical method for the dmd patient with a moderate spinal curve needing correction . success rates are highest if surgery is performed early , when the spine is still mobile at a cobb angle of 2040 and the cardiac and respiratory function is , in part , preserved . the rate of complications is so reduced and if there is a pelvic obliquity , the fixation to the pelvis is always indicated . the search for a safe and resistant surgical technique has led to the advent of pedicle screws and hook system . overall , the described instrumentations appear to provide and maintain an optimal degree of correction at medium to long - term follow - up but the advantages of lowest blood loss and least surgical time without the need for pelvic fixation seem to swing the verdict in favor of the pedicle screw system . independently of the surgical technique , patients operated with a scoliotic curve less than 40 had better results , even in time , than patients operated with a more severe scoliotic curve ( 40 or more ) . patients with dmd develop a restrictive respiratory pattern with reduction of maximal respiratory pressures and forced vital capacity ( fvc ) that eventually causes respiratory insufficiency and death ( figure 2 ) . in these patients , lung function increases up to the age of 1012 years hitting its plateau ; then lung function decreases with an estimated loss of 10% per year of fvc . no correlation has been found between grade and progression of respiratory dysfunction and severity of scoliosis , because intrinsic respiratory muscle 's weakness is actually the main determinant of decline in respiratory function in dmd . , in a study on spinal surgery and lung function , reported no differences between surgical group and nonsurgical group in the rate of deterioration of % fvc which was 35% per year concluding that spinal stabilization in dmd does not alter the decline in pulmonary function nor does it improve survival . in addition , no difference was found in the rate of vital capacity decrease between operated or nonoperated patients and , in operated patients ' subgroup , between patients with scoliotic curve less than 40 and patients with more severe scoliotic patterns ( > 40 ) . in spite of these progresses , spinal stabilization in patients with dmd remains a demanding surgery because of dystrophic involvement of the paraspinal muscles and vascular smooth fibers , abnormal platelet function with a higher risk of intraoperatory bleeding requiring an adequate surgical technique , and a proper availability of blood units . intraoperative bleeding is the main risk factor ; however , several other complications are common in spinal surgery in dmd patients : intraoperative infection , implant failure , vertebral fracture , pullout of the screws , sacral decubitus , and death [ 46 , 47 ] . this surgery should be performed only by experienced surgeons and highly specialized centers ; moreover , it does not influence the natural course of respiratory decline . the first scientific evidence on the beneficial effect of steroids in duchenne muscular dystrophy was documented over 40 years ago by drachman et al . ; since then , several other studies have demonstrated the efficacy of corticosteroid therapy in delaying the loss of independence and autonomous ambulation and in maintaining an adequate pulmonary function . prednisone or deflazacort has been demonstrated to have a beneficial effect on muscle strength and function in boys with dmd and should be offered as treatment [ 15 , 48 ] . currently , corticosteroids are the gold standard treatment for muscle weakness in ambulant children with dmd . other studies , using steroids with various combinations of daily , alternate - day , or cyclical prednisone treatment [ 13 , 4951 ] , have also demonstrated benefit in functional parameters . however , despite the fact that corticosteroids are routinely prescribed to dmd patients in most countries , there is no consensus on the optimal age to initiate treatment , optimal dose , and optimal dose schedule [ 6 , 15 ] . although most of the different schedules / dosages claim to be effective at improving muscle strength and function , none has been shown to be able to maintain this result with time . the effect of corticosteroid treatment , at its best , is only able to slow the progressive course of the disease . this has been documented in a long - term study of alternate - day corticosteroids in five 2- to 4-year - old dmd patients [ 13 , 14 , 52 ] . four patients , aged 16 to 18 were fully ambulant , and 3 of them could still climb stairs . short stature and delayed puberty this and other studies [ 15 , 48 ] suggest that long - term corticosteroid treatment is effective in prolonging function but not in recovering lost function and , therefore , its early use seems appropriate . various possibilities have been proposed based mainly on observations in mouse models of muscular dystrophy and a limited number of studies in patients . the effects of steroids in animal models include attenuating muscle fiber necrosis ; decreasing the entry of calcium into cells [ 55 , 56 ] ; regulating gene expression ; reducing cytotoxic t lymphocytes ; increasing laminin expression and myogenic repair ; decreasing muscle apoptosis and cellular infiltration ; protecting against mechanically induced fiber damage ( possibly by stabilizing the muscle fiber membranes ) ; increasing muscle levels of taurine and creatine ; reducing muscle degeneration and increasing survival ; alleviating myofiber pathology by activation of the calcineurin / nf - at pathway ; increasing the number of myoblasts ; enhancing the myogenesis of satellite cells ; and increasing dystrophin - related protein expression . it is unlikely that the effect of prednisone results from its immunosuppressive action given that azathioprine decreases mononuclear subsets infiltrating muscle to a similar degree as does prednisone , although azathioprine - treated patients do not show a clinical improvement . it is important to support parents with adequate dietary counseling to limit weight gain , to cut down on high calorie foods , and to maintain a healthy diet . long - term daily use of steroids has an effect on a loss of final height at the end of growth . bone mineral density is one of the most important side effects related to the steroids ; however , it was also demonstrated that the bone density reduction occurs in dmd even before steroid use . cataract is another complication suffered by children with dmd , and , for this reason , ophthalmological examination is necessary to monitor for the development of cataracts and increased intraocular pressure . other common side effects include cushingoid appearance , hirsutism , acne , behavioral changes consisting of irritability and hyperactivity , osteonecrosis , and hypertension . the positive effect of corticosteroid treatment in the prevention / delay of the development of scoliosis deformity has been recognized by all long - term studies [ 11 , 7578 ] . of particular interest is the canadian deflazacort study involving 54 dmd boys who were followed for 15 years [ 12 , 75 , 7981 ] . fifty - four dmd boys aged 7 to 10 and able to walk were enrolled in a nonrandomized comparative study ; thirty patients were treated with deflazacort ( treatment group ) , and twenty - four were not ( control group ) . patients in the treatment group had a better pulmonary function ; they were able to walk longer and to climb stairs for a mean of 1.5 years longer compared to patients in the control group . at the last follow - up ( fifteen years ) , six ( 20% ) in the deflazacort group and twenty - two ( 92% ) in the control group developed scoliosis and underwent spinal surgery . , in another study of deflazacort use , report that scoliosis was much less severe in treated ( 14 2.5 ) than in untreated boys ( 46 24 ) . examining the orthopedic outcomes of long - term daily corticosteroid treatment in dmd , showed that treated boys had a significantly lower prevalence of scoliosis than the untreated group ( 31 versus 91% ) . the average scoliotic curve was also significantly milder in the treated group ( 11.6 ) compared with the untreated group ( 33.2 ) . moreover , in another recent cohort study that analyzed the effect of prednisone or deflazacort , boys who had received steroid therapy were significantly less likely to undergo spinal surgery . showed that both deflazacort and prednisolone had a beneficial long - term effect on pulmonary function . a recent similar study also showed that cs can stabilize or delay the loss of lung function even in nonambulant patients or patients older than 10 years and in those treated after 7 years of age . the most promising therapies for dmd are gene therapy , exon skipping , and stop codon read - through , all aiming at restoring the expression of dystrophin . a drisapersen phase iii clinical trial ( nct01254019 ) , with 186 patients , aiming to induce skipping of exon 51 and de novo dystrophin production in patient muscle , failed to show significant improvement of the primary outcome measure , the 6-minute - walk test . the ataluren trial ( stop codon read - through ) with 174 patients showed a marginally significant improvement in the 6-minute - walk test compared to placebo . the failure of the only phase iii study of antisense oligonucleotide ( drisapersen ) performed so far has raised much discussion about the validity of dystrophin as a biomarker and the 6-minute - walk test as an outcome measure [ 8688 ] . it has also been suggested to optimize aspects of the clinical trial design , including younger age of treatment , because older boys have fewer myofibers left to rescue . first , the inclusion of patients on cs , both in the treatment and in the control groups , may be problematic . this setting supposes that if the active treatment has a positive effect , it will be added to the already known positive effect of cs . second , the choice of the clinical outcome measures should match the type of improvement that is expected . in a rapidly progressive disease like dmd muscle strength ( maximal isometric muscle force ) and muscle function ( 6-minute - walk test ) are not appropriate endpoints for any intervention that has an impact limited to slow progression . muscle strength and muscle function , in this situation , may have a transient improvement at some point in time ; however , they are expected to deteriorate with time , although at a lower rate compared to no treatment . some endpoints suitable to demonstrate slowing progression of the disease are ( 1 ) survival to death ; ( 2 ) survival to death or any respiratory intervention ; and ( 3 ) prolongation of independent walking . it is evident that all these endpoints require a long period of treatment particularly if treatment is started early in the course of the disease . in dmd patients , the development of spinal deformity has been dramatically changed by the progressive diffusion of cs treatment . there is now a general consensus that long - term cs therapy ( 1 ) prolongs ambulation , ( 2 ) reduces the need for spinal surgery , ( 3 ) reduces cardiopulmonary dysfunction , ( 4 ) delays the need for mechanical ventilation , and ( 5 ) increases survival and quality of life of dmd patients . along this road , the new emerging and promising treatments are nonsense suppression therapies for boys with premature stop codon mutations and exon skipping by means of antisense oligonucleotides . there are plenty of lessons to be learnt from the recent failure of a phase iii exon skipping clinical trial , which should help to overcome the roadblock . first , there is the urgent need to standardize methods for dystrophin quantification and optimize several aspects of the clinical trial design . second , approvals of exon skipping and splice modulation as therapies for dmd require that a correlation be shown between dystrophin expression and clinical outcomes . in addition placebo controlled trials will be unfeasible if a decade or more of blindness is needed to show slowing of disease progression in the treated group compared to placebo .

modified nucleosides are relatively rare from marine organisms , but they have a long history . early pioneering work by bergmann on caribbean sponges of the genera tethya and cryptotethya resulted in the isolation of spongosine , spongothymidine , and spongouridine ( the first arabino - nucleosides ) and others . predating the modern era of marine natural products chemistry , these seminal discoveries were the inspiration for development of the clinically important antitumor drugs ara a and ara c. in our investigations of antifungal and antitumor compounds using nanomole - scale techniques , we examined several extracts of rare tunicates that displayed antifungal activity against a panel of candida spp . and cryptococcus spp . here , we report salvadenosine ( 1 ) , an uncommon 5-deoxy-5-(methylthio ) nucleoside from an encrusting deep - water tunicate didemnum sp . in addition , we revise the structure of the known compound , hamiguanosinol ( 2 ) , reported by proksch and co - workers from the pacific sponge hamigera hamigera . salvadenosine ( 1 ) joins the family of rare marine - derived nucleosides that include bergmann s arabino - nucleosides from cryptotethya sp . and the antiproliferative trachycladine a ( 3 ) from the sponge trachycladus laevispirulifer . structures of marine nucleosides ( 13 ) , the tautomers of guanosine ( 4a , 4b ) , and original and revised structures of hamiguanisinol ( 2 and 5 ) . the n - buoh soluble partition of the methanol extract of didemnum sp . was separated by reversed - phase hplc to give 1 in addition to tryptamine and the known natural product 6-bromotryptamine . the molecular formula of 1 , c11h15n5o4s , established from hrms ( esi - tof m / z 312.0777 [ m h ] ) , was isomeric with 2 ( table 1 ) . cross peaks arising from a modified ribose corresponded to the following contiguous spin system : anomeric proton h-1 ( h 5.87 , d , j = 4.9 hz ) to h-2 ( h 5.12 , t , j = 4.9 hz ) to h-3 ( h 4.42 , t , j = 4.9 hz ) to h-4 ( h 4.07 , ddd , j = 4.9 , 5.5 , 7.2 hz ) and the diastereotopic protons h-5a and h-5b of a methylene attached to s instead of o ( h 2.88 , dd , j = 14.0 , 5.5 hz ; 2.81 , dd , j = 14.0 , 7.2 hz ) . an hmbc correlation from the three - proton singlet ( h 2.10 , s , c 16.1 , jch = 138.4 hz ) to c-5 established the location of the mes group . distinct differences were apparent between the h and c chemical shifts of 1 and those published for hamiguanosinol ( 2 , table 2 ) , particularly the spc nmr signals . an hsqc correlation from the sole downfield nonexchangeable aromatic signal ( h 8.00 , c 150.8 , jch = 203.4 hz ) revealed a c chemical shift that was deshielded ( 15.8 ppm ) compared to that of the h-8 of 2 ( h 7.90 , c 135.0 ) . another notable difference was observed for the most shielded spc signal of 1 ( c 103.6 , s ) and 2 ( c 118.0 , s , c-8 ; 14.4 ppm ) . hmbc correlations of 1 were observed from the anomeric proton h-1 signal , and spc signals were also inconsistent with guanine ; for example , no correlation was observed between the downfield h nmr signal ( h 8.08 , s , 1h ) and the anomeric carbon , c-1 , a correlation common to guanosine nucleosides . the hsqc ( dmso - d6 ) showed the aforementioned downfield proton was attached to a carbon with a c signal ( c 151.7 , s ) more compatible with an imidazolone c = o group than the imidazole c-8 chemical shift of 2 ( c 135.0 , s ) . a better match for the nmr data of 1 was obtained by replacement of guanine with 8-oxoadenine , a nucleobase isomeric with the former . the downfield h nmr signal ( h 8.08 , s , 1h ) was assigned to h-2 , and all inconsistencies were resolved . for example , the expected long - range correlation between c-1 and h-8 for a guanine ring system but missing in 2is replaced by j correlations of h-2 to c-4 and c-5 in an 8-oxoadenine , respectively . the c chemical shifts ( dmso - d6 , table 2 ) of the quaternary ring junction carbons c-4 and c-5 ( c 146.7 , s ; 103.6 , s ) of 1 are more polarized ( [ c-4 c-5 ] = 43.1 ppm ) than the corresponding signals of 2 ( = 35 ppm ) , guanosine , or adenosine but closer in magnitude than the 8-oxoadenosines , aplidiamine , erinacean , and caissarone . additionally , we measured the heteronuclear coupling constants of the downfield sph nmr singlets in 1 and several purine nucleosides ( table 3 ) , revealing a better match between 8-oxoadenosine and the natural product ( 1 : h-2 , jch = 203.4 hz ; 8-oxoadenosine : h-2 jch = 201.5 hz ; guanosine : h-8 jch = 213.5 hz ) . the natural products salvadenosine ( 1 ) and hamiguansinol are clearly not identical , but isomeric ( figure 1 ) . naturally , the question of validity of the structure of 2 arises . enol form of guanosine 4a , figure 1 ) based on a c shift for c-6 of c 157.2 instead of a ca.170 ppm for a keto amide function ; however , we find this less convincing for three reasons . keto groups in purines ( guanine , inosine , etc . ) are not electronically equivalent to simple amides and often exhibit relatively high field chemical shifts in the range observed for 3 ; the c-6 c chemical shift reported for hamiguanosinol is not incompatible with the 6-keto tautomer 4b . numerous studies have shown the keto form of guanosine ( 4b ) and deoxyguanosine are the naturally stable tautomers in protic solvents : in fact , watson given that electronic differences between guanosine and 2 are insignificant , there are no compelling reasons to expect that 2 would be locked in the enol form and unable to spontaneously tautomerize to the keto form in protic solvent . lastly , compound 5 has been synthesized by van tilburg and co - workers who characterized it as the keto tautomer . we repeated the synthesis of 5 ( scheme 1 ) from guanosine ( 4b ) and showed the c chemical shifts of the product were essentially the same as those reported for natural hamiguanosinol ( supporting information , table s2 ) . therefore , the structure of hamiguanosinol is the keto tautomer 5 , not the enol 2 : synthetic 5 and hamiguanosinol are identical . in order to verify the structure of salvadenosine ( 1 ) , the natural product was synthesized by extension of the sequence of reactions used to prepare 5 . n , o - protected adenosine was subjected to bromination ( scheme 2 , saturated br2h2o , ph 4 naoac buffer ) , but only the cyclized product 7 was formed presumably through intramolecular attack by the 5-oh group after bromination at c-8 . the same result was observed under aprotic conditions ( nbs , dmf ; 5,5-dimethyl-1,3- dibromohydantoin , dbh , dmf ) ; consequently , we turned to a protecting - group free strategy ( scheme 2 ) . adenosine ( 8) was converted to 8-bromoadenosine ( 9 ) ( saturated br2h2o , ph 4 buffer ) followed by an efficient conversion to the 8-oxoadenosine ( 10 ) under chatgilialoglus conditions . compound 10 was converted to primary chloride 11 with improved yield ( socl2 , dmpu , 70% ) ; the latter , in turn , was subjected to nucleophilic substitution with sodium thiomethoxide to yield 1 in four steps and 17% yield from adenosine . since the sample of natural 1 was purified as the formate salt , synthetic 1 the h and c nmr spectra ( supporting information , table s3 ) as well as uv , ir , and cd data of the two samples 1hco2h matched in every way . co - injection of natural 1 with synthetic 1 by hplc resulted in a single peak ( see the supporting information , figure s1 ) . the nature of the 5-(methylthio ) group in 1 and 5 deserves some comment . one plausible origin of 1 is from s - adenosyl methionine ( sam ) . the two ch2 groups and one ch3 group bonded to the s in sam are electrophilic in nature . common biological methylation involves sn2 nucleophilic substitution of the electrophilic me - s bond through attack by c- , n- , o- , s - centered nucleophiles , generating s - adenosylhomocysteine : the latter is cycled back to adenosine and homocysteine . in the biosynthesis of both the chlorinated antitumor drug salinosporamide a and rare fluorinated natural products , substantial biochemical and structural evidence supports participation of the 5-ch2s bond of sam in sn2 substitutions by halide ions ( figure 2 ) . for example , streptomyces cattleya produces 5-fluoroadenosine through nucleophilic attack at the 5-ch2 of sam by f ( path a ) , catalyzed by the enzyme fluorinase ; the former in turn is catabolized to fluoroacetate . a homologous chlorinase catalyzes displacement at 5-ch2 of sam by cl ( path a ) in salinosporamide a biosynthesis . the gene duf62 , represented in nature in about 100 bacterial and archael genomes , expresses a protein , duf62 , that has high structural homology to the halogenases . lacks halogenase activity , but carries out hydrolysis of sam by nucleophilic attack at the 5-ch2 group of adenosine group by ho ( path b ) to liberate l - methionine and adenosine . attack at the 5-ch2 of sam is similarly rare , with the best - characterized example occurring in the biosynthesis of nocardicin . transfer of the 3-amino-3-carboxypropyl group from sam has also been proposed in the biosynthesis of modified bases for bacterial and yeast trnaphe , the natural product discadenine , nicotianamine ( a precursor of plant siderophores ) , and homoserine - based betaine lipids . in addition , transfer of the 3-aminopropyl group from decarboxylated sam is involved in polyamine biosynthesis . common to these biological reactions is the release of 5-deoxy-5-(methylthio)adenosine . hypothetical biosynthetic origin of 1 from s - adenosylmethionine ( sam ) . we speculate that 1 may arise through displacement of 5-deoxy-5-(methylthio)adenosine from sam ( path b ) , possibly via a luxi - type mechanism with release of homoseryl lactone . the product may then be converted to 1 by electrophilic or free - radical attack at c-8 with reactive oxygen species ( ros ) , a reaction known from purine metabolism and the biology of dna damage . the anomaly is 5 ; no guanosine analogue of sam has been demonstrated yet , but 5 has been detected in human urine as a byproduct of nucleotide catabolism . in conclusion , we confirmed the structure of a new nucleoside salvadenosine ( 1 , 5-deoxy-5-(methylthio)-8-oxoadenosine ) from the tunicate didemnum sp . through integrated analysis of spectroscopic data and total synthesis . re - evaluation of the published structure of hamiguanosinol and its synthesis from guanosine requires revision of the structure of hamiguanosinol from the enol tautomer 2 to the keto form 5 . inverse detected 2d nmr spectra were measured on a 500 mhz spectrometer equipped with a 5 mm h{c } 5 mm probe or a 600 mhz nmr spectrometer with a 1.7 mm h{c } microcryoprobe . c nmr spectra were collected on a 125 mhz spectrometer equipped with a 5 mm c{h } cryoprobe . nmr spectra were referenced to residual solvent signals , cd3od ( h 3.31 , c 49.00 ) , ( cd3)2so ( h 2.50 , c 39.52 ) . low - resolution ms measurements were made on a uhplc coupled to an msd single - quadrupole detector . automated medium - pressure chromatography was carried out using a 30 g c18 cartridges under specified gradient elution conditions . cd spectra were measured in quartz cells ( 1 or 2 mm path length ) . other solvents were dried by passage through activate alumina or molecular sieves under ar . ( 11 - 25 - 039 and 11 - 14 - 018 ) were collected in july 2011 off little san salvador island , bahamas , at a depths of 28 and 32 m. voucher samples of the tunicates ( 11 - 25 - 039 and 11 - 14 - 018 ) are archived at uc san diego . the meoh extract ( 5 ml ) of didemnum sp . ( 1114018 , 1.21 g dry ) was separated by progressive solvent partition . the meoh extract was adjusted to approximately 1:9 h2o / meoh and was extracted repeatedly with hexane ( 5 ml 3 ) to yield fraction a ( 7.7 mg ) . the aqueous layer was then adjusted to 2:3 h2o / meoh followed by extraction with ch2cl2 ( 5 ml 3 ) to yield fraction b ( 7.2 mg ) . the aqueous layer was adjusted to approximately 9:1 h2o / meoh before the solution was extracted with n - buoh ( 5 ml 3 ) to yield fraction c ( 18.5 mg ) . removal of the volatiles from the remaining aqueous phase provided fraction d ( 202 mg ) . fraction c was further purified by reversed - phase hplc ( phenylhexyl column , 10 250 mm , linear gradient ( initial conditions 90:10 h2o ( 0.1% hco2h)ch3cn to 50:50 over 20 min ) to yield 5-deoxy-5-(methylthio)-8-oxoadenosine ( 1hco2h , 0.8 mg , tr = 10.2 min ) , 6-bromotryptamine ( 2.2 mg , tr = 12.6 min ) , and tryptamine ( 0.43 mg , tr = 9.4 min ) . fraction b was purified under identical conditions to obtain additional 1 ( 0.46 mg ) and 6-bromotryptamine ( 0.34 mg ) . the combined samples of 1 were repurified by hplc over the same column ( 90:10 h2o meoh , 0.5% hco2h ) to yield 1hco2h ( 0.42 mg , calculated through nmr quantification of c satellite peaks ) . extraction of sample 11 - 14 - 039 and purification was carried out in a similar manner to provide additional 1hco2h ( 0.3 mg ) . pale yellow powder , hco2 salt ; [ ]d + 11.3 ( c 0.3 , meoh ) ; uv ( meoh ) max 210 nm ( log10 4.49 ) , 255 ( 3.95 ) , 270 ( 4.00 ) ; ftir ( atr , znse plate ) : 3356 , 3188 , 2920 , 2850 , 1710 , 1662 , 1633 , 1590 , 1379 , 1350 , 1131 , 1092 , 1030 , 1006 cm ; h and c nmr see table 1 ( cd3od ) and supporting information , table s1 ( dmso - d6 ) ; esi - tof m / z 312.0777 [ m h ] ( calcd for c11h14n5o4s 312.0767 ) . colorless powder ; h , c , and hrms data were consistent with previously published data . colorless powder ; ms and h nmr spectra were identical to those of an authentic sample . samples of natural and synthetic 1 ( see below ) were prepared in meoh as equimolar solutions ( 0.025 mg / ml ) , and aliquots of each solution , along with an admixture of both ( equivolume ) , were analyzed by hplc ( polar - rp column , 4 m , 80 , 150 4.6 mm , gradient elution ; ( h2o + 0.1% hco2h / ch3cn , 05 min hold at 5% ch3cn , 518 min ramp to 50% ch3cn , 1 ml / min , 40 c column oven ) . the following retention times were obtained : natural 1 , tr = 10.48 min ; synthetic 1tr = 10.47 min ; combined natural and synthetic samples , tr = 10.48 min , single peak . guanosine ( 0.60 g , 2.12 mmol ) was suspended in dry dmpu ( 10.6 ml ) and dissolved upon heating . after the mixture was cooled in an ice bath , thionyl chloride ( 770 l , 10.6 mmol , 5 equiv ) was slowly added with stirring and the mixture was warmed to 23 c over 2 h. the mixture was cooled in an ice bath , diluted with cold h2o ( 10 ml ) , and absorbed onto a column of dowex 50 2400 resin ( 200400 mesh , h form ) . the column was washed with water and the compound eluted with 5% aqueous ammonia . the volatiles were removed from the eluate under reduced pressure to yield 6 ( 478 mg , 1.58 mmol , 75% yield ) . 5-chloro-5-deoxyguanosine ( 0.10 g , 0.33 mmol ) was dissolved in dry dmf with sodium thiomethoxide ( 232 mg , 3.3 mmol , 10 equiv ) at 23 c . to this , naome in dmf ( 1.8 mg , 33 mol , 0.1 equiv ) was added and the mixture stirred for 3 h. the reaction was neutralized with hcl ( 1 m ) and extracted with ether three times . the aqueous layer was dried , and the residue was preabsorbed on c18 stationary phase for solid - phase loading and purified by automated medium - pressure chromatography ( gradient elution : 0.1% hco2h / meoh / h2o , 1050% over 20 min ) . the volatiles were evaporated under reduced pressure , and the aqueous residue was lyophilized to yield 5 ( 60 mg , 0.19 mmol , 56% yield ) as a fluffy , white powder . a solution of n-(tert - butoxycarbonyl)-2,3-o - isopropylideneadenosine ( 1.30 g , 3.19 mmol ) in meoh ( 25 ml ) and naoac buffer ( 25 ml , 0.5 m , ph 4 ) was treated by slow addition of saturated br2water ( 32.5 ml ) and the resulting mixture stirred at room temperature for 48 h. the mixture was decolorized by addition of nahso3 ( 5 m ) and adjusted to ph 7 with naoh aqueous ( 2 m ) to give a precipitate which was filtered , washed with water , and dried under reduced pressure . the residue was preabsorbed onto c18 stationary phase for solid - phase loading and purified by automated medium - pressure chromatography ( gradient elution , 1080% 0.1% hco2h / meoh / h2o over 20 min ) . the volatiles were evaporated under reduced pressure and the aqueous phase was lyophilized to yield 7 ( 290 mg , 0.72 mmol , 23% yield ) as an off - white powder : [ ]d 33.9 ( c 0.1 , meoh ) ; uv ( meoh ) max 266 nm ( , log10 4.24 ) , 210 ( 4.42 ) ; ftir ( atr , znse plate ) 3336 , 3187 , 2990 , 2932 , 2363 , 2337 , 1710 , 1645 , 1606 , 1462 , 1377 , 1292 , 1084 , 849 cm ; h nmr ( cd3od ) h 8.51 ( s , 1h ) , 6.33 ( s , 1h ) , 5.18 ( d , 1h , j = 5.7 hz ) , 4.87 ( d , 1h , j = 5.7 hz ) , 4.77 ( d , 1h , j = 1.0 hz ) , 4.70 ( dd , 1h , j = 2.1 , 13.0 hz ) , 4.30 ( d , 1h , j = 13.0 hz ) , 1.57 ( s , 9h ) , 1.53 ( s , 3h ) , 1.36 ( s , 3h ) ; c nmr ( cd3od ) c 157.3 , 152.6 , 152.4 , 151.2 , 149.4 , 120.0 , 114.1 , 88.3 , 87.5 , 86.7 , 82.6 , 82.6 , 76.4 , 28.5 , 26.4 , 24.7 ; esi - tof m / z 406.1723 [ m + h ] ( calcd for c18h24n5o6 406.1721 ) . attempted bromination of n-(tert - butoxycarbonyl)-2,3-o - isopropylideneadenosine ( 18.0 mg , 0.058 mmol ) in dmf ( 300 l ) with 5,5-dimethyl-1,3- dibromohydantoin ( dbh , 25.3 mg , 0.089 mmol , 1.5 equiv ) or nbs ( 16.2 mg , 0.089 mmol , 1.5 equiv ) resulted in rapid loss of starting material ( 30 min , tlc ) , no detection of starting material , and only 7 as the product ( lcms ) . kohyama s protocol was used to yield 9 ( 1.04 g , 3.01 mmol , 85% yield ) . h nmr , c nmr , and ms data of 9 were identical to literature values . the title compound was prepared according to a published method and purified by automated flash chromatography ( 0.1% hco2h / meoh / h2o , gradient elution 1050% over 25 min ) to yield 10 ( 428 mg , 1.51 mmol , 70% yield ) . h nmr , c nmr , and ms data of 10 were identical with literature values . 8-oxoadenosine ( 0.32 g , 1.1 mmol ) was dissolved in dry dmpu ( 5.7 ml ) . the solution was cooled in an ice bath before slow addition of socl2 ( 400 l , 5.51 mmol , 5 equiv ) . the reaction was warmed to 23 c , stirred for 2 h , cooled in an ice bath , diluted with cold water ( 10 ml ) , absorbed on dowex 50 2400 resin ( 200400 mesh , h form ) , washed with water , and eluted with 5% aqueous ammonia . the eluant was dried under reduced pressure and lyophilized to yield 11 ( 136 mg , 0.45 mmol , 70% yield ) as an off - white solid . h , c , and ms data were consistent with literature data . following the protocol described in the synthesis of 5 , compound 11 ( 0.10 g , 0.33 mmol ) and sodium thiomethoxide ( 232 mg , 3.3 mmol , 10 equiv ) were dissolved in dry dmf at 23 c and treated with naome in dmf ( 1.8 mg , 0.033 mmol , 0.1 equiv ) . the mixture was stirred for 3 h and then neutralized with hcl ( 1 m ) and extracted with diethyl ether ( 3 ) . the aqueous layer was concentrated under reduced pressure and the residue triturated with hot , anhydrous ch3cn to yield , after removal of solvent , 1 free base ( 42 mg , 0.13 mmol , 41% yield ) as a colorless powder : [ ]d 147.0 ( c 0.1 , meoh ) ; uv ( meoh ) max 210 nm ( log10 4.45 ) , 255 ( 3.89 ) , 270 ( 3.96 ) ; uv ( meoh , naoh , ph 12 ) max 210 nm ( log10 4.43 ) , 277 ( 4.03 ) ; ftir ( atr , znse plate ) 3390 , 3141 , 2920 , 1696 , 1628 , 1566 , 1355 , 1126 , 1030 , 1011 cm ; h nmr ( cd3od ) h 8.06 ( s , 1h ) , 5.88 ( d , 1h , j = 4.9 hz ) , 5.13 ( t , 1h , j = 4.9 hz ) , 4.42 ( t , 1h , j = 4.9 hz ) , 4.07 ( ddd , 1h , j = 7.2 , 5.5 , 0.9 hz ) , 2.88 ( dd , 1h , j = 14.0 , 5.5 hz ) , 2.80 ( dd , 1h , j = 14.0 , 7.2 hz ) , 2.10 ( s , 3h ) ; c nmr ( cd3od ) c 154.5 , 152.1 , 148.8 , 148.8 , 105.9 , 87.9 , 85.0 , 74.5 , 72.0 , 37.4 , 16.1 ; esi - tof m / z 312.0773 [ m h ] ( calcd for c11h14n5o4s 312.0767 ) . free base 1 ( 10.88 mg ) was dissolved in cd3od ( 500 l ) , hco2h ( 90% aq , 1 equiv ) was added , and the solution was dried under nitrogen to give 1hco2h : white powder ; [ ]d + 20.6 ( c 0.1 , meoh ) ; ftir ( atr , znse plate ) 3343 , 3212 , 1710 , 1658 , 1592 , 1475 , 1429 , 1364 , 1129 , 1096 , 1037 cm ; uv vis was identical to free base 1 ; h and c nmr , see the supporting information , table s3 .

salvadenosine , ( 1 ) a rare 5-deoxy-5-(methylthio ) nucleoside , was isolated from the deep - water bahaman tunicate didemnum sp . the structure was solved by integrated analysis of ms and 1d and 2d nmr data . we revise the structure of the known natural product , hamiguanosinol , which is a constitutional isomer of 1 , to 5 by interpretation of the spectroscopic data and comparison with synthesized nucleosides .

Introduction Results Discussion Experimental Section

modified nucleosides are relatively rare from marine organisms , but they have a long history . early pioneering work by bergmann on caribbean sponges of the genera tethya and cryptotethya resulted in the isolation of spongosine , spongothymidine , and spongouridine ( the first arabino - nucleosides ) and others . predating the modern era of marine natural products chemistry , these seminal discoveries were the inspiration for development of the clinically important antitumor drugs ara a and ara c. in our investigations of antifungal and antitumor compounds using nanomole - scale techniques , we examined several extracts of rare tunicates that displayed antifungal activity against a panel of candida spp . here , we report salvadenosine ( 1 ) , an uncommon 5-deoxy-5-(methylthio ) nucleoside from an encrusting deep - water tunicate didemnum sp . in addition , we revise the structure of the known compound , hamiguanosinol ( 2 ) , reported by proksch and co - workers from the pacific sponge hamigera hamigera . salvadenosine ( 1 ) joins the family of rare marine - derived nucleosides that include bergmann s arabino - nucleosides from cryptotethya sp . and the antiproliferative trachycladine a ( 3 ) from the sponge trachycladus laevispirulifer . the n - buoh soluble partition of the methanol extract of didemnum sp . was separated by reversed - phase hplc to give 1 in addition to tryptamine and the known natural product 6-bromotryptamine . the molecular formula of 1 , c11h15n5o4s , established from hrms ( esi - tof m / z 312.0777 [ m h ] ) , was isomeric with 2 ( table 1 ) . cross peaks arising from a modified ribose corresponded to the following contiguous spin system : anomeric proton h-1 ( h 5.87 , d , j = 4.9 hz ) to h-2 ( h 5.12 , t , j = 4.9 hz ) to h-3 ( h 4.42 , t , j = 4.9 hz ) to h-4 ( h 4.07 , ddd , j = 4.9 , 5.5 , 7.2 hz ) and the diastereotopic protons h-5a and h-5b of a methylene attached to s instead of o ( h 2.88 , dd , j = 14.0 , 5.5 hz ; 2.81 , dd , j = 14.0 , 7.2 hz ) . an hmbc correlation from the three - proton singlet ( h 2.10 , s , c 16.1 , jch = 138.4 hz ) to c-5 established the location of the mes group . distinct differences were apparent between the h and c chemical shifts of 1 and those published for hamiguanosinol ( 2 , table 2 ) , particularly the spc nmr signals . an hsqc correlation from the sole downfield nonexchangeable aromatic signal ( h 8.00 , c 150.8 , jch = 203.4 hz ) revealed a c chemical shift that was deshielded ( 15.8 ppm ) compared to that of the h-8 of 2 ( h 7.90 , c 135.0 ) . another notable difference was observed for the most shielded spc signal of 1 ( c 103.6 , s ) and 2 ( c 118.0 , s , c-8 ; 14.4 ppm ) . hmbc correlations of 1 were observed from the anomeric proton h-1 signal , and spc signals were also inconsistent with guanine ; for example , no correlation was observed between the downfield h nmr signal ( h 8.08 , s , 1h ) and the anomeric carbon , c-1 , a correlation common to guanosine nucleosides . a better match for the nmr data of 1 was obtained by replacement of guanine with 8-oxoadenine , a nucleobase isomeric with the former . the downfield h nmr signal ( h 8.08 , s , 1h ) was assigned to h-2 , and all inconsistencies were resolved . for example , the expected long - range correlation between c-1 and h-8 for a guanine ring system but missing in 2is replaced by j correlations of h-2 to c-4 and c-5 in an 8-oxoadenine , respectively . the c chemical shifts ( dmso - d6 , table 2 ) of the quaternary ring junction carbons c-4 and c-5 ( c 146.7 , s ; 103.6 , s ) of 1 are more polarized ( [ c-4 c-5 ] = 43.1 ppm ) than the corresponding signals of 2 ( = 35 ppm ) , guanosine , or adenosine but closer in magnitude than the 8-oxoadenosines , aplidiamine , erinacean , and caissarone . additionally , we measured the heteronuclear coupling constants of the downfield sph nmr singlets in 1 and several purine nucleosides ( table 3 ) , revealing a better match between 8-oxoadenosine and the natural product ( 1 : h-2 , jch = 203.4 hz ; 8-oxoadenosine : h-2 jch = 201.5 hz ; guanosine : h-8 jch = 213.5 hz ) . the natural products salvadenosine ( 1 ) and hamiguansinol are clearly not identical , but isomeric ( figure 1 ) . naturally , the question of validity of the structure of 2 arises . enol form of guanosine 4a , figure 1 ) based on a c shift for c-6 of c 157.2 instead of a ca.170 ppm for a keto amide function ; however , we find this less convincing for three reasons . we repeated the synthesis of 5 ( scheme 1 ) from guanosine ( 4b ) and showed the c chemical shifts of the product were essentially the same as those reported for natural hamiguanosinol ( supporting information , table s2 ) . therefore , the structure of hamiguanosinol is the keto tautomer 5 , not the enol 2 : synthetic 5 and hamiguanosinol are identical . in order to verify the structure of salvadenosine ( 1 ) , the natural product was synthesized by extension of the sequence of reactions used to prepare 5 . compound 10 was converted to primary chloride 11 with improved yield ( socl2 , dmpu , 70% ) ; the latter , in turn , was subjected to nucleophilic substitution with sodium thiomethoxide to yield 1 in four steps and 17% yield from adenosine . since the sample of natural 1 was purified as the formate salt , synthetic 1 the h and c nmr spectra ( supporting information , table s3 ) as well as uv , ir , and cd data of the two samples 1hco2h matched in every way . the nature of the 5-(methylthio ) group in 1 and 5 deserves some comment . one plausible origin of 1 is from s - adenosyl methionine ( sam ) . common biological methylation involves sn2 nucleophilic substitution of the electrophilic me - s bond through attack by c- , n- , o- , s - centered nucleophiles , generating s - adenosylhomocysteine : the latter is cycled back to adenosine and homocysteine . in the biosynthesis of both the chlorinated antitumor drug salinosporamide a and rare fluorinated natural products , substantial biochemical and structural evidence supports participation of the 5-ch2s bond of sam in sn2 substitutions by halide ions ( figure 2 ) . for example , streptomyces cattleya produces 5-fluoroadenosine through nucleophilic attack at the 5-ch2 of sam by f ( path a ) , catalyzed by the enzyme fluorinase ; the former in turn is catabolized to fluoroacetate . transfer of the 3-amino-3-carboxypropyl group from sam has also been proposed in the biosynthesis of modified bases for bacterial and yeast trnaphe , the natural product discadenine , nicotianamine ( a precursor of plant siderophores ) , and homoserine - based betaine lipids . in addition , transfer of the 3-aminopropyl group from decarboxylated sam is involved in polyamine biosynthesis . hypothetical biosynthetic origin of 1 from s - adenosylmethionine ( sam ) . in conclusion , we confirmed the structure of a new nucleoside salvadenosine ( 1 , 5-deoxy-5-(methylthio)-8-oxoadenosine ) from the tunicate didemnum sp . through integrated analysis of spectroscopic data and total synthesis . re - evaluation of the published structure of hamiguanosinol and its synthesis from guanosine requires revision of the structure of hamiguanosinol from the enol tautomer 2 to the keto form 5 . inverse detected 2d nmr spectra were measured on a 500 mhz spectrometer equipped with a 5 mm h{c } 5 mm probe or a 600 mhz nmr spectrometer with a 1.7 mm h{c } microcryoprobe . nmr spectra were referenced to residual solvent signals , cd3od ( h 3.31 , c 49.00 ) , ( cd3)2so ( h 2.50 , c 39.52 ) . cd spectra were measured in quartz cells ( 1 or 2 mm path length ) . ( 11 - 25 - 039 and 11 - 14 - 018 ) were collected in july 2011 off little san salvador island , bahamas , at a depths of 28 and 32 m. voucher samples of the tunicates ( 11 - 25 - 039 and 11 - 14 - 018 ) are archived at uc san diego . the meoh extract ( 5 ml ) of didemnum sp . the meoh extract was adjusted to approximately 1:9 h2o / meoh and was extracted repeatedly with hexane ( 5 ml 3 ) to yield fraction a ( 7.7 mg ) . removal of the volatiles from the remaining aqueous phase provided fraction d ( 202 mg ) . the combined samples of 1 were repurified by hplc over the same column ( 90:10 h2o meoh , 0.5% hco2h ) to yield 1hco2h ( 0.42 mg , calculated through nmr quantification of c satellite peaks ) . colorless powder ; ms and h nmr spectra were identical to those of an authentic sample . the following retention times were obtained : natural 1 , tr = 10.48 min ; synthetic 1tr = 10.47 min ; combined natural and synthetic samples , tr = 10.48 min , single peak . the column was washed with water and the compound eluted with 5% aqueous ammonia . the volatiles were removed from the eluate under reduced pressure to yield 6 ( 478 mg , 1.58 mmol , 75% yield ) . to this , naome in dmf ( 1.8 mg , 33 mol , 0.1 equiv ) was added and the mixture stirred for 3 h. the reaction was neutralized with hcl ( 1 m ) and extracted with ether three times . a solution of n-(tert - butoxycarbonyl)-2,3-o - isopropylideneadenosine ( 1.30 g , 3.19 mmol ) in meoh ( 25 ml ) and naoac buffer ( 25 ml , 0.5 m , ph 4 ) was treated by slow addition of saturated br2water ( 32.5 ml ) and the resulting mixture stirred at room temperature for 48 h. the mixture was decolorized by addition of nahso3 ( 5 m ) and adjusted to ph 7 with naoh aqueous ( 2 m ) to give a precipitate which was filtered , washed with water , and dried under reduced pressure . the residue was preabsorbed onto c18 stationary phase for solid - phase loading and purified by automated medium - pressure chromatography ( gradient elution , 1080% 0.1% hco2h / meoh / h2o over 20 min ) . the volatiles were evaporated under reduced pressure and the aqueous phase was lyophilized to yield 7 ( 290 mg , 0.72 mmol , 23% yield ) as an off - white powder : [ ]d 33.9 ( c 0.1 , meoh ) ; uv ( meoh ) max 266 nm ( , log10 4.24 ) , 210 ( 4.42 ) ; ftir ( atr , znse plate ) 3336 , 3187 , 2990 , 2932 , 2363 , 2337 , 1710 , 1645 , 1606 , 1462 , 1377 , 1292 , 1084 , 849 cm ; h nmr ( cd3od ) h 8.51 ( s , 1h ) , 6.33 ( s , 1h ) , 5.18 ( d , 1h , j = 5.7 hz ) , 4.87 ( d , 1h , j = 5.7 hz ) , 4.77 ( d , 1h , j = 1.0 hz ) , 4.70 ( dd , 1h , j = 2.1 , 13.0 hz ) , 4.30 ( d , 1h , j = 13.0 hz ) , 1.57 ( s , 9h ) , 1.53 ( s , 3h ) , 1.36 ( s , 3h ) ; c nmr ( cd3od ) c 157.3 , 152.6 , 152.4 , 151.2 , 149.4 , 120.0 , 114.1 , 88.3 , 87.5 , 86.7 , 82.6 , 82.6 , 76.4 , 28.5 , 26.4 , 24.7 ; esi - tof m / z 406.1723 [ m + h ] ( calcd for c18h24n5o6 406.1721 ) . kohyama s protocol was used to yield 9 ( 1.04 g , 3.01 mmol , 85% yield ) . following the protocol described in the synthesis of 5 , compound 11 ( 0.10 g , 0.33 mmol ) and sodium thiomethoxide ( 232 mg , 3.3 mmol , 10 equiv ) were dissolved in dry dmf at 23 c and treated with naome in dmf ( 1.8 mg , 0.033 mmol , 0.1 equiv ) . the mixture was stirred for 3 h and then neutralized with hcl ( 1 m ) and extracted with diethyl ether ( 3 ) . the aqueous layer was concentrated under reduced pressure and the residue triturated with hot , anhydrous ch3cn to yield , after removal of solvent , 1 free base ( 42 mg , 0.13 mmol , 41% yield ) as a colorless powder : [ ]d 147.0 ( c 0.1 , meoh ) ; uv ( meoh ) max 210 nm ( log10 4.45 ) , 255 ( 3.89 ) , 270 ( 3.96 ) ; uv ( meoh , naoh , ph 12 ) max 210 nm ( log10 4.43 ) , 277 ( 4.03 ) ; ftir ( atr , znse plate ) 3390 , 3141 , 2920 , 1696 , 1628 , 1566 , 1355 , 1126 , 1030 , 1011 cm ; h nmr ( cd3od ) h 8.06 ( s , 1h ) , 5.88 ( d , 1h , j = 4.9 hz ) , 5.13 ( t , 1h , j = 4.9 hz ) , 4.42 ( t , 1h , j = 4.9 hz ) , 4.07 ( ddd , 1h , j = 7.2 , 5.5 , 0.9 hz ) , 2.88 ( dd , 1h , j = 14.0 , 5.5 hz ) , 2.80 ( dd , 1h , j = 14.0 , 7.2 hz ) , 2.10 ( s , 3h ) ; c nmr ( cd3od ) c 154.5 , 152.1 , 148.8 , 148.8 , 105.9 , 87.9 , 85.0 , 74.5 , 72.0 , 37.4 , 16.1 ; esi - tof m / z 312.0773 [ m h ] ( calcd for c11h14n5o4s 312.0767 ) . free base 1 ( 10.88 mg ) was dissolved in cd3od ( 500 l ) , hco2h ( 90% aq , 1 equiv ) was added , and the solution was dried under nitrogen to give 1hco2h : white powder ; [ ]d + 20.6 ( c 0.1 , meoh ) ; ftir ( atr , znse plate ) 3343 , 3212 , 1710 , 1658 , 1592 , 1475 , 1429 , 1364 , 1129 , 1096 , 1037 cm ; uv vis was identical to free base 1 ; h and c nmr , see the supporting information , table s3 .

staphylococcus epidermidis is a coagulase - negative staphylococcus ( cns ) that often colonizes the skin and mucous membranes of the human body , representing an important part of its normal microflora [ 1 , 2 ] . however , these staphylococci have emerged in the last years as the most frequently isolated pathogen in nosocomial sepsis , associated with implanted medical devices [ 3 , 4 ] , namely , prosthetic heart valves and joints , central venous catheters , urinary catheters , contact lenses , and hip prostheses . s. epidermidis has the ability to adhere to biomaterials surface and develop as biofilm , which constitutes an important virulence factor and the most important pathogenic mechanism of staphylococcal infection . therefore , initial adhesion of bacteria to the biomaterial surface is thought to be a key step in the colonization of indwelling medical devices . it is a complex process , affected by numerous aspects , such as surface properties of bacteria , material surface properties , and environmental factors . the better understanding of these features is of extreme importance for the development of effective adhesion control mechanisms that will ultimately prevent biofilm formation and thus , the infection of medical devices . during the adhesion process these comprise cell surface hydrophobicity [ 11 , 12 ] and charge as well as the hydrophobicity , charge , roughness , and chemical composition of the biomaterial surface itself . surface hydrophobicity , in particular , has been described as one of the most important properties involved in the adhesion phenomenon [ 1416 ] . according to van oss and giese , in biological systems , hydrophobic interactions are normally the strongest of the long - range noncovalent interactions and can be defined as the attraction among apolar , or slightly polar , cells or other molecules themselves , when immersed in an aqueous solution . biomaterial surface roughness is another property relevant for the bacterial adhesion process , with the irregularities of the polymeric surfaces normally promoting bacterial adhesion and biofilm accumulation [ 18 , 19 ] . this is due to the increased surface area and depressions that provide more favourable and additional sites for colonization , as such crevices protect bacterial cells from the shear forces . however , the accumulation of bacteria in such locations depends largely on their size , cell dimension , and division mode . in fact , according to some authors [ 23 , 24 ] , a linear relation of bacterial adhesion with surface roughness is not always verified . a small increase in roughness can lead to a significant increase in bacterial adhesion , while a larger increase in roughness can have no significant effect on cellular attachment . the aim of the present work was to study the ability of eight strains of s. epidermidis to adhere to acrylic and to silicone , two materials commonly used in the manufacture of medical devices , in relation to the surface properties of these materials . s. epidermidis 9142 is a known producer of the surface polysaccharide intercellular adhesin ( pia ) , which was identified as one of the main responsible factors for biofilm formation . the strain s. epidermidis 9142-m10 is an isogenic mutant with a transposon inserted in the ica locus that encodes the proteins involved in pia production and thus does not form biofilm . the pia - positive s. epidermidis 1457 was isolated from an infected central venous catheter , while s. epidermidis 1457-m10 is a pia - negative isogenic mutant of s. epidermidis 1457 , also obtained by insertion of a transposon into the ica locus . s. epidermidis ie186 , s. epidermidis ie214 and s. epidermidis ie75 were previously isolated from blood of patients with infective endocarditis , while s. epidermidis le7 was isolated from the skin of a healthy individual . g. b. pier , channing laboratory , department of medicine , brigham and women 's hospital , harvard medical school , boston . for all the assays , cells were firstly grown for approximately 36 hours in plates of tryptic soy agar ( tsa ; merck , germany ) , and then for 24 hours in 15 ml of tryptic soy broth ( tsb , merck ) , at 37c under a constant agitation of 120 rpm . after this period , 50 l of each suspension were transferred into 30 ml of fresh tsb and incubated for 18 hours , under the same conditions . then , the cells were centrifuged for 5 minutes at 10 500 g and 4c , washed twice with a saline solution ( 0.9% nacl ( merck ) in distilled water ) and sonicated ( ultrasonic processor , cole - parmer , ill , usa ) during 10 seconds , with an amplitude of 22% ( previously optimized to avoid cell disruption ) . the cellular suspension was adjusted to a final concentration of approximately 1 10 cells / ml , determined by optical density at 640 nm , prior to usage in the adhesion assays . 2 cm 2 cm squares of commercial acrylic , specifically poly(methyl methacrylate ) ( pmma ) ( repsol , brnderslev , denmark ) and silicone ( leewood elastomer ab , sweden ) , both with 2 mm in thickness , were used as substrata in the adhesion assays . this acrylic is the synthetic polymer of methyl methacrylate , containing small amounts of a uv - absorber and release agent and with a molecular weight in the range 1.21.4 million da . concerning silicone , it consists mainly of cross - linked polydimethylvinylmethylsiloxanes with the chain length of ca . 200 000 da , but also of low chain length polymers in the amount up to typically 2% and not exactly defined amount of fumed silica . prior to use , the coupons were washed several times with sterile distilled water and let to soak overnight . next , they were transferred to a new container with sterile distilled water and washed for 5 minutes under agitation , followed by a 30 minutes immersion period in a 70% ethanol / sterile distilled water solution . finally , the coupons were aseptically and individually washed with ultra - pure sterile water and let to dry overnight at 60c . briefly , the acrylic and silicone squares were placed in 6-well tissue - culture plates containing 4 ml of bacterial suspension ( 1 10 cells / ml ) in saline solution ( nacl , 0.9% ( w / v ) ) . initial adhesion to each substrate was allowed to occur for 2 hours at 37c , in a shaker rotating at 120 rpm . negative controls were obtained by placing the coupons in a saline solution without bacterial cells . each coupon was then carefully removed and washed by immersion , in order to remove loosely attached cells . this procedure was gently undertaken and involved their transference to a glass beaker containing 50 ml of distilled water , where they were kept for about 10 seconds . afterwards , a new transfer was made to an additional beaker with 50 ml of distilled water , followed by a third transfer 10 seconds later . the dried coupons were stained with a 4-6-diamidino-2-phenylindole ( dapi ; sigma - aldrich , st louis , mo , usa ) solution ( 0.1 subsequently , each coupon was rinsed with distilled water in order to remove excess stain and let to air - dry in the dark for 30 minutes . adhered cells were visualised under an epifluorescence microscope ( carl zeiss , germany ) with a filter sensitive to dapi fluorescence and coupled with a 3ccd video camera . for each coupon , at least 20 images , with an 820 560 resolution and 1000 magnification , were taken . enumeration of adhered cells was performed with automated enumeration software ( sigmascan pro 5 , spss inc . , chicago , ill , usa ) and the results presented as number of adhered cells / cm . the coupons with adhered bacteria were dehydrated by a 15-minute immersion in increasing ethanol concentration solutions : 10 , 25 , 40 , 50 , 60 , 70 , 80 , 90 and 100% ( v / v ) , having then been placed in a sealed desiccator . samples were then mounted on aluminium stubs with carbon tape , sputter - coated with gold and observed with a leica cambridge s-360 scanning electron microscope ( leo , cambridge , uk ) . in order to assess the extent of bacterial adhesion in each sample , three fields were used for image analysis . all photographs were taken using a magnification of 3000. hydrophobicity parameters of substrata and bacteria surface were determined through the sessile drop contact angle technique , using an automated contact angle measurement apparatus ( oca 15 plus ; dataphysics , germany ) . cleaned and dried substratum surfaces were used for determining the hydrophobicity parameters of acrylic and silicone . in the particular case of bacteria , briefly , a 30 ml suspension of s. epidermidis cells , adjusted to a concentration of approximately 1 10 cells / ml in saline solution ( nacl , 0.9% ( w / v ) ) , was deposited onto a 0.45 m cellulose filter ( pall - life sciences , usa ) , previously wetted with 10 ml of distilled water . to standardise the moisture content , the filters with the resultant lawn of cells deposited were then let to dry onto petri dishes containing 1% ( w / v ) agar ( merck ) and 10% ( v / v ) glycerol ( sigma - aldrich ) , for at least 3.5 hours . all measurements ( at least 25 determinations for each material and bacterial strain ) were performed at room temperature and water , formamide and -bromonaphtalene , with known surface tension components , were used as reference liquids for standardized contact angles measurements . contact angle measurements allowed the calculation of substrata and bacteria hydrophobicity parameters , using the van oss approach [ 17 , 30 ] . according to it , the absolute degree of hydrophobicity of a given material ( i ) is defined in terms of the variation of the free energy of interaction ( g ) between two moieties of that material , when immersed in water ( w ) , that is , giwi . when giwi is negative , the free energy of interaction between molecules is attractive , existing a smaller affinity for water than among molecules themselves , making ( i ) ( the microbial cell or material surface ) hydrophobic . in an opposite way , acrylic and silicone surfaces topography was assessed by atomic force microscopy ( afm ) , using a picoplus scanning probe microscope from molecular imaging ( usa ) . surface imaging was performed in tapping mode and the samples were analysed in air at room temperature . the acrylic surfaces were analysed using a silicon ( si ) tip with a spring constant 42 n / m , while for silicone surfaces the si tip had a spring constant 2.8 n / m . the roughness measurements were performed under a scan range of 2.5 2.5 m , using the spip version 4.2.2.0 software . results from all the assays were compared using one - way analysis of variance ( anova ) by applying levene 's test of homogeneity of variances and the tukey multiple comparisons test , using software statistical package for the social sciences inc . , ( spss ) ( chicago , ill , usa ) . the initial adhesion of the s. epidermidis strains to the acrylic and silicone surfaces is presented in figure 1 . as it can be seen , almost all s. epidermidis strains adhered at a significantly ( p < .05 ) higher extent to the silicone substrate than to acrylic . the only exceptions were observed for strains 1457-m10 , ie75 , and le7 , which also adhered more to silicone than to acrylic but in a nonsignificant way ( p > .05 ) . in fact , the extent of adhesion of s. epidermidis 9142 to silicone was approximately 2.5 times greater than to acrylic . for strains 9142-m10 , ie186 , and ie214 , this difference ranged between 1.8 and 2.0 times . concerning acrylic , strains ie214 and ie186 were the ones that most extensively , and significantly ( p < .05 ) adhered to the coupons . in opposition , strains ie75 and le7 showed the lowest levels of initial binding to this material , being markedly different from all the other strains ( p < .05 ) . s. epidermidis ie214 and s. epidermidis ie186 were the strains showing the highest number of cells adhered ( p < .05 ) to this substrate , likewise to acrylic . scanning electron microscope ( sem ) images of bacteria adhered to acrylic ( a ) and silicone ( b ) squares are presented in figure 2 . it is also visible the higher extent of adhesion to silicone than to acrylic , especially for strains ie214 , ie186 , 9142 , 9142-m10 , and 1457 . in addition , it can be seen , both for acrylic and for silicone , how strain ie214 cells ( figure 2(a)-a and figure 2(b)-a ) grew highly aggregated , quite differently from the mode of growth of the remaining strains . in fact , this strain formed flocculent suspensions in liquid medium . in figure 2(b)-g , it must be noted ( arrow ) how cells adhered to the silicone along the depression on its surface . hydrophobicity of substrata and bacteria was evaluated through contact angle measurements , using the van oss approach [ 17 , 30 ] . contact angles , surface tension parameters , and hydrophobicity of acrylic and silicone are presented in table 1 . water contact angles can be used as a qualitative indication of the surface material hydrophobicity , with higher values indicating a more hydrophobic surface . as it can be seen , the water contact angles obtained for both surfaces are high , a fact that is indicative of their hydrophobicity . the values of giwi also showed that both materials are hydrophobic ( giwi < 0 ) , with silicone holding a more hydrophobic character . from table 1 , it can also be seen that both acrylic and silicone surfaces are predominantly electron donors ( higher values of ) , with low electron acceptor parameters ( ) . in fact , acrylic does not have an electron acceptor parameter ( = 0 ) but is only electron donor ( ) . cell surface hydrophobicity parameters of s. epidermidis strains , as well as the contact angles obtained using the three liquids tested , are presented in table 2 . the eight strains studied showed similar values of water contact angles , lower than 65 , which in accordance to vogler is indicative of a hydrophilic surface , ranging from 21.6 ( strain ie186 ) to 31.8 ( s. epidermidis 1457 ) . these values are quite similar to those obtained for formamide , also polar , with exception of strain le7 that presented a formamide contact angle much lower than that of water . the contact angles determined by using the apolar liquid , -bromonaphtalene , showed small variation between strains with values higher than 49.6 ( s. epidermidis 1457-m10 ) . also , all strains showed positive values of giwi and so , can be considered hydrophilic . in what concerns surface tension components , all strains predominantly showed electron donation , with higher values of electron donor parameters ( ) compared to the low values of the electron acceptor parameters ( ) . strain ie214 showed the highest values of electron acceptor and electron donor parameters of the acid - base component of the surface tension , while strains ie186 and le7 revealed the lowest values of electron acceptor and electron donor parameters , respectively . the surface topography of acrylic and silicone was analyzed by afm in tapping mode ( figure 3 ) . silicone surface displays higher roughness with an average value ( ra ) of 4.237 nm and a maximum ( rmax ) of 44.367 nm , in opposition to ra = 0.789 nm and rmax = 15.683 nm for acrylic surface . ra indicates the average distance of the roughness profile to the centre plane of the profile , while rmax represents the maximum height measured within the screened area . s. epidermidis is strongly associated with infections related to implants and medical devices , such as joint prosthesis , prosthetic heart valves , vascular catheters and contact lenses [ 3 , 32 , 33 ] . given the fact that acrylic and silicone are materials normally used in the production of some of these devices , it is of major importance to study the adhesion of s. epidermidis to these polymers . thus , the primary intention of this study was to attempt to correlate the adhesion capability of 8 s. epidermidis strains with the hydrophobicity parameters of cells and these materials surfaces . surface roughness of acrylic and silicone was also assessed . as the present results indicated , all s. epidermidis strains adhered at a higher extent to silicone substrate than to acrylic ( figure 1 ) . these results are in accordance with other studies that refer silicone rubber as being especially prone to being colonized by candida , streptococci , pseudomonas species , and also by staphylococci and other cns , depending on the site of implantation [ 3436 ] . taking into consideration water contact angle values ( 114.5 2.3 ) and the hydrophobicity degree parameter , giwi = 67.1 mj / m , the silicone assayed was found to be more hydrophobic than acrylic ( water contact angle = 85.3 2.2 ; giwi = 62.5 mj / m ) ( table 1 ) , despite this difference was more pronounced if only water contact angles values were considered . these results are in accordance with values previously obtained [ 12 , 37 ] and clearly demonstrate the importance of the material hydrophobic effect in initial adhesion , since acrylic and silicone have both a hydrophobic character . a higher surface hydrophobicity of silicone is probably responsible for the highest levels of initial binding to this substrate . this fact is corroborated by the work of oliveira et al . where the attachment of s. epidermidis to four polymeric materials ( including silicone ) , commonly used in indwelling medical devices , was assayed . all materials were considered hydrophobic ( giwi < 0 ) and an increase in the degree of hydrophobicity was linearly correlated with the number of attached cells . a point also to be noted is that acrylic stands solely as an electron - donor ( ) . given the fact that a microorganism may adhere to a substratum via the hydrophobic effect , that is , if hydrophobic areas are available for interactions with hydrophobic sites on substrata , the lower densities of apolar areas in acrylic ( = 0 ) help to justify the preferential adhesion of s. epidermidis cells to a more hydrophobic material such as silicone . in what concerns cell surface hydrophobicity parameters determined , all strains were considered to be hydrophilic ( giwi > 0 ) ( table 2 ) . the most hydrophilic s. epidermidis strain , ie186 , ( giwi = 32.5 mj / m ) was the second most adherent strain to both materials , while the least adherent , strain le7 , was the one with the weakest hydrophilic character . thus , contrary to what was found for materials hydrophobicity , no correlation was found between cell surface hydrophobicity of the s. epidermidis strains and their ability to adhere to both hydrophobic surfaces . this fact is corroborated by previous studies [ 37 , 39 ] and suggests that other cell surface factors can as well contribute to the initial attachment to biomaterials surfaces , such as the production of exopolysaccharides like extracellular polysaccharide adhesins and autolysins with adhesive properties like atle and aae . in fact , some authors attribute cell surface hydrophobicity to covalently bound cell - wall - associated proteins [ 4245 ] . furthermore , it was observed ( table 2 ) that all cell surfaces were predominantly electron donors ( higher values of ) , with low electron acceptor parameters ( ) . this polar character can be due to the presence of residual water of hydration or polar groups . however , the high value of the electron acceptor parameter of strain ie214 can justify its highest number of cells adhered to both materials by increasing the interactions between the electron - donor groups of the substrata and the electron - acceptor groups of cells . these slightly higher values of surface tension parameters comparing to the other strains can also indicate that acid - base interactions between cells of this strain are more favoured than in the other s. epidermidis strains studied . therefore , when the first s. epidermidis ie214 cells approach the surface , they have good conditions to adhere to it , but as long as more cells approximate , they tend to adhere to another close cell instead to the surface itself . this leads to the formation of prominent cell aggregates that can be seen in figure 2 , which were still firmly adhered to the surface . the polysaccharide intercellular adhesin ( pia ) , a polymer of n - acetyl glucosamine synthesised by enzymes encoded by the ica operon , is crucial to the cell - to - cell adhesion process and biofilm accumulation [ 48 , 49 ] . according to the hemagglutination ability , which reflects the level of pia expression , s. epidermidis ie214 is a strong producer of pia ( hemagglutination titer of 1:16 ) . therefore , the high levels of pia production by s. epidermidis ie214 , along with its physicochemical properties , namely , surface tension , aid to support the unique behaviour of s. epidermidis ie214 , compared to the remaining strains . these specific physicochemical properties are most probably due to ie214 being the only strain capable of producing a 148 kda cell wall protein ( atle ) ( data not shown ) , which has been described as determinant in the adhesion to unmodified polymer surfaces . in addition to hydrophobicity and surface tension parameters , the material surface roughness is another factor that has been pointed out as capable of influencing bacterial adhesion to a given material . this is probably due to the fact that rough surfaces have greater surface areas and that depressions in the roughened surfaces provide more favourable sites for colonisation . in fact , according to van hoogmoed et al . , there is a microorganism 's preference for adherence to scratches or grooves , which could be seen in figure 2(b)-g . the afm results obtained showed that the average roughness is higher for silicone than for acrylic . however , it is difficult to ascertain the possible effect of this parameter in cocci adhesion , since for both surfaces the roughness is at a nanoscale , meaning that there are no microcrevices in the surfaces to act as niches for the microbial cells . nevertheless , according to katainen et al . , while in particles smaller than the surface features the interaction is limited to one contact between the particle and a single asperity , being the strength of adhesion determined by this only contact , particles larger than the surfaces features ( which is the present case for silicone ) have several contacts with the surface . this thus allows a higher level of interaction leading to a major influence on the adhesion phenomenon . therefore , a stable bacterial adhesion to a biomaterial requires a high degree of hydrophobicity , as well as a certain degree of roughness between other physicochemical properties of the substratum . silicone is widely used as a biomaterial but it has the disadvantages of being more hydrophobic and rougher than acrylic , thus becoming a more prone material to s. epidermidis adherence . bacterial adhesion to the less hydrophobic material ( acrylic ) was significantly lower than to the more hydrophobic ( silicone ) . these results showed the importance of the hydrophobic effect of the biomaterial surface in initial adhesion . the higher roughness of silicone seems also to exert some effect in bacterial adhesion . on the other hand , bacteria surface physicochemical properties seem to have less effect in their binding to substrata , highlighting the importance of other cell surface factors to the initial adhesion process . nevertheless , s. epidermidis strain ie214 revealed completely distinct adherence behaviour compared to the remaining strains , probably as a consequence of its unique surface features , as displayed by its flocculence ability .

the aim of the present study was to compare the ability of eight staphylococcus epidermidis strains to adhere to acrylic and silicone , two polymers normally used in medical devices manufacture . furthermore , it was tried to correlate that with the surface properties of substrata and cells . therefore , hydrophobicity and surface tension components were calculated through contact angle measurements . surface roughness of substrata was also assessed by atomic force microscopy ( afm ) . no relationship was found between microbial surface hydrophobicity and adhesion capability . nevertheless , staphylococcus epidermidis ie214 showed very unique adhesion behaviour , with cells highly aggregated between them , which is a consequence of their specific surface features . all strains , determined as being hydrophilic , adhered at a higher extent to silicone than to acrylic , most likely due to its more hydrophobic character and higher roughness . this demonstrates the importance of biomaterial surface characteristics for bacterial adhesion .

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

staphylococcus epidermidis is a coagulase - negative staphylococcus ( cns ) that often colonizes the skin and mucous membranes of the human body , representing an important part of its normal microflora [ 1 , 2 ] . s. epidermidis has the ability to adhere to biomaterials surface and develop as biofilm , which constitutes an important virulence factor and the most important pathogenic mechanism of staphylococcal infection . therefore , initial adhesion of bacteria to the biomaterial surface is thought to be a key step in the colonization of indwelling medical devices . it is a complex process , affected by numerous aspects , such as surface properties of bacteria , material surface properties , and environmental factors . the better understanding of these features is of extreme importance for the development of effective adhesion control mechanisms that will ultimately prevent biofilm formation and thus , the infection of medical devices . during the adhesion process these comprise cell surface hydrophobicity [ 11 , 12 ] and charge as well as the hydrophobicity , charge , roughness , and chemical composition of the biomaterial surface itself . surface hydrophobicity , in particular , has been described as one of the most important properties involved in the adhesion phenomenon [ 1416 ] . according to van oss and giese , in biological systems , hydrophobic interactions are normally the strongest of the long - range noncovalent interactions and can be defined as the attraction among apolar , or slightly polar , cells or other molecules themselves , when immersed in an aqueous solution . biomaterial surface roughness is another property relevant for the bacterial adhesion process , with the irregularities of the polymeric surfaces normally promoting bacterial adhesion and biofilm accumulation [ 18 , 19 ] . this is due to the increased surface area and depressions that provide more favourable and additional sites for colonization , as such crevices protect bacterial cells from the shear forces . in fact , according to some authors [ 23 , 24 ] , a linear relation of bacterial adhesion with surface roughness is not always verified . a small increase in roughness can lead to a significant increase in bacterial adhesion , while a larger increase in roughness can have no significant effect on cellular attachment . the aim of the present work was to study the ability of eight strains of s. epidermidis to adhere to acrylic and to silicone , two materials commonly used in the manufacture of medical devices , in relation to the surface properties of these materials . s. epidermidis 9142 is a known producer of the surface polysaccharide intercellular adhesin ( pia ) , which was identified as one of the main responsible factors for biofilm formation . s. epidermidis ie186 , s. epidermidis ie214 and s. epidermidis ie75 were previously isolated from blood of patients with infective endocarditis , while s. epidermidis le7 was isolated from the skin of a healthy individual . then , the cells were centrifuged for 5 minutes at 10 500 g and 4c , washed twice with a saline solution ( 0.9% nacl ( merck ) in distilled water ) and sonicated ( ultrasonic processor , cole - parmer , ill , usa ) during 10 seconds , with an amplitude of 22% ( previously optimized to avoid cell disruption ) . the cellular suspension was adjusted to a final concentration of approximately 1 10 cells / ml , determined by optical density at 640 nm , prior to usage in the adhesion assays . 2 cm 2 cm squares of commercial acrylic , specifically poly(methyl methacrylate ) ( pmma ) ( repsol , brnderslev , denmark ) and silicone ( leewood elastomer ab , sweden ) , both with 2 mm in thickness , were used as substrata in the adhesion assays . concerning silicone , it consists mainly of cross - linked polydimethylvinylmethylsiloxanes with the chain length of ca . briefly , the acrylic and silicone squares were placed in 6-well tissue - culture plates containing 4 ml of bacterial suspension ( 1 10 cells / ml ) in saline solution ( nacl , 0.9% ( w / v ) ) . for each coupon , at least 20 images , with an 820 560 resolution and 1000 magnification , were taken . in order to assess the extent of bacterial adhesion in each sample , three fields were used for image analysis . all photographs were taken using a magnification of 3000. hydrophobicity parameters of substrata and bacteria surface were determined through the sessile drop contact angle technique , using an automated contact angle measurement apparatus ( oca 15 plus ; dataphysics , germany ) . cleaned and dried substratum surfaces were used for determining the hydrophobicity parameters of acrylic and silicone . to standardise the moisture content , the filters with the resultant lawn of cells deposited were then let to dry onto petri dishes containing 1% ( w / v ) agar ( merck ) and 10% ( v / v ) glycerol ( sigma - aldrich ) , for at least 3.5 hours . all measurements ( at least 25 determinations for each material and bacterial strain ) were performed at room temperature and water , formamide and -bromonaphtalene , with known surface tension components , were used as reference liquids for standardized contact angles measurements . contact angle measurements allowed the calculation of substrata and bacteria hydrophobicity parameters , using the van oss approach [ 17 , 30 ] . according to it , the absolute degree of hydrophobicity of a given material ( i ) is defined in terms of the variation of the free energy of interaction ( g ) between two moieties of that material , when immersed in water ( w ) , that is , giwi . in an opposite way , acrylic and silicone surfaces topography was assessed by atomic force microscopy ( afm ) , using a picoplus scanning probe microscope from molecular imaging ( usa ) . the initial adhesion of the s. epidermidis strains to the acrylic and silicone surfaces is presented in figure 1 . as it can be seen , almost all s. epidermidis strains adhered at a significantly ( p < .05 ) higher extent to the silicone substrate than to acrylic . the only exceptions were observed for strains 1457-m10 , ie75 , and le7 , which also adhered more to silicone than to acrylic but in a nonsignificant way ( p > .05 ) . in fact , the extent of adhesion of s. epidermidis 9142 to silicone was approximately 2.5 times greater than to acrylic . concerning acrylic , strains ie214 and ie186 were the ones that most extensively , and significantly ( p < .05 ) adhered to the coupons . s. epidermidis ie214 and s. epidermidis ie186 were the strains showing the highest number of cells adhered ( p < .05 ) to this substrate , likewise to acrylic . scanning electron microscope ( sem ) images of bacteria adhered to acrylic ( a ) and silicone ( b ) squares are presented in figure 2 . it is also visible the higher extent of adhesion to silicone than to acrylic , especially for strains ie214 , ie186 , 9142 , 9142-m10 , and 1457 . in addition , it can be seen , both for acrylic and for silicone , how strain ie214 cells ( figure 2(a)-a and figure 2(b)-a ) grew highly aggregated , quite differently from the mode of growth of the remaining strains . in figure 2(b)-g , it must be noted ( arrow ) how cells adhered to the silicone along the depression on its surface . hydrophobicity of substrata and bacteria was evaluated through contact angle measurements , using the van oss approach [ 17 , 30 ] . contact angles , surface tension parameters , and hydrophobicity of acrylic and silicone are presented in table 1 . water contact angles can be used as a qualitative indication of the surface material hydrophobicity , with higher values indicating a more hydrophobic surface . as it can be seen , the water contact angles obtained for both surfaces are high , a fact that is indicative of their hydrophobicity . the values of giwi also showed that both materials are hydrophobic ( giwi < 0 ) , with silicone holding a more hydrophobic character . from table 1 , it can also be seen that both acrylic and silicone surfaces are predominantly electron donors ( higher values of ) , with low electron acceptor parameters ( ) . cell surface hydrophobicity parameters of s. epidermidis strains , as well as the contact angles obtained using the three liquids tested , are presented in table 2 . these values are quite similar to those obtained for formamide , also polar , with exception of strain le7 that presented a formamide contact angle much lower than that of water . also , all strains showed positive values of giwi and so , can be considered hydrophilic . in what concerns surface tension components , all strains predominantly showed electron donation , with higher values of electron donor parameters ( ) compared to the low values of the electron acceptor parameters ( ) . strain ie214 showed the highest values of electron acceptor and electron donor parameters of the acid - base component of the surface tension , while strains ie186 and le7 revealed the lowest values of electron acceptor and electron donor parameters , respectively . the surface topography of acrylic and silicone was analyzed by afm in tapping mode ( figure 3 ) . silicone surface displays higher roughness with an average value ( ra ) of 4.237 nm and a maximum ( rmax ) of 44.367 nm , in opposition to ra = 0.789 nm and rmax = 15.683 nm for acrylic surface . ra indicates the average distance of the roughness profile to the centre plane of the profile , while rmax represents the maximum height measured within the screened area . given the fact that acrylic and silicone are materials normally used in the production of some of these devices , it is of major importance to study the adhesion of s. epidermidis to these polymers . thus , the primary intention of this study was to attempt to correlate the adhesion capability of 8 s. epidermidis strains with the hydrophobicity parameters of cells and these materials surfaces . surface roughness of acrylic and silicone was also assessed . as the present results indicated , all s. epidermidis strains adhered at a higher extent to silicone substrate than to acrylic ( figure 1 ) . these results are in accordance with other studies that refer silicone rubber as being especially prone to being colonized by candida , streptococci , pseudomonas species , and also by staphylococci and other cns , depending on the site of implantation [ 3436 ] . taking into consideration water contact angle values ( 114.5 2.3 ) and the hydrophobicity degree parameter , giwi = 67.1 mj / m , the silicone assayed was found to be more hydrophobic than acrylic ( water contact angle = 85.3 2.2 ; giwi = 62.5 mj / m ) ( table 1 ) , despite this difference was more pronounced if only water contact angles values were considered . these results are in accordance with values previously obtained [ 12 , 37 ] and clearly demonstrate the importance of the material hydrophobic effect in initial adhesion , since acrylic and silicone have both a hydrophobic character . a higher surface hydrophobicity of silicone is probably responsible for the highest levels of initial binding to this substrate . where the attachment of s. epidermidis to four polymeric materials ( including silicone ) , commonly used in indwelling medical devices , was assayed . all materials were considered hydrophobic ( giwi < 0 ) and an increase in the degree of hydrophobicity was linearly correlated with the number of attached cells . given the fact that a microorganism may adhere to a substratum via the hydrophobic effect , that is , if hydrophobic areas are available for interactions with hydrophobic sites on substrata , the lower densities of apolar areas in acrylic ( = 0 ) help to justify the preferential adhesion of s. epidermidis cells to a more hydrophobic material such as silicone . in what concerns cell surface hydrophobicity parameters determined , all strains were considered to be hydrophilic ( giwi > 0 ) ( table 2 ) . the most hydrophilic s. epidermidis strain , ie186 , ( giwi = 32.5 mj / m ) was the second most adherent strain to both materials , while the least adherent , strain le7 , was the one with the weakest hydrophilic character . thus , contrary to what was found for materials hydrophobicity , no correlation was found between cell surface hydrophobicity of the s. epidermidis strains and their ability to adhere to both hydrophobic surfaces . in fact , some authors attribute cell surface hydrophobicity to covalently bound cell - wall - associated proteins [ 4245 ] . furthermore , it was observed ( table 2 ) that all cell surfaces were predominantly electron donors ( higher values of ) , with low electron acceptor parameters ( ) . this polar character can be due to the presence of residual water of hydration or polar groups . however , the high value of the electron acceptor parameter of strain ie214 can justify its highest number of cells adhered to both materials by increasing the interactions between the electron - donor groups of the substrata and the electron - acceptor groups of cells . these slightly higher values of surface tension parameters comparing to the other strains can also indicate that acid - base interactions between cells of this strain are more favoured than in the other s. epidermidis strains studied . therefore , when the first s. epidermidis ie214 cells approach the surface , they have good conditions to adhere to it , but as long as more cells approximate , they tend to adhere to another close cell instead to the surface itself . this leads to the formation of prominent cell aggregates that can be seen in figure 2 , which were still firmly adhered to the surface . according to the hemagglutination ability , which reflects the level of pia expression , s. epidermidis ie214 is a strong producer of pia ( hemagglutination titer of 1:16 ) . therefore , the high levels of pia production by s. epidermidis ie214 , along with its physicochemical properties , namely , surface tension , aid to support the unique behaviour of s. epidermidis ie214 , compared to the remaining strains . these specific physicochemical properties are most probably due to ie214 being the only strain capable of producing a 148 kda cell wall protein ( atle ) ( data not shown ) , which has been described as determinant in the adhesion to unmodified polymer surfaces . in addition to hydrophobicity and surface tension parameters , the material surface roughness is another factor that has been pointed out as capable of influencing bacterial adhesion to a given material . this is probably due to the fact that rough surfaces have greater surface areas and that depressions in the roughened surfaces provide more favourable sites for colonisation . , there is a microorganism 's preference for adherence to scratches or grooves , which could be seen in figure 2(b)-g . the afm results obtained showed that the average roughness is higher for silicone than for acrylic . however , it is difficult to ascertain the possible effect of this parameter in cocci adhesion , since for both surfaces the roughness is at a nanoscale , meaning that there are no microcrevices in the surfaces to act as niches for the microbial cells . nevertheless , according to katainen et al . , while in particles smaller than the surface features the interaction is limited to one contact between the particle and a single asperity , being the strength of adhesion determined by this only contact , particles larger than the surfaces features ( which is the present case for silicone ) have several contacts with the surface . this thus allows a higher level of interaction leading to a major influence on the adhesion phenomenon . therefore , a stable bacterial adhesion to a biomaterial requires a high degree of hydrophobicity , as well as a certain degree of roughness between other physicochemical properties of the substratum . silicone is widely used as a biomaterial but it has the disadvantages of being more hydrophobic and rougher than acrylic , thus becoming a more prone material to s. epidermidis adherence . bacterial adhesion to the less hydrophobic material ( acrylic ) was significantly lower than to the more hydrophobic ( silicone ) . these results showed the importance of the hydrophobic effect of the biomaterial surface in initial adhesion . the higher roughness of silicone seems also to exert some effect in bacterial adhesion . on the other hand , bacteria surface physicochemical properties seem to have less effect in their binding to substrata , highlighting the importance of other cell surface factors to the initial adhesion process . nevertheless , s. epidermidis strain ie214 revealed completely distinct adherence behaviour compared to the remaining strains , probably as a consequence of its unique surface features , as displayed by its flocculence ability .

theories of career guidance and counseling that exist in china were mainly adopted from europe and the united states , particularly in the period since 1990 . however , one institution in china , tsinghua university , actually started work in this field as early as 1916 , and established a vocational guidance committee in 1923 . this initiative can probably be regarded as the beginning of career guidance in china ( liu 2006 ) . more recently , the country has been developing rapidly and there has been a significant increase in the need for career guidance . at a national level , new policies have been issued and implemented , and there has been expansion in the provision of training courses on careers matters for teachers . an increase in the publishing of teaching materials for career guidance curricula is also evident ( ma 2009 ) . over the years , many career guidance centers have been set up in universities , accumulating some useful experiences in implementation of career guidance and counseling ( li 2002 ; ma 2009 ) . the following sections summarise some of the significant achievements and the state of current research on career guidance in china . in reviewing the chinese literature , the terms zhiye guihua ( career planning ) or jiuye zhidao ( employment guidance ) are used when referring to career guidance , career education and vocational guidance . wang ( 2008 ) defined the difference between these two terms in the chinese educational context , with jiuye zhidao ( employment guidance ) being concerned with issues related to finding jobs for prospective graduates . issues addressed in this domain typically include : familiarization with employment policy , providing employment information , coaching on resum preparation , and developing interview skills . zhiye guihua ( career planning ) , on the other hand , means guidance given to students according to their personal circumstances to assist them in planning for lifelong career development . here , typically , students are made aware of the options available to them in the world of work , and they are led to realize the many important decisions that have to be made . in the situation existing at present in most chinese universities , employment guidance still dominates over career planning ( wu 2008 ) . effective career planning is implemented in only a few universities , but currently other colleges and universities are beginning to make significant efforts to extend their employment guidance and counseling more in the direction of career planning ( bian 2008 ) . in the complex and evolving situation existing in china , it is insufficient to address all aspects of career development by using only employment guidance strategies or by dealing only with career planning - both elements are required . for convenience , in this paper the term as a result of national policy ( state council of china 1999 ) , chinese universities began to expand their enrolments in 1999 , and this has ultimately created many problems in the current job market . the average rate of increase of new graduates in every year has been 30% since 2002 , so graduating students have faced more and more severe employment difficulties ( geng 2007 ) . in 2008 , the total number of national university graduates reached 5.59 million ; but according to official statistics the initial employment rate of graduates is only 70% , so there would be at least 1.7 million university graduates faced with a problem in gaining employment ( gao 2009 ) . indeed , difficulty getting a job has emerged as an ongoing focus of personal concern and anxiety , leading to an increased need for career counseling services ( wu 2008 ) . according to gao ( 2009 ) and wu ( 2008 ) , the problem of employment for graduates is exacerbated by many factors , an important one of which is that they have limited long - term career planning and personal goal - setting strategies . when faced later with employment demands , they generally exhibit such psychological problems as confusion , anxiety and panic . wen ( 2009 ) interpreted the significance of career guidance in university contexts from two perspectives , ( i ) the needs of students , and ( ii ) development of the university . for students , career planning can help them set personal goals and decide upon current and future directions . such career guidance is helpful to them in overcoming any misunderstandings in choosing careers , in selecting their study paths , and in identifying their potential strengths to enhance their competitiveness for positions . effective career guidance provides guidelines with a long - term vision for career planning , from which students can benefit by realizing their true potential in life ( guo 2009 ) . for universities , career guidance can help promote necessary reforms in teaching and can improve employment rates for their graduates , thus enhancing the reputation of a university . solving the problem of employment is not only related to university students and their families , but also to the universities reputations , and even to the country 's politics , construction of the economy , and maintaining a harmonious society ( li and ye 2001 ) . despite its very early start in tsinghua university , career guidance and counseling within most universities in china is still at a fairly elementary stage . this is reflected in the fact that chinese papers and articles on career guidance have been mostly written after 2004 . key words searched in the chinese journal data - base in relation to this study were career guidance in titles or descriptors between 1979 and 2001 . within the decade from 2001 to 2011 , approximately 250 papers about career guidance for university students were published in chinese core journals . of these , nearly 200 were written after 2005 . it is almost impossible to find any paper on this issue in china written before 2001 . this suggests that research on career guidance in china started late , but then developed very rapidly . in the material that does exist , employment guidance in universities , with just a few writers addressing career guidance in high schools ( e.g. , liu and tian 2008 ) . fang and tan ( 2010 ) claimed that papers on career counseling for university students in china are even fewer in number , and generally lack practical strategies or data from empirical studies . articles tend to be confined to investigations of students ' demand for career counseling ( e.g. , zhao and shen 2008 ) , analysis of existing conditions in career guidance ( e.g. , yu 2002 ) , and describing the duties of career counselors ( e.g. , fang and tan 2010 ) . in the areas of both guidance and counseling , the content of many published papers mainly describes possible provisions , often based on models from other countries ( e.g. , li 2002 ) , identifies potential problems ( e.g. , lan and wang 2010 ) , and suggests possible solutions ( e.g. , yu 2008 ) . there is very little reported research about the combination of career planning and selecting appropriate courses , or examples of empirical research on career guidance or counseling strategies . this situation suggests that the theoretical basis for career guidance in china needs to be strengthened ( liu and li 2007 ) . on the other hand , there is evidence in some reports that career guidance and counseling endeavours in some chinese universities appear to have achieved very good results ( e.g. , yu 2002 ) , and these examples could provide a reference model for other universities in china to emulate ( wan and wang 2006 ) . in addition to presenting foreign career theories , a number of papers focus on the current situation regarding career guidance in chinese universities in different regions of china ( e.g. , fang 2007 ; zhou 2008 ) . these studies tend to highlight a need to develop an indigenous career theory ( or theories ) to suit the actual situations existing in those regions . in terms of the methods of career guidance currently in operation , over 90% of 16 universities investigated in beijing have open employment guidance courses for introducing job - hunting skills and employment policy ( liu 2006 ) . other methods of delivering career guidance content to students include ad hoc lectures , campus recruitment fairs , and individual counseling as required . only 25% of universities in beijing provided career assessment services , and in zhejiang province , only three of the surveyed 21 universities provided such assessment ( hong 2007 ) . at the present time , career guidance in universities is mostly delivered via career centres, and through departmental career tutors ( long and song 2007 ) . however , many practitioners consider this traditional model usually fails to satisfy the many needs that students have for career guidance and career counseling . to change the traditional mode of vocational guidance , some writers ( e.g. , long and song 2007 ; sun 2009 ) suggest developing more comprehensive career guidance programmes . at present , a comprehensive approach to employment guidance has begun mainly in beijing normal university , shanghai jiaotong university , renmin university of china , fudan university and guangxi university ( yu 2002 ) . for instance , the model of comprehensive career planning in beijing normal university conducts preliminary guidance about " how to be successful " for freshman , all - round student development education for sophomores , guidance on vocational selection in junior years , with guidance for employment policies and techniques for application in the senior year . in anhui industry university , a three - direction education model comprising ( i ) education for direction of majors when students enter university , ( ii ) education for situation awareness while at university itself , and , finally ( iii ) education for choosing careers when students graduate , operates . so far , the effects of these new models appear to be positive ( yu 2008 ) . the approach to career guidance needs to be flexible and responsive to students needs and interests , and to local conditions . it is necessary to create a network of resources and tools that make career guidance more in - depth , relevant and accessible . one example of this is the occupational information network in the united states ( peterson et al . 2001 ) . students at fudan can view 50 different video sessions as part of a career guidance course on - line by entering their individual login names and passwords . this on - line course is in four parts : career assessment , vocations introduction , curriculum study , and career counseling ( sun 2009 ) . tianjin normal university provides a good example of using software applications for vocational assessment and planning . that establishment uses careersky software to offer self - service for students in career education . the careersky online career planning system is the most widely used and appears to be reasonably effective ( beisen career assessment 2007 ; sun 2009 ) . based on the problem of insufficient teaching personnel to service the area of career guidance , some universities have built independent learning channels for students , such as a website providing relevant information on career matters , making available appropriate print resources free of charge , and organizing relevant forums ( zhao and shen 2008 ) . yan ( 2008 ) reported that experiential - style guidance is used in some universities , including simulated job interviews and the provision of firsthand vocational experiences . internships and work fellowships in companies can also provide valuable opportunities for university students to observe at firsthand and practice the jobs in which they may be interested . naturally , providing this form of off - campus experiential learning is not easy to arrange for large numbers of students . it also relies heavily on the willingness of local companies and worksites to cooperate and provide placements . at the moment there appears to be too little theory underpinning most of what universities and colleges attempt do in the way of career guidance . the institutions do not seem to establish career planning systems on any scientific or proactive basis , but instead respond to needs in an ad hoc manner , as and when they arise . the core duties of most career guidance centers are confined to providing information and processing employment procedures , such as registering employment whereabouts , guidance on signing contracts , and employment statistics . the service mostly emphasizes introduction to employment policy and analysis of the current employment situation . professional career counseling is rarely provided automatically to all students ; and even some counseling services that are offered are not individualized and focused enough to satisfy students ' needs ( li 2009 ) . there is a lack of well - structured and purposeful guidance to assist students career planning in a practical and personalized way . since there is still a scarcity of teaching materials and teachers texts designed specifically to match existing chinese employment contexts , it is usual for universities to copy western ideas . while some general principles may hold true in both cultures , western policies and practices do not necessarily mesh with chinese national conditions ( zhao and shen 2008 ) . similarly , assessment tools relevant for career guidance in china have basically adopted three authoritative western theories and measurement scales , namely , holland 's vocational interest theory ( holland 1959 ) , myers - briggs type indicator ( myers 1962 ) , and edgar schein s career anchor ( schein 1978 ) . due to the cultural differences between the west and china such assessment tools often lack local relevance for chinese realities ; and the data they produce are , therefore , of doubtful validity , reliability and usefulness ( zhao and shen 2008 ) . another problem stems from the fact that the number of career guidance professionals currently available is not enough to meet actual demands . also , lan and wang 2010 ) , by 2006 , there were more than 26,000 career counselors in china , but the majority of them worked in social organizations , such as beisen career , with only a small number in universities . in addition , the distribution of career counselors is not balanced across different regions , with more counselors employed in cities . the employment structure in this field shows a trend toward younger counselors , many of who may lack work experience and may not possess the relevant background knowledge in psychology and pedagogy . based on a survey in beijing , jin and fan ( 2002 ) found that , on average , there was less than one career teacher for every 1,000 undergraduates in universities . in one context they examined , among the 70 staff engaged in career guidance , only 12.9% possessed a relevant background in career guidance , 45.7% had worked in career guidance for less than 5 years , and only 17.1% had worked for over 10 years . an additional problem occurs because the concept of career planning is not presented explicitly to students early in their university lives . they do not have a clear understanding of what career planning actually entails , and they have not recognized its importance for their own life and future employment ( li 2009 ) . in order to develop effective and adaptive strategies to improve career guidance in chinese universities , authorities ( e.g. , chen 2007 ; liu and li 2007 ) have pointed out that career guidance and career counseling should not merely try to copy policies and practices from foreign countries . ideally , chinese theories and strategies for career guidance and counseling should be created , with due reference to chinese society 's characteristics and practical problems . indigenous factors that need to be considered include : job market , employment system , educational philosophy , and the social value system ( chen 2007 ) . since these factors will be distinctive for chinese communities , local theories and strategies for career guidance and counseling should reflect , or at least acknowledge , these features this refers to the regular collection of data showing students ' study achievements and their continuing progress . when students are first enrolled in colleges , a dynamic database should be set up for each individual ( sun 2009 ) . electro - portfolio assessment. they suggest that through analyzing and evaluating the data in the system , teachers can get a comprehensive understanding of students development and can guide and support them in accordance with their specific aptitudes and aspirations . a second improvement suggested by qian and fang ( 2009 ) is that universities should introduce more participatory and interactive forms of career guidance , such as a festival of college students ' career planning , including career planning contests , career assessment , interview - style interactive forums , experts and alumni seminars , mock recruitment , and visits to various firms . peer interdependent training course , which involves university associations , and dormitory , friendship and community groups . thirdly , vital to the improvement of career guidance and counseling , is the need to establish a team of career guidance teachers who have high - quality professional knowledge and expertise relevant for college students . the number of professional teachers should be increased , and training of existing staff should be reinforced . department tutors need to be replaced by full - time guidance teachers for fulfilling the functions of employment guidance . experts in career counseling from outside college settings should be hired as necessary to supplement existing teaching staff ( ruan 2009 ) . the existing system of career guidance tends only to emphasize the role of university staff and ignores the impact and importance of family and friends on students career choices . effective career planning systems must be established that include family and parental influences , and also the influence of senior students and classmates . effective systems should also collect ongoing data and feedback of information regarding the later employment histories of their graduated students ( yao and zhen 2009 ) . this information can highlight strengths and weaknesses in the career guidance system and can lead to improvements in the content and delivery of information and personal guidance . gao ( 2008 ) pointed out that career counseling within class is an effective way of providing true contextualized career guidance . it brings career planning into daily teaching activities in class and provides many opportunities for students to ask questions , seek information , and share their concerns and experiences within a social group . university tutors should play an important role in career guidance in collaboration with subject teachers . they can help students acquire comprehensive understanding of curriculum and their major subject in relation to requirements for specific careers ( gao and huang 2009 ) . in general terms , current research and practice in career guidance and counseling in china remains at an early stage of development , lacking a sound basis in theory , still learning from foreign experiences , and still drawing upon western methods to a large extent . however , through day - to - day experiences in those institutions where dedicated practitioners are now designing and implementing innovative models , rapid progress is being made and is achieving some positive results . based on the literature reviewed in this paper , several suggestions can be made for promoting future research and practical efforts in this domain : educational systems , policies and practices are deeply embedded in local histories and cultures ( watts et al . 2010 ) . in view of unique educational and social situations in china , foreign experiences and models of career guidance and counseling therefore , the key focus of research on career guidance and counseling in the future needs to explore how best to tailor systems and practices specifically to local chinese contexts.a supportive public policy is critical to the development of career guidance services . such policy is usually determined largely at national level , though it may be devolved to regional , local or institutional level ( watts 2009 ; watts et al . 2010 ) . at the national level , career guidance should be supported by policy and resources in order that the development of career guidance can be accelerated . at a local level , based on the different characteristics and needs of students in different regions in china , researchers should liaise closely with different types of schools , colleges and universities in different regions and share their findings and opinions with career practitioners . this process could help universities design and implement courses that really meet students needs.career counseling , as an indispensable part and strategy of career guidance , needs to be implemented more professionally and made available more widely . as proposed by watts and van esbroeck ( 2000 ) , there is a general recognition that all counselors will need to use new technologies , including computer - assisted assessment in the diagnostic and self - assessment process , as well as computer - assisted career guidance and counseling via the internet . in the context of china , the use of new technologies is an extremely positive strategy for integrating and making good use of resources in career counseling.other asian regions , such as singapore , japan , hong kong , and taiwan , have set up career guidance systems that are sound in terms of local applicability and in their underpinning theory . through exchanges and dialogue , program designers , researchers and career practitioners in chinese mainland and in these regions educational systems , policies and practices are deeply embedded in local histories and cultures ( watts et al . 2010 ) . in view of unique educational and social situations in china , foreign experiences and models of career guidance and counseling therefore , the key focus of research on career guidance and counseling in the future needs to explore how best to tailor systems and practices specifically to local chinese contexts . such policy is usually determined largely at national level , though it may be devolved to regional , local or institutional level ( watts 2009 ; watts et al . , career guidance should be supported by policy and resources in order that the development of career guidance can be accelerated . at a local level , based on the different characteristics and needs of students in different regions in china , researchers should liaise closely with different types of schools , colleges and universities in different regions and share their findings and opinions with career practitioners . career counseling , as an indispensable part and strategy of career guidance , needs to be implemented more professionally and made available more widely . as proposed by watts and van esbroeck ( 2000 ) , there is a general recognition that all counselors will need to use new technologies , including computer - assisted assessment in the diagnostic and self - assessment process , as well as computer - assisted career guidance and counseling via the internet . in the context of china , the use of new technologies is an extremely positive strategy for integrating and making good use of resources in career counseling . other asian regions , such as singapore , japan , hong kong , and taiwan , have set up career guidance systems that are sound in terms of local applicability and in their underpinning theory . through exchanges and dialogue , program designers , researchers and career practitioners in chinese mainland and in these regions , could gain many useful shared insights in policies , research and practices . to summarize briefly the main points and implications from this review career guidance and career counseling in china started late , but there is now scope for improvement , both in the theory and in practice . significant progress has been made already in some universities and regions , but overall china still lags behind many other nations . not only is it necessary to continue to develop strong and effective career guidance services in universities , more action is also required in schools to provide the first steps in preparing youngsters for later career choices and decisions . all forms of career guidance , career counseling , and career assessment in china must take into account indigenous characteristics of the students , and the realities of local and regional employment opportunities . similarly , indigenous factors need to be considered by researchers investigating or evaluating career services in chinese mainland settings .

in recent years , various forms of career guidance and career counseling have become more prominent and better serviced in most universities throughout the world . such services are obviously to the benefit of the students themselves and for society . after an initially slow start , researchers and practitioners in china have now begun to focus on the localization of guidance and counselling theory and strategies in order to match more exactly actual employment situations in different regions of the country . this should result in a service that meets students ' needs more effectively . using mainly core literature examining the context of career guidance and counseling in china from 2001 to the present , this paper elaborates on the current situation and summarizes the progress that has been made . the authors detail the content , implementation , problems that exist , and ways of improving projects of this kind in chinese universities . conclusions and suggestions for further research on career guidance and counseling are provided .

Introduction Recent and Current Employment Situation Significance of Career Guidance Current Situation and Results of Research Implementation of Career Guidance Problems Existing in Career Guidance Improving Career Guidance Conclusion and Implications

theories of career guidance and counseling that exist in china were mainly adopted from europe and the united states , particularly in the period since 1990 . however , one institution in china , tsinghua university , actually started work in this field as early as 1916 , and established a vocational guidance committee in 1923 . this initiative can probably be regarded as the beginning of career guidance in china ( liu 2006 ) . more recently , the country has been developing rapidly and there has been a significant increase in the need for career guidance . at a national level , new policies have been issued and implemented , and there has been expansion in the provision of training courses on careers matters for teachers . an increase in the publishing of teaching materials for career guidance curricula is also evident ( ma 2009 ) . over the years , many career guidance centers have been set up in universities , accumulating some useful experiences in implementation of career guidance and counseling ( li 2002 ; ma 2009 ) . the following sections summarise some of the significant achievements and the state of current research on career guidance in china . in reviewing the chinese literature , the terms zhiye guihua ( career planning ) or jiuye zhidao ( employment guidance ) are used when referring to career guidance , career education and vocational guidance . wang ( 2008 ) defined the difference between these two terms in the chinese educational context , with jiuye zhidao ( employment guidance ) being concerned with issues related to finding jobs for prospective graduates . issues addressed in this domain typically include : familiarization with employment policy , providing employment information , coaching on resum preparation , and developing interview skills . zhiye guihua ( career planning ) , on the other hand , means guidance given to students according to their personal circumstances to assist them in planning for lifelong career development . here , typically , students are made aware of the options available to them in the world of work , and they are led to realize the many important decisions that have to be made . in the situation existing at present in most chinese universities , employment guidance still dominates over career planning ( wu 2008 ) . effective career planning is implemented in only a few universities , but currently other colleges and universities are beginning to make significant efforts to extend their employment guidance and counseling more in the direction of career planning ( bian 2008 ) . in the complex and evolving situation existing in china , it is insufficient to address all aspects of career development by using only employment guidance strategies or by dealing only with career planning - both elements are required . for convenience , in this paper the term as a result of national policy ( state council of china 1999 ) , chinese universities began to expand their enrolments in 1999 , and this has ultimately created many problems in the current job market . the average rate of increase of new graduates in every year has been 30% since 2002 , so graduating students have faced more and more severe employment difficulties ( geng 2007 ) . indeed , difficulty getting a job has emerged as an ongoing focus of personal concern and anxiety , leading to an increased need for career counseling services ( wu 2008 ) . when faced later with employment demands , they generally exhibit such psychological problems as confusion , anxiety and panic . wen ( 2009 ) interpreted the significance of career guidance in university contexts from two perspectives , ( i ) the needs of students , and ( ii ) development of the university . such career guidance is helpful to them in overcoming any misunderstandings in choosing careers , in selecting their study paths , and in identifying their potential strengths to enhance their competitiveness for positions . effective career guidance provides guidelines with a long - term vision for career planning , from which students can benefit by realizing their true potential in life ( guo 2009 ) . for universities , career guidance can help promote necessary reforms in teaching and can improve employment rates for their graduates , thus enhancing the reputation of a university . solving the problem of employment is not only related to university students and their families , but also to the universities reputations , and even to the country 's politics , construction of the economy , and maintaining a harmonious society ( li and ye 2001 ) . despite its very early start in tsinghua university , career guidance and counseling within most universities in china is still at a fairly elementary stage . this is reflected in the fact that chinese papers and articles on career guidance have been mostly written after 2004 . key words searched in the chinese journal data - base in relation to this study were career guidance in titles or descriptors between 1979 and 2001 . within the decade from 2001 to 2011 , approximately 250 papers about career guidance for university students were published in chinese core journals . it is almost impossible to find any paper on this issue in china written before 2001 . this suggests that research on career guidance in china started late , but then developed very rapidly . in the material that does exist , employment guidance in universities , with just a few writers addressing career guidance in high schools ( e.g. fang and tan ( 2010 ) claimed that papers on career counseling for university students in china are even fewer in number , and generally lack practical strategies or data from empirical studies . articles tend to be confined to investigations of students ' demand for career counseling ( e.g. , zhao and shen 2008 ) , analysis of existing conditions in career guidance ( e.g. , yu 2002 ) , and describing the duties of career counselors ( e.g. , fang and tan 2010 ) . in the areas of both guidance and counseling , the content of many published papers mainly describes possible provisions , often based on models from other countries ( e.g. , li 2002 ) , identifies potential problems ( e.g. , lan and wang 2010 ) , and suggests possible solutions ( e.g. , yu 2008 ) . there is very little reported research about the combination of career planning and selecting appropriate courses , or examples of empirical research on career guidance or counseling strategies . this situation suggests that the theoretical basis for career guidance in china needs to be strengthened ( liu and li 2007 ) . on the other hand , there is evidence in some reports that career guidance and counseling endeavours in some chinese universities appear to have achieved very good results ( e.g. , yu 2002 ) , and these examples could provide a reference model for other universities in china to emulate ( wan and wang 2006 ) . in addition to presenting foreign career theories , a number of papers focus on the current situation regarding career guidance in chinese universities in different regions of china ( e.g. in terms of the methods of career guidance currently in operation , over 90% of 16 universities investigated in beijing have open employment guidance courses for introducing job - hunting skills and employment policy ( liu 2006 ) . other methods of delivering career guidance content to students include ad hoc lectures , campus recruitment fairs , and individual counseling as required . only 25% of universities in beijing provided career assessment services , and in zhejiang province , only three of the surveyed 21 universities provided such assessment ( hong 2007 ) . at the present time , career guidance in universities is mostly delivered via career centres, and through departmental career tutors ( long and song 2007 ) . however , many practitioners consider this traditional model usually fails to satisfy the many needs that students have for career guidance and career counseling . to change the traditional mode of vocational guidance , some writers ( e.g. , long and song 2007 ; sun 2009 ) suggest developing more comprehensive career guidance programmes . at present , a comprehensive approach to employment guidance has begun mainly in beijing normal university , shanghai jiaotong university , renmin university of china , fudan university and guangxi university ( yu 2002 ) . for instance , the model of comprehensive career planning in beijing normal university conducts preliminary guidance about " how to be successful " for freshman , all - round student development education for sophomores , guidance on vocational selection in junior years , with guidance for employment policies and techniques for application in the senior year . in anhui industry university , a three - direction education model comprising ( i ) education for direction of majors when students enter university , ( ii ) education for situation awareness while at university itself , and , finally ( iii ) education for choosing careers when students graduate , operates . the approach to career guidance needs to be flexible and responsive to students needs and interests , and to local conditions . it is necessary to create a network of resources and tools that make career guidance more in - depth , relevant and accessible . one example of this is the occupational information network in the united states ( peterson et al . students at fudan can view 50 different video sessions as part of a career guidance course on - line by entering their individual login names and passwords . this on - line course is in four parts : career assessment , vocations introduction , curriculum study , and career counseling ( sun 2009 ) . that establishment uses careersky software to offer self - service for students in career education . the careersky online career planning system is the most widely used and appears to be reasonably effective ( beisen career assessment 2007 ; sun 2009 ) . based on the problem of insufficient teaching personnel to service the area of career guidance , some universities have built independent learning channels for students , such as a website providing relevant information on career matters , making available appropriate print resources free of charge , and organizing relevant forums ( zhao and shen 2008 ) . internships and work fellowships in companies can also provide valuable opportunities for university students to observe at firsthand and practice the jobs in which they may be interested . naturally , providing this form of off - campus experiential learning is not easy to arrange for large numbers of students . it also relies heavily on the willingness of local companies and worksites to cooperate and provide placements . at the moment there appears to be too little theory underpinning most of what universities and colleges attempt do in the way of career guidance . the core duties of most career guidance centers are confined to providing information and processing employment procedures , such as registering employment whereabouts , guidance on signing contracts , and employment statistics . the service mostly emphasizes introduction to employment policy and analysis of the current employment situation . professional career counseling is rarely provided automatically to all students ; and even some counseling services that are offered are not individualized and focused enough to satisfy students ' needs ( li 2009 ) . there is a lack of well - structured and purposeful guidance to assist students career planning in a practical and personalized way . since there is still a scarcity of teaching materials and teachers texts designed specifically to match existing chinese employment contexts , it is usual for universities to copy western ideas . similarly , assessment tools relevant for career guidance in china have basically adopted three authoritative western theories and measurement scales , namely , holland 's vocational interest theory ( holland 1959 ) , myers - briggs type indicator ( myers 1962 ) , and edgar schein s career anchor ( schein 1978 ) . due to the cultural differences between the west and china such assessment tools often lack local relevance for chinese realities ; and the data they produce are , therefore , of doubtful validity , reliability and usefulness ( zhao and shen 2008 ) . another problem stems from the fact that the number of career guidance professionals currently available is not enough to meet actual demands . also , lan and wang 2010 ) , by 2006 , there were more than 26,000 career counselors in china , but the majority of them worked in social organizations , such as beisen career , with only a small number in universities . in addition , the distribution of career counselors is not balanced across different regions , with more counselors employed in cities . the employment structure in this field shows a trend toward younger counselors , many of who may lack work experience and may not possess the relevant background knowledge in psychology and pedagogy . in one context they examined , among the 70 staff engaged in career guidance , only 12.9% possessed a relevant background in career guidance , 45.7% had worked in career guidance for less than 5 years , and only 17.1% had worked for over 10 years . an additional problem occurs because the concept of career planning is not presented explicitly to students early in their university lives . they do not have a clear understanding of what career planning actually entails , and they have not recognized its importance for their own life and future employment ( li 2009 ) . in order to develop effective and adaptive strategies to improve career guidance in chinese universities , authorities ( e.g. , chen 2007 ; liu and li 2007 ) have pointed out that career guidance and career counseling should not merely try to copy policies and practices from foreign countries . ideally , chinese theories and strategies for career guidance and counseling should be created , with due reference to chinese society 's characteristics and practical problems . indigenous factors that need to be considered include : job market , employment system , educational philosophy , and the social value system ( chen 2007 ) . since these factors will be distinctive for chinese communities , local theories and strategies for career guidance and counseling should reflect , or at least acknowledge , these features this refers to the regular collection of data showing students ' study achievements and their continuing progress . when students are first enrolled in colleges , a dynamic database should be set up for each individual ( sun 2009 ) . electro - portfolio assessment. they suggest that through analyzing and evaluating the data in the system , teachers can get a comprehensive understanding of students development and can guide and support them in accordance with their specific aptitudes and aspirations . a second improvement suggested by qian and fang ( 2009 ) is that universities should introduce more participatory and interactive forms of career guidance , such as a festival of college students ' career planning , including career planning contests , career assessment , interview - style interactive forums , experts and alumni seminars , mock recruitment , and visits to various firms . peer interdependent training course , which involves university associations , and dormitory , friendship and community groups . thirdly , vital to the improvement of career guidance and counseling , is the need to establish a team of career guidance teachers who have high - quality professional knowledge and expertise relevant for college students . the number of professional teachers should be increased , and training of existing staff should be reinforced . department tutors need to be replaced by full - time guidance teachers for fulfilling the functions of employment guidance . experts in career counseling from outside college settings should be hired as necessary to supplement existing teaching staff ( ruan 2009 ) . the existing system of career guidance tends only to emphasize the role of university staff and ignores the impact and importance of family and friends on students career choices . effective career planning systems must be established that include family and parental influences , and also the influence of senior students and classmates . this information can highlight strengths and weaknesses in the career guidance system and can lead to improvements in the content and delivery of information and personal guidance . gao ( 2008 ) pointed out that career counseling within class is an effective way of providing true contextualized career guidance . it brings career planning into daily teaching activities in class and provides many opportunities for students to ask questions , seek information , and share their concerns and experiences within a social group . university tutors should play an important role in career guidance in collaboration with subject teachers . in general terms , current research and practice in career guidance and counseling in china remains at an early stage of development , lacking a sound basis in theory , still learning from foreign experiences , and still drawing upon western methods to a large extent . based on the literature reviewed in this paper , several suggestions can be made for promoting future research and practical efforts in this domain : educational systems , policies and practices are deeply embedded in local histories and cultures ( watts et al . in view of unique educational and social situations in china , foreign experiences and models of career guidance and counseling therefore , the key focus of research on career guidance and counseling in the future needs to explore how best to tailor systems and practices specifically to local chinese contexts.a supportive public policy is critical to the development of career guidance services . at the national level , career guidance should be supported by policy and resources in order that the development of career guidance can be accelerated . at a local level , based on the different characteristics and needs of students in different regions in china , researchers should liaise closely with different types of schools , colleges and universities in different regions and share their findings and opinions with career practitioners . this process could help universities design and implement courses that really meet students needs.career counseling , as an indispensable part and strategy of career guidance , needs to be implemented more professionally and made available more widely . as proposed by watts and van esbroeck ( 2000 ) , there is a general recognition that all counselors will need to use new technologies , including computer - assisted assessment in the diagnostic and self - assessment process , as well as computer - assisted career guidance and counseling via the internet . in the context of china , the use of new technologies is an extremely positive strategy for integrating and making good use of resources in career counseling.other asian regions , such as singapore , japan , hong kong , and taiwan , have set up career guidance systems that are sound in terms of local applicability and in their underpinning theory . through exchanges and dialogue , program designers , researchers and career practitioners in chinese mainland and in these regions educational systems , policies and practices are deeply embedded in local histories and cultures ( watts et al . 2010 ) . in view of unique educational and social situations in china , foreign experiences and models of career guidance and counseling therefore , the key focus of research on career guidance and counseling in the future needs to explore how best to tailor systems and practices specifically to local chinese contexts . such policy is usually determined largely at national level , though it may be devolved to regional , local or institutional level ( watts 2009 ; watts et al . , career guidance should be supported by policy and resources in order that the development of career guidance can be accelerated . at a local level , based on the different characteristics and needs of students in different regions in china , researchers should liaise closely with different types of schools , colleges and universities in different regions and share their findings and opinions with career practitioners . career counseling , as an indispensable part and strategy of career guidance , needs to be implemented more professionally and made available more widely . as proposed by watts and van esbroeck ( 2000 ) , there is a general recognition that all counselors will need to use new technologies , including computer - assisted assessment in the diagnostic and self - assessment process , as well as computer - assisted career guidance and counseling via the internet . in the context of china , the use of new technologies is an extremely positive strategy for integrating and making good use of resources in career counseling . other asian regions , such as singapore , japan , hong kong , and taiwan , have set up career guidance systems that are sound in terms of local applicability and in their underpinning theory . through exchanges and dialogue , program designers , researchers and career practitioners in chinese mainland and in these regions , could gain many useful shared insights in policies , research and practices . to summarize briefly the main points and implications from this review career guidance and career counseling in china started late , but there is now scope for improvement , both in the theory and in practice . significant progress has been made already in some universities and regions , but overall china still lags behind many other nations . not only is it necessary to continue to develop strong and effective career guidance services in universities , more action is also required in schools to provide the first steps in preparing youngsters for later career choices and decisions . all forms of career guidance , career counseling , and career assessment in china must take into account indigenous characteristics of the students , and the realities of local and regional employment opportunities . similarly , indigenous factors need to be considered by researchers investigating or evaluating career services in chinese mainland settings .

sunlight is the most abundant renewable energy resource and underpins life on our planet . nature has evolved complex photosynthetic processes to harvest this light for the generation of chemical energy . while chemical energy underpins the natural world , electrical energy is a more easily transmittable form in human societies . consequently , harvesting of sunlight for the generation of electrical energy is highly desirable , and great developments in the conversion efficiency of photovoltaic cells have been made over the last 50 years . a desire for low - cost , low toxicity , large - area , thin - film technologies has led to organic photovoltaic cells showing promise to satisfy all of these properties . in organic photovoltaics , the absorbing material is a thin ( 100 nm ) layer of organic semiconducting material that is sandwiched between two electrical contacts . light absorbed in the organic layer forms tightly bound excitons that with clever choices of materials and device architectures are split into free electrons and holes , which are then extracted at electrodes to give useful electrical power . they enable simple fabrication either by vacuum sublimation , printing from solution , or spray - coating . thin films of organic semiconductors show a high absorption coefficient of 10 cm , which is very attractive for photovoltaic applications because very little material is needed : layers of only 100 nm can absorb nearly 90% of the incident light in a double pass using reflection from the metal electrode . thin and lightweight photovoltaic panels can be made on a wide range of substrates , including flexible ones . flexibility also enables roll - to - roll printing , easy transportation , and simple installation . chemical synthesis presents almost endless opportunities to tune optical , electronic , and mechanical properties of organic materials through molecular engineering , miscibility , and self - assembly . organic photovoltaic materials and devices for solar energy conversion have been researched extensively in the last 2 decades , and remarkable progress has been achieved , with recorded power conversion efficiencies over 10% for organic solar cells . in order to harvest sunlight for the generation of electrical energy , the active layer of a photovoltaic solar cell has to perform several functions : it has to absorb the solar light that then has to generate free charge carriers , and these carriers need to reach the electrodes to give photovoltage and photocurrent . the highest occupied molecular orbital ( homo ) of a neutral organic molecule in the ground electronic state contains two electrons with opposite spins . upon absorption of a photon ( figure 1a ) , one electron is promoted to the lowest unoccupied molecular orbital ( lumo ) and its spin is conserved ; therefore , the primary photoexcitations have singlet ( spin zero ) character . as they consist of a bound electron a key difference from inorganic semiconductors is that primary photoexcitations in organic semiconductors are strongly bound frenkel and charge transfer excitons . typical binding energies of singlet excitons have been reported to be between 0.1 and 0.4 ev . in order to split an exciton into a charge pair , a heterojunction of electron donor and acceptor materials is used in which the homo and lumo energies of the acceptor are lower than those of the donor ( figure 1b , c ) . this energy offset drives electron transfer from the excited electron donor to acceptor and hole transfer from the excited electron acceptor to donor . its function is analogous to a type ii heterojunction ( staggered gap ) in inorganic semiconductors . for simplicity in organic photovoltaics schematic illustration of charge generation in organic photovoltaic materials which involves ( a ) light absorption and the generation of a singlet exciton with opposite up and down spins , followed by energy transfer to a type ii heterojunction of an electron donor and acceptor and then ( b ) electron or ( c ) hole transfer at the heterojunction . charge transfer can also occur from triplet excitons if the energy level offset is sufficient . it has been shown experimentally that the rate of photoinduced electron transfer ( ket ) decreases exponentially with the distance ( rda ) between donor and acceptor1here k0 is the electron transfer rate at the donor acceptor contact and is the attenuation factor . acceptor systems was found to be bridge - specific and also to depend on the orientation between donor and acceptor as well as their electronic properties . for dispersed donors and acceptors in solution or in organic glasses , as well as for pairs linked through nonconjugated bridges , values of > 1 are usually observed . these results indicate that electron transfer is limited to subnanometer distances between the donor and acceptor . excitons must migrate to a heterojunction between the electron donor and the electron acceptor in order to generate charge carriers . excitons can be transported to a heterojunction by frster resonance energy transfer ( fret ) between the electron donor and electron acceptor and by exciton diffusion in the donor or acceptor materials . two organic photovoltaic architectures of donor and acceptor materials are generally used to harvest sunlight . in the first , shown on the left in figure 2 , the donor and acceptor materials are deposited as a simple bilayer structure , while on the right , the two are mixed throughout the active layer to form a bulk heterojunction . the two main architectures of organic photovoltaic devices and the processes occurring in them ( not to scale ) . on the left is a cross section of a planar heterojunction ; on the right is a magnified region of a bulk heterojunction . in both diagrams , the donor region is light blue and the acceptor region is red , and the key defines the other elements . sunlight incident on the devices leads to the formation of excitons that then diffuse ( and/or transfer energy by fret ) to the heterojunction where charge separation occurs . these processes are discussed in more detail throughout the review . in the bilayer structure excitons can only be harvested at a limited distance from the heterojunction that is determined by the sum of a fret distance ( lfret ) and of an exciton diffusion length ( ld ) . here and throughout this review we use the term light harvesting to mean the successful conversion of absorbed photons into free charge carriers . we emphasize this definition as if any one element of all the processes fails or leads to a bottleneck then the ultimate device performance is determined by it , not the many other steps that are well - optimized ; thus , the concept of light harvesting is important when one wants to connect the fundamental processes occurring in devices with the ultimate performance . in a bilayer , both the diffusion length and fret distance are limited by a finite exciton lifetime to distances typically less than 20 nm in organic films ; therefore , a maximum useful thickness of donor or acceptor layers in a bilayer is < 40 nm , even with an optimized exciton diffusion length and fret . because of a limited width of the absorption spectra of organic materials , for the best coverage of the solar spectrum , one has to use donor and acceptor materials with distinct absorption spectra . a maximum absorption coefficient in organic materials of 10 cm means that a layer of 40 nm can absorb only up to 55% of the incident light in a double pass at the peak and less far away from the absorption maximum . power conversion efficiencies ( pce ) of around 6% have been achieved in research solar cells with 1 cm active area using organic bilayers . it is worth noting that high - efficiency bilayer solar cells typically have a rough interface between donor and acceptor layers ( about 16 nm rms ) that increases the interface area and helps to harvest excitons from donor and acceptor layers . the roughness occurs naturally by vacuum sublimation of polycrystalline films or by sequential deposition of donor and acceptor layers from solution using different solvents for each layer . the limited number of excitons reaching the acceptor gives poor light harvesting in bilayers and so severely limits their pce . an elegant solution to improve light harvesting in planar heterojunctions is to use multilayer architectures of materials with efficient fret between layers . recently , a pce of 8.4% has been achieved with a three - layer structure comprising two electron acceptors and a donor and utilizing fret between the acceptor layers . we describe this approach in more detail in section 2.5 . an alternative way to improve light harvesting is to increase the interface area of the heterojunction even more than the roughness of the layers allows . this has been successfully implemented by blending electron donor and acceptor materials , where spontaneous separation of donor and acceptor phases forms a nanostructure that is known as a bulk heterojunction . this approach ( figure 2 , right ) enables light harvesting from materials with a short exciton diffusion length . blends can be deposited from solution using a common solvent or a mixture of solvents or by coevaporation of donor and acceptor materials . in the overall schematic shown in figure 2 , the bulk heterojunction is shown at a nanoscale level , with a donor phase ( light blue ) surrounded by an acceptor phase ( light red ) . absorption of light in the donor phase leads to the formation of excitons ( dark blue ellipse ) that may be some distance from the acceptor phase . if the two components mix well , then every single donor molecule is close enough to an acceptor molecule and almost every exciton can migrate to the donor this can proceed by site - to - site exciton diffusion within the donor phase until the exciton reaches the interface with the acceptor , or it can occur by direct fret from the donor to the acceptor . the small length scale of the bulk heterojunction phase separation enables good harvesting of excitons , with only a few example tracks for the drawn excitons in figure 2 being lost , with most harvested ( some easily and some only just , depending on the initial position and the path followed ) . this contrasts with the bilayer case , where many are lost inside the donor layer unless it is significantly thinner than needed for light absorption , as already discussed . in the bulk heterojunction , once the exciton is at the donor this charge pair has to overcome coulomb attraction in order to separate into free charges . the holes are transported in the donor phase and electrons in the acceptor phase ; therefore , the charge transport pathways in the planar heterojunction solar cell are straightforward provided there is a sufficient built - in electric field from the difference of the work functions of the electrodes . recent studies of bulk heterojunctions have shown that the blend morphology has to be just right for the charge pair to dissociate into free carriers , and we discuss this in more detail in section 4.2 . the free carrier generation and their transport to the correct electrodes in bulk heterojunctions relies on continuous and uninterrupted donor and acceptor phases . even with continuous charge transport pathways , both electrons and holes must navigate the labyrinth of the complex morphology to the electrodes . if a free electron encounters a hole , they form a nongeminate pair that can recombine or dissociate again into free carriers . the rate of nongeminate charge recombination in bulk heterojunctions depends on its morphology . in a heterojunction with small donor and acceptor domains and very high interface area , a charge is more likely to encounter the interface with the other component of the blend and hence more likely to recombine with its opposite charge . this complex series of steps involved in light harvesting has led to intensive research efforts on bulk heterojunction solar cells , and recently , multiple 1 cm active area research cells have been demonstrated with pce above 10% . the progress was achieved by rational design of donor and acceptor materials with efficient light absorption and good charge transport , morphology optimization of the active layers , and development of new charge transporting layers . recent advances in bulk heterojunction solar cells using conjugated polymers as electron donors are described in a review by yu and co - workers . in summation , the key steps of converting light into electrical power in organic photovoltaics are ( a ) light absorption , ( b ) energy transfer ( by exciton diffusion and/or fret ) to a heterojunction , ( c ) exciton splitting into an electron hole pair , ( d ) dissociation of bound charge pairs into free carriers , and ( e ) charge extraction to the electrodes . in this review we discuss recent advances toward understanding of processes b , c , and d and improving their efficiencies in planar and bulk heterojunctions . we present an overview of energy transfer mechanisms , methods to measure exciton transport , and recent efforts to improve light harvesting in planar heterojunctions by enhancing the exciton diffusion length , by encouraging interlayer fret , and by directing exciton diffusion . we briefly discuss harvesting of triplet excitons , which now attracts substantial interest when used in conjunction with singlet fission , where one high - energy singlet exciton can generate two triplet excitons and so double the charge carrier yield . then we review the main experimental findings and inferred mechanisms of charge pair generation and dissociation into free carriers . in particular , we discuss the role of the morphology of bulk heterojunctions , which has to be just right for the best solar conversion efficiency . we introduce the main techniques for measuring the morphology of bulk heterojunctions and discuss optimized morphologies for photovoltaic performance . charge extraction and nongeminate recombination are not discussed here , and we refer the reader to recent reviews on these topics . light capture by microstructures and plasmonics are also excluded from this review but have been covered in detail recently by others . because electron transfer in organic materials occurs only at subnanometre distances between molecules , excitons must migrate to a heterojunction between the electron donor and the electron acceptor in order to generate charge carriers . in this section we discuss the mechanisms of energy transfer , the techniques to measure exciton diffusion , and recent efforts to enhance the exciton harvesting range by increasing their diffusion length and by directing exciton transport . , energy transfer involves emission of a photon from one molecule and absorption of that photon by another molecule . nonradiative energy transfer occurs by two different types of intermolecular interactions : ( 1 ) coulombic interaction of a transition dipole moment responsible for the luminescence of an exciton with the absorption transition dipole of a nearby molecule and ( 2 ) electron exchange interactions , which involve simultaneous exchange of electrons in homos and lumos between nearest neighbors ( figure 3 ) . schematic illustration of nonradiative frster resonance energy transfer of singlet exciton s1 and dexter transfer of triplet exciton t1 . the up and down arrows illustrate electron spins , and s0 denotes a chromophore in the ground state . energy transfer by coulombic coupling is usually referred to as frster resonance energy transfer , in recognition of the contribution of the german scientist theodor frster to the theory . in the weak coupling limit the rate of fret can be calculated using the fermi golden rule2where v is the electronic coupling energy between the exciton and energy - accepting chromophore ; s is the dielectric screening of the interaction by the surrounding medium ; j is the spectral overlap between the homogeneous spectral profiles of exciton luminescence f(e ) and absorption of energy - accepting chromophore a(e ) , which have each been normalized to unit area on an energy scale , ; and emax is the upper energy of the luminescence and absorption spectra . in a dipole approximation , s = 1/n , where n is the refractive index of the surrounding medium , typically in the range of 1.52 for organic semiconductors . when the emitting and absorbing dipoles can be approximated as point dipoles , the energy transfer rate has a simple expression3where is the luminescence lifetime of the excited chromophore in the absence of energy transfer , r is the distance between the exciton emission dipole and the energy - accepting dipole , and r0 is the frster radius , which depends on the photoluminescence quantum yield of the excited chromophore , the angle between the donor emission and acceptor absorption transition dipole moments , and the spectral overlap j , which was defined above . the point dipole approximation is only accurate when the distance between the interacting chromophores is much larger than the size of the exciton . when these distances are comparable , calculations of the electrostatic interaction between transition dipoles require more sophisticated methods , such as transition density calculations or multicentric monopole expansion . for elongated chromophores , such as conjugated polymers , while fluorescent singlet excitons move predominantly by fret , triplet excitons have an electronic spin of 1 and therefore are spin - forbidden from emitting light . the absorption of neutral molecules from a singlet ground state to a triplet excited state is also spin - forbidden . as efficient fret requires strong spectral overlap of absorption and emission , fret from the triplet to singlet state is inefficient , and triplet the dominant transport mechanism of triplet excitons is by electron exchange , which is often referred to as dexter transfer . this requires an overlap of molecular orbitals of neighboring molecules ; therefore , the transfer rate decreases exponentially with a distance between molecules and operates effectively only over a distance of less than 2 nm . localized excitons move by spontaneously hopping from one site to another by fret or dexter transfer . however , in an ideal molecular aggregate , when the interaction energy between molecules is higher than the energetic disorder and the dissipation of electronic excitation energy is slow , the exciton is delocalized between participating units and can transfer energy coherently in a wavelike motion . recent experiments suggested that coherent energy transfer takes place in photosynthetic pigment protein complexes . macroscopic spatial coherence reaching tens of micrometers has been observed in ordered single chains of a conjugated polymer polydiacetylene at cryogenic temperatures . several experimental studies suggested that primary excitons in conjugated polymers are delocalized along the chain in the first 100 fs after excitation , even at room temperature . theoretically it has been shown that correlated fluctuations of electronic transition energy in molecular aggregates can help to preserve the electronic coherence between excitonic states and only uncorrelated fluctuations result in loss of coherence . correlated exciton relaxation has been reported in poly(3-hexylthiophene ) , which suggests that indeed coherent energy transfer can occur in conjugated polymers on a 100 fs time scale . the signatures of delocalized primary excitons have also been reported in helical -stacks of self - assembled perylene bisimides and in star - shaped molecules . generally , exciton localization on fewer units of a polymer or an aggregate is observed in 100300 fs at room temperature and is attributed to exciton self - trapping driven by structural relaxation of excited units . however , recent observations of efficient exciton transport over micrometer - scale distances in individual self - assembled nanofibers suggest the existence of delocalized singlet excitons at room temperature in well - ordered h - type aggregates which significantly enhance the distance of energy transfer . nevertheless , in most circumstances , excitons hop from chromophore to chromophore in the material , and methods to measure this diffusion are the topics under discussion in the next sections . in this section , we review the main techniques to measure the distances over which singlet and triplet excitons can be transported in their finite lifetime . for excitons , which move by random walk , the convention is to quote their average diffusion length , where d is the exciton diffusivity , is the exciton lifetime , and z = 1 , 2 , or 3 for one - dimensional , two - dimensional , and three - dimensional diffusion , respectively . in the case of time - dependent exciton diffusivity , the integral of d over the time interval should be used . a film of the organic material under study is deposited on a surface that quenches the luminescence of the organic material , as depicted in figure 4 . for this measurement , the advantage of this technique is that it measures energy transfer to the donor acceptor interface in a bilayer and is directly linked to light harvesting in planar heterojunctions . the quenching can be measured by either steady - state or time - resolved photoluminescence ( pl ) . in steady - state measurements , one has to take into account a variation of the amount of light absorbed because of the optical interference , which in turn requires a detailed knowledge of optical constants and a calculation . the overall quenching dynamics involves two consecutive processes : exciton transport to the surface and quenching by charge transfer to a quencher or by fret . to measure exciton transport it is very important that the exciton is quenched very quickly at the interface so that the quenching dynamics are transport - limited . when quenching at the interface is slower than exciton transport , the overall quenching kinetics are monoexponential and very different from transport - limited quenching . time - resolved measurements have an advantage in being able to distinguish between the transport - limited quenching and surface - limited quenching . ( a ) varying thicknesses of the material of interest ( blue block in the stack ) are deposited on top of a quencher material ( red block ) , and the time - resolved pl decays are measured ( schematically shown in panel b ) and compared against a reference film of the material prepared without the quencher layer . the thickness dependence of the time - resolved quenching can be used to determine the exciton diffusion coefficient . shown in panel c are experimental data of surface quenching in 6.5 , 12 , and 32 nm thick films of the polymer p3ht deposited on titania ( the quencher ) . if exciton transport is studied in an electron donor material , then it is best to use a layer of an electron acceptor as a quencher and vice versa . the luminescence intensity at time t is proportional to the concentration of excitons , n(x , t ) , integrated over the thickness of the film ; here x is the distance from the exciton to the quencher surface . it is assumed that excitons move by a random walk , and the results can then be modeled using a one - dimensional diffusion equation4where g(x , t ) is the exciton generation rate , d is the exciton diffusivity in the direction perpendicular to the quenching interface , kq(x ) is the rate constant of surface quenching , which depends on the distance to the quenching interface , and ks is the rate constant of spontaneous exciton decay measured in the reference film of the same thickness but on a nonquenching surface ( for example , fused silica ) . the fluorescence intensity at time t is proportional to the integral of n(x , t ) over the thickness of the film . in the simplest analysis and estimation of the exciton diffusion length , it is usually assumed that exciton diffusivity is time - independent ; however , different models have been proposed to describe time - dependent exciton diffusivity in materials with significant energetic disorder , and we discuss this in more detail in section 2.3 . to study exciton diffusion in electron donor materials it is common to use a layer of fullerene molecules as a quencher , which can quench by electron transfer and fret . when using a vacuum - deposited c60 fullerene as a quencher , care must be taken to avoid interdiffusion of c60 molecules with the donor molecules , which would lead to an overestimation of the exciton diffusion length ( figure 5 ) . for this purpose , fluorescence quenching efficiency in the 26 nm polymer film as a function of time after evaporation of c60 on top . the inset shows the increase of the apparent exciton diffusion distance with time , which indicates intermixing of the evaporated c60 molecules with the soft polymer layer . copyright 2005 american chemical society . when a long - distance fret to the quencher is active as a quenching mechanism , it has to be included in the rate constant kq(x ) in eq 4 to get an accurate measurement of the exciton diffusion length . for example , luhman and holmes have obtained consistent values of the singlet exciton diffusion length in films of boron subphthalocyanine chloride using three different electron acceptors with very different frster radii r0 for fret to the acceptor , which they measured independently . sometimes it is referred to as bulk quenching because it uses dispersed quenchers of pl in the bulk of the film and therefore measures three - dimensional exciton diffusion . very low concentrations of quenchers are used to ensure that they are homogeneously dispersed throughout the film . the measurement involves recording time - integrated or time - resolved pl intensity with varying concentration of the quencher , as depicted in figure 6 . schematic illustration of the volume - quenching technique in which quenchers are dispersed throughout the layer of the material to be studied . shown in panel a from left to right are three films of the material under investigation ( blue block ) with increasing concentrations of a dispersed quencher ( red solid circles ) . excitons are more readily quenched with increasing quencher concentration , and this is measured with time - resolved pl and compared against a reference film without any dispersed quenchers , as shown in panel b schematically and in panel c as experimentally measured data of the polymer pcdtbt mixed with the quencher pc71bm . time - resolved pl measurements have several advantages over steady - state measurements : they enable the quenching mechanisms and time - dependence of exciton diffusion to be studied , they are less susceptible to thin - film interference effects , and modeling of the data is strongly constrained , as an entire decay curve is obtained for each concentration of quencher . the pl intensity at time t is proportional to the exciton concentration n(t ) . the decay rate of excitons in a blend is the sum of the spontaneous decay rate with the rate constant ks and a quencher - induced decay rate with the rate constant kq5where g is the exciton generation rate and q is the quencher concentration , which is , of course , time - independent . for such a monomolecular quenching process , the pl quenching rate constant kq is easily isolated from the rate constant of the unquenched , spontaneous decay of excitons by differentiating the natural logarithm of the pl ratio using6where ndoped is the exciton density in the film doped with quenchers and npristine is the exciton density in the pristine reference film . with this technique , it is particularly important that the exciton is quenched on the first encounter with the quencher and to include all active quenching mechanisms in the analysis . for example , long - distance fret to the quencher has to be taken into account , as well as exciton diffusion to the quencher , and analytical expressions for kq exist for both processes . alternatively , monte carlo simulation of 3d exciton diffusion in the blend can be used to fit the quenching dynamics and to determine exciton diffusivity . this approach can be used even when the quencher distribution is not uniform but it has to be included in the model . showed that the quenching rate of pl in films of the conjugated polymer ptb7 by electron transfer is at a maximum for an energy offset of 0.4 ev between the electron affinities of ptb7 and acceptor molecules . this dependence was described well by the marcus theory of electron transfer . for the optimum energy offset , the exciton is quenched on the first encounter with the quencher and the above - described analysis can be used . however , when the energy level offset is not an optimum and the rate of electron transfer is lower , the exciton can escape quenching by diffusing away . in some cases , it is possible to include an exciton escape rate into the analysis . if that is not possible , then the measured exciton diffusivity and diffusion length can only be considered as a lower limit . this process occurs at high excitation densities when one exciton transfers its energy to another exciton and brings it to a higher - energy excited state . the higher - energy excited state relaxes rapidly , and overall an exciton is lost . in so far as quenching occurs throughout the volume of the sample , this measurement resembles volume quenching , but with excitons acting as their own quenchers . in these experiments , typically high light intensities are used , far higher than used under solar illumination conditions , and the consequences of this have to be taken into account when analyzing the data . for example , there is a finite probability for a singlet exciton in the higher - energy excited state to split into an electron hole pair or into two triplet excitons ; in that case , both singlet excitons are lost . hole pairs and triplets can also quench singlet excitons ; hence , very high light intensities should be avoided . the process can be measured using time - resolved photoluminescence , as its intensity is proportional to exciton population ( figure 7 ) , or transient absorption , which can probe stimulated emission from an exciton or its absorption to higher excited states . schematic illustration of exciton exciton annihilation measurement methodology to determine the exciton diffusion coefficient . ( a ) the material under investigation ( blue block ) is excited with a laser of increasing power ( left to right ) . at higher powers as they diffuse they will meet and annihilate , producing a time dependence of the pl intensity as shown schematically in panel b. if this is compared with a reference pl decay taken at low power , then the exciton diffusion coefficient can be derived . experimental data from films of p3ht are shown in panel c and are reprinted with permission from ref ( 65 ) . when excitons can be treated as point particles and energy transfer occurs by incoherent hopping , the exciton concentration n is described by a kinematic rate equation7where g is the exciton generation rate , ks is the exciton decay rate in the absence of annihilation , and is the annihilation rate . exciton annihilation involves two consecutive processes : multistep exciton hopping from an excited to an unexcited chromophore ( exciton diffusion ) and energy transfer to an excited chromophore ( annihilation ) . when exciton diffusion is restricted to one dimension ( 1d ) a time dependence of t is observed . the same time dependence is observed in a three - dimensional system if excitons are immobile and exciton exciton annihilation occurs by direct frster energy transfer onto an excited chromophore ( static annihilation ) . the strong time - dependence of in both cases can be explained by a nonuniform spatial distribution of excitons because of the fast annihilation of excitons at the nearest distance ; in that case , the annihilation process depends on the history of excitation dynamics and is therefore non - markovian . in the case of fast exciton diffusion in three - dimensional ( 3d ) systems , a uniform distribution of excitons is quickly restored by exciton diffusion and the annihilation rate is time - independent on a long time scale . when excitons annihilate on the first encounter , the annihilation rate is limited by diffusion and in 3d systems can be described by the solution of the smoluchowski equation8where d is exciton diffusivity and ra is the annihilation radius . here it is assumed that only one exciton is lost per encounter . for typical values of d 10 cm s and ra 1 nm , the time - dependent component becomes negligible for t > 16 ps and eq 8 is reduced to the time - independent form of 3d = 4rad . a time - independent annihilation rate has been observed at long times in films of poly(3-hexylthiophene ) ( p3ht ) as well as other polythiophenes and poly(phenylenevinylene ) derivatives , which indicates that annihilation is controlled by 3d exciton diffusion . at early times after excitation for example , masri et al . observed a decrease of the value in the first 20 ps after the excitation pulse in p3ht films and fitted it to an equation derived for diffusion - limited annihilation in the case of preferentially 1d diffusion and a spherical annihilation volume with a radius ra(86)9here dz is a high diffusivity in a preferred direction and dp is a much lower diffusivity in a direction perpendicular to z. they obtained dz 100dp and attributed dz to exciton diffusion along the -stack of p3ht chromophores ( figure 8) . time dependence of exciton population n(t ) in p3ht film measured for different excitation densities by ( a ) time - resolved fluorescence after excitation at 2.2 ev and ( b ) transient absorption after excitation at 2.4 ev . ( c ) annihilation rate obtained from time - resolved fluorescence data using eq 7 and ( d ) annihilation rate obtained from transient absorption data using eq 7 ; solid line and dotted lines are simulated rates using eq 9 with dp and dz values given in the legend . reprinted with permission from ref ( 86 ) . exciton annihilation in p3ht films but used selective excitation of the crystalline regions of the polymer with low - energy photons at 2 ev . they observed the t time - dependence up to 100 ps and attributed it to strictly one - dimensional exciton diffusion in the crystalline domains ( figure 9 ) . this time - dependence of has been observed in the quasi - one - dimensional organic semiconductor phthalocyanines and in 4,9,10-perylenetetracarboxylic dianhydride . annihilation rate for a p3ht film excited at 2 ev obtained using eq 7 . the red line represents the fitting curve using t. the blue and green lines represent the annihilation rate coefficient calculated by the 3d ( eq 8) and 2d models , respectively . these results indicate that the technique is useful not only to estimate the exciton diffusivity but also to evaluate the dimensionality of energy transfer . like other techniques for measuring exciton diffusion , exciton exciton annihilation has various advantages and disadvantages . an important advantage is that the measurements can be made on a single sample . a serious disadvantage is that in order to determine the diffusion coefficient , the annihilation radius needs to be known and there are not convenient ways of determining it independently . the vast majority of measurements of exciton diffusion have used experiments such as those described above ; however , under some circumstances , it has been possible to image the motion of excitons directly . the basic idea of these experiments is very simple : excite a small spot , image the resulting fluorescence , and watch it spread out . of course in practice it is challenging , because the exciton diffusion length is small compared with the excitation spot . this type of experiment has mainly been applied to materials with high diffusion coefficients , such as molecular crystals . ern et al . studied triplet diffusion in anthracene crystals , by detecting delayed blue fluorescence resulting from triplet triplet annihilation . by varying the spatial distribution of the exciting red light , they were able to deduce a diffusion constant of 2 10 cm / s for triplet diffusion in the ab plane on anthracene crystals at room temperature . direct imaging of the motion of excitons and trions ( negatively charged excitons ) was studied by sanvitto et al . and pulizzi et al . in gaas / algaas quantum wells at a temperature of 4.2 k. they used a 40 microscope objective to focus the beam of a ti : sapphire laser to a spot of 1.6 m diameter , and the same objective imaged the spot through a spectrograph to give spatial information on one axis and spectral information on the other . the neutral excitons were found to be more diffusive than the trions and spread out to a total spatial extent of 10 m , corresponding to a diffusion coefficient of 120 cm s. it should be remembered , however , that this value is for an inorganic semiconductor at low temperature ; it serves to demonstrate the technique , though it is not in itself relevant to opv . excitation from a steady - state laser was focused using a microscope objective , and the same objective imaged pl onto a ccd camera . triplet annihilation , with many of the triplets forming as a result of singlet fission . in contrast to the work of sanvitto and pulizzi , spectral selection was provided by a filter , thereby allowing exciton diffusion throughout the plane ( rather than just in one direction ) to be imaged . the upper panel shows the excitation spot , and the lower panel shows the pl detected . the emission has spread much more in the b direction than in the a direction : the exciton diffusion is highly anisotropic and the elliptical shape of the lower panel directly shows this . the spread to 2 m in each direction actually corresponds to triplets diffusing 4 m from the center because the measurement detects delayed fluorescence due to triplet triplet annihilation , which is a bimolecular process and therefore follows the square of the triplet density . these results show exciton diffusion over micron distances at room temperature in steady - state measurements on the organic crystal , rubrene . however , these authors noted they were not able to observe exciton diffusion directly in tetracene . direct imaging of exciton diffusion . contour plot of the intensity distribution of the excitation light ( a ) and the of the pl at the surface of a rubrene crystal ( b ) . reprinted with permission from ref ( 99 ) . refined the above measurement in two ways and then applied it to image exciton transport in tetracene . the refinements were to increase the magnification to 500 , thereby improving the spatial resolution , and to make time - resolved measurements . mller and bardeen had shown that a streak camera could be used to image the motion of molecular excited states with picosecond time resolution and 150 nm spatial resolution , but akselrod used a simpler approach of translating an avalanche photodiode ( apd ) across the image of the emission . although the excitation spot is diffraction - limited , its spread can be imaged with subwavelength resolution . at first this seems surprising because of the abbe criterion , which gives a wavelength - scale limit on resolving two spots . however , monitoring exciton diffusion does not require two spots to be distinguished , so more accurate measurements are possible and indeed are now commonly demonstrated in super - resolution microscopy . by translating the apd across the image of the emission spot , the spreading of the emission could be resolved in time , leading to the results shown in figure 11 . panel a shows the emission pattern as it evolves in space and time along the crystal a axis . the distribution at a particular time has been normalized to emphasize changes in the distribution width . panel b shows cross sections of the emission intensity map at four time points showing spatial broadening of the intensity distribution . panel c shows the time evolution of the mean square displacement of triplet excitons . the initial intensity distribution was described by a gaussian with a width of 229 nm that rapidly broadened in the first 2 s , followed by a slower expansion to 701 nm at 7 s . in the first 2 s , the process could be described by a diffusion coefficient ( in the a direction ) of ( 1.35 0.01 ) 10 cm / s . when combined with the triplet lifetime of 1.37 s for the crystal studied , this leads to a diffusion length (2dt ) of 0.61 m . the authors note that ultrapure tetracene can have lifetimes up to 58 s , which for the same diffusion coefficient would enable diffusion up to 4 m . as in the case of other exciton diffusion measurements , time - resolved measurements provide additional information , in particular how the excitons spread as a function of time . this is summarized in panel c of figure 11 , where it is found that exciton diffusion becomes slower at longer time , and this is attributed to trapping associated with energetic disorder . the anisotropy of exciton diffusion is also reported by making the same measurement along different crystal directions . for the b direction , the diffusion coefficient is ( 2.28 0.07 ) 10 cm / s , and for the c direction , it is ( 0.31 0.02 ) 10 cm / s . a transient absorption microscope has also been used to image exciton diffusion in tetracene . in this experiment , the pump beam is held at a fixed position and the probe beam is scanned to form an image . this measurement works best on picosecond and few nanosecond time scales and so allows both singlet and triplet dynamics to be studied . for delay times between 1 and 7 ns , the triplet diffusion coefficients appeared to be more than an order of magnitude higher than those deduced from transient luminescence . the authors propose that this effective enhancement of triplet exciton transport on picosecond and nanosecond time scales is due to the interconversion of triplet and singlet excitons enabling singlets to assist in triplet diffusion . modeling of their data suggests that the singlet - mediated process alone would give a diffusion length for triplets of 1.9 m . pure triplet diffusion alone would give a diffusion length of 5.4 m if a lifetime of 62.5 s is assumed , and the two processes together would give a diffusion length of 5.6 m . however , so far they have been only been applied to a very restricted range of organic semiconductors with rather high order . it would be much more demanding to apply them to the most widely used opv materials , including conjugated polymers , so there is a continuing need for the many other techniques described in this section . as explained at the start of this review , charge generation in an organic solar cell arises primarily from the migration of photogenerated excitons to a heterojunction ; hence , device characteristics can be used to learn about exciton diffusion . in a planar heterojunction , only excitons within a diffusion length of the heterojunction will reach it and contribute to photocurrent . the situation is very similar to surface quenching measurements described above , except that the loss of the exciton is detected by current instead of as a loss of fluorescence . by studying the influence of layer thickness and excitation wavelength it is possible to estimate the exciton diffusion length , though typically it is necessary to assume that all the charges generated at the interface are extracted from the device . this type of approach was applied to molecular crystals in the 1960s . in an extensive and detailed study of anthracene , mulder used steady - state excitation spectra of fluorescence and of photocurrent as methods for measuring exciton diffusion lengths . the results were analyzed in terms of a diffusion model that also included reabsorbance of fluorescence . the results of the exciton diffusion length deduced from fluorescence and from photocurrent were compared . the latter appeared to depend on the choice of electrode , but it was found that consistent results could be obtained by the use of alkaline electrode solutions . the resulting exciton diffusion length was in the region of 30 nm and was consistent with values obtained from fluorescence studies . the exact value depended on the method of preparation of the crystal and the orientation measured . higher ( but unreliable ) values were obtained for nonalkaline electrode solutions , which was attributed to charge generation not only occurring at the surface ( as assumed in the exciton diffusion model ) but also at deeper centers such as oxygen molecules and hydrogen ions penetrating from the ( acidic ) electrode solution into a thin layer of the crystal next to the electrode . photocurrent studies were applied to study the effect of introducing dopants on exciton diffusion and to study other materials , obtaining a diffusion length of 29 nm for tetracene and 10 nm for -perylene . ghosh and feng also derived diffusion equations and used photocurrent excitation spectra to study exciton diffusion . in their case , they studied a solar cell consisting of a layer of merocyanine in between aluminum and silver contacts . the main differences from the earlier work are the materials studied , the contacts used , and the fact that the built - in potential of the contacts rather than an applied external field was used to extract charge . using the spectrum from 440 nm to longer wavelengths , an exciton diffusion length of 6 nm was deduced . the same approach was used by bulovic and forrest to study excitons in 3,4,9,10-perylenetetracarboxylic dianhydride ( ptcda ) . one was assigned to a self - trapped charge transfer state with a diffusion length of 225 15 nm ; the other was a triplet with a diffusion length of 88 6 nm . modeling of the photocurrent in an early polymer solar cell consisting of the polymer poly(p - phenylenevinylene ) [ ppv ] and a perylene acceptor layer was performed by halls and friend . it was assumed that all excitons excited within a diffusion length of the interface are ionized at the interface , lead to separated charges , and are then extracted . the resulting exciton diffusion length was 9 1 nm . a fuller model of the photocurrent excitation spectrum of a polymer solar cell was described by pettersson et al . the treatment of exciton diffusion and photocurrent generation is similar to that of the earlier papers , but the paper pays particular attention to modeling the electric field distribution inside the device , which of course also determines the spatial profile of photogenerated excitons . the optical model used complex indices of refraction and layer thicknesses determined by spectroscopic ellipsometry . the work also found that it was important to take into account the optical field distribution and found that both layers in their device contributed to the photocurrent . the resulting values of the diffusion length were 4.7 nm for poly(3-(4-(1,4,7-trioxaoctyl)phenyl)thiophene ) ( peopt ) and 7.7 nm for c60 . we note , however , that the latter value is lower than the value of 20 nm in another report . many subsequent studies have followed the approach of using a full optical and diffusion model of photocurrent excitation spectra to deduce exciton diffusion length . yoo et al . reported an exciton diffusion length of at least 60 nm . subsequently it has become clear that pentacene undergoes singlet fission , leading to the generation of two triplets . after taking account of this effect , tabachnyk et al . measured a triplet exciton diffusion length of 40 nm in pentacene . exciton transport in nonluminescent materials can be studied using a planar heterojunction to generate charge carriers and to detect them instantly with electrodeless techniques , so that the response time is limited by energy transfer to heterojunction . researchers at delft university of technology have used a microwave probe to measure an increase in conductivity in the sensing layer when an exciton reaches it and injects charge . only excitons within a diffusion length of the heterojunction or fret distance a singlet diffusion length of 7 nm has been found in evaporated c60 films and a triplet diffusion length of 28 nm in palladium tetrakis(4-carboxyphenyl)porphyrin ( pdtppc ) films . alternatively , transient absorption can be used to detect radical cations or anions in a planar heterojunction that is within an exciton diffusion length of a heterojunction . because of the conformational and positional disorder present in organic semiconductors , as well as dispersive interactions between molecules that are caused by rapid fluctuations of electron densities , the same electronic state has slightly different energies on different chromophores . singlet exciton transport occurs predominantly by fret - controlled hopping from chromophore to chromophore . according to eqs 2 and 3 , in order to get fast exciton hopping , one requires a high oscillator strength of luminescence and absorption , a short distance between chromophores , and high spectral overlap of homogeneous spectral profiles of luminescence and absorption . two factors reduce the spectral overlap : the reorganization energy of individual chromophores in the excited state and energetic disorder . the spectral overlap is high for excitations hopping downhill in transition energy , whereas it is much smaller for excitations hopping uphill and often needs thermal activation to get to the resonant transition energy . many theoretical and experimental studies have explored the influence of energetic disorder on exciton diffusion . more than 30 years ago movaghar et al . derived an analytical theory to describe the time dependence of exciton diffusion by incoherent hopping by assuming uncorrelated free energies of adjacent sites and a gaussian distribution of site energies.figure 12 shows the diffusivity as a function of time for different values of the disorder width normalized to the thermal energy . the theory predicts two different time regimes of exciton diffusion : an initial dispersive regime , where exciton diffusivity is decreasing with time when excitations migrate downhill in energy , and an equilibrium regime with a constant diffusivity d , where transport occurs through low energy sites . they found that the crossover time tr to long - time behavior ( marked by an arrow on each curve ) and diffusivity at long time d roughly obey the relation trd const with = 0.45 . the analytical theory was compared with monte carlo simulations in a cubic lattice , and very good agreement was obtained . using a simple thermodynamic argument , movaghar et al . predicted that the mean long - time energy is lower than the peak energy of the gaussian density of states by a value e = a / kt , where a is the half - width of the gaussian disorder and kt is the thermal energy . plot of log10 of the diffusivity d(t ) versus log10 of time for different values of a / kt , where a is the half - width of the gaussian disorder and kt is the thermal energy ; the crossover time to long - time behavior d is denoted by an arrow on each curve ; the time axis is scaled with 0 , which is the hopping rate downhill in energy . the inset shows the long - time ( equilibrium ) value d(t ) and the crossover time tr plotted versus ( a / kt ) . the gaussian disorder model described above has been very successful in describing the time evolution of photoluminescence and phosphorescence spectra at different temperatures and the temperature dependence of the time - averaged exciton diffusivity . for example , mikhnenko et al . measured the time - averaged diffusivity of singlet exciton in films of the poly(p - phenylenevinylene ) derivative mdmo - ppv using surface quenching of the photoluminescence and found that the exciton diffusivity decreased by a factor of 2 when cooling the sample from room temperature to 150 k ( figure 13 ) . they explained this result by a decrease of the thermally activated hopping rate at lower temperature . below 150 k , exciton diffusivity was almost temperature - independent , suggesting that thermally activated hopping is inefficient at very low temperature and excitons migrated only downhill in energy . they also found a strong correlation between the temperature dependence of exciton diffusivity and the pl ( 00 ) vibronic peak position , suggesting that the width of energetic disorder can be estimated simply by measuring the temperature dependence of the pl spectrum . observed a similar temperature dependence of the time - averaged hopping rate of triplet excitons in phosphorescent iridium - cored metal ( a ) temperature dependence of the exciton diffusion length ld ( red circles ) and the time - averaged diffusivity d ( green squares ) in films of the poly(p - phenylenevinylene ) derivative mdmo - ppv . ( b ) temperature dependence of the time - integrated photoluminescence spectrum vibronic ( 00 ) peak position . two temperature regimes are observed : a low - temperature regime ( up to 150 k ) highlighted in blue and a high - temperature regime . later models have adopted the gaussian disorder model but used eq 2 or 3 to calculate the hopping rate with a time - dependent frster radius r0 or spectral overlap term j used instead of a boltzmann term . this approach better describes an exciton hopping downhill in energy on an early time scale after excitation . the one - dimensional diffusion length of singlet excitons in organic films typically ranges from 5 to 20 nm . to absorb most of the incident light , the thickness of donor and acceptor layers has to be at least 100 nm ; therefore , there is a strong drive to increase the exciton diffusion length in order to improve light harvesting in planar heterojunctions . in addition to the strength of electronic coupling between molecules , two other main factors influence exciton diffusivity . these are the spectral overlap integral of the absorption and fluorescence spectra ( term j in eq 2 ) and energetic disorder . . showed a direct correlation between the spectral overlap integral ( term j ) and the exciton diffusivity in triphenylamine derivatives with different side arms . they observed an enhancement of diffusivity by a factor of 5 and have obtained a good agreement with monte carlo simulations using the measured optical properties of the films . generally longer diffusion lengths are observed in crystalline materials , and this can be explained by lower energetic disorder that results in higher exciton diffusivity on a long time scale where energy transport is thermally activated . sometimes it is possible to increase the exciton lifetime and maintain high diffusivity . menke et al . showed an increase of the exciton diffusion length from 11 to 15 nm in the electron donor boron subphthalocyanine chloride by diluting it into a wide - energy - gap host material ugh2 . the main reason for the increased exciton diffusion length was a 6-fold increase of the exciton lifetime . using this approach , they made planar - heterojunction solar cells with the fullerene c60 as an electron acceptor and obtained a power conversion efficiency of 4.4% , which is 30% higher than that of the control cell made using undiluted subphthalocyanine molecules . however , dilution of chromophores does not always help to increase exciton lifetime . for example , the dilution of metal - free phthalocyanine molecules in ugh2 to 25% resulted in a 3-fold decrease of the exciton lifetime , showing that the nonradiative decay of singlet excitons is very sensitive to intermolecular interactions . as the diffusion proceeds in a donor material by a random walk , the range is limited , as excitations are as likely to hop away from the acceptor as toward it . there is therefore a strong desire to direct energy transport to planar heterojunctions between electron donors and acceptors . in contrast to exciton diffusion , fret between electron donor and acceptor materials is directional and can bring excitons directly to a heterojunction . fret can enhance the exciton harvesting distance in bulk heterojunctions ; however , much bigger enhancement can be gained in planar heterojunctions . the general equation to describe the rate of fret from a point donor to a slab of acceptor molecules of thickness is10where z is the distance between the excited donor and the acceptor film , is the pl decay time of the donor in the absence of energy transfer , and d0 is the critical transfer distance ( figure 14 ) . the parameter d0 is typically 23 times larger than the frster radius r0 and is proportional to the density of energy of accepting chromophores in the acceptor layer . the transfer rate decreases much more slowly with distance than in the case of energy transfer between single chromophores , so interlayer fret can be efficient over long distances . it has been shown that 90% of excitons can be transferred over 20 nm distance and 50% of excitons over 35 nm . schematic of fret from an exciton in a donor layer to a slab of acceptor molecules . cnops et al . designed a three - layer planar heterojunction solar cell using an -sexithiophene ( -6 t ) electron donor , a boron subnaphthalocyanine chloride ( subnc ) electron acceptor , and an additional light - harvesting layer of boron subphthalocyanine chloride ( subpc ) ( figure 15 ) . in this structure , excitons generated in subpc transfer their energy to subnc and subsequently dissociate at both interfaces by hole or electron transfer . they have demonstrated a pce of 8.4% ( 7.77% certified ) in a 1 cm active area cell with an external quantum efficiency in the range of 5080% over a broad wavelength ranging from 400 to 720 nm . studied the same structure using x - ray photoemission spectroscopies and found that there is a small energy barrier between subnc and subpc , which may limit the efficiency of electron extraction in this configuration . these results suggest that even higher efficiencies could be attained by aligning the electron affinities of these materials . reid and rumbles used experimentally verified monte carlo simulations to evaluate the maximum achievable efficiencies in planar multilayer heterojunctions with energy cascades . they found that a pce of 10% is plausible and 15% is theoretically possible , assuming a typical 0.6 ev energy loss at the heterojunction . schematic representation of the device architecture with three active layers and the energy - level diagram of the active layers illustrating an interlayer fret from subpc to subnc followed by hole transfer to -6 t and charge extraction . reprinted with permission from ref ( 28 ) . these results show that multilayered structures with interlayer fret are very promising for light harvesting in organic photovoltaics . this approach allows optimization of the ordering and thicknesses of different layers to maximize light absorption in each one by tuning optical interference . in combination with long - range interlayer fret , use of multilayer heterojunctions with energy cascades discussed in the previous section is one of the ways to achieve directional exciton transport and efficient light harvesting . menke et al . have explored a different approach to direct energy transport from a diluted to a neat layer of the same organic semiconductor subpc . in such a structure , there is no energy gradient between layers and energy transfer occurs in both directions . however , as there is a higher concentration of energy - accepting molecules in the neat layer , forward energy transfer to the neat layer is faster than back energy transfer . they demonstrated a light - harvesting efficiency of 50% in a trilayer made of a diluted 20-nm - thick outer layer of subpc , a 10-nm - thick layer of neat subpc , and a layer of the electron acceptor ntcda . although this efficiency is lower than that in energy - cascade structures , it can potentially give a higher open circuit voltage because there is no loss of excitation photon energy in its transfer between layers . a high level of control of the energetic disorder and directional energy transport has been demonstrated by inclusion of fluorescent molecules into ordered one - dimensional nanochannels made of optically inert host materials . host and guest guest interactions , as well as molecular dynamics in confined geometries , and they can enable exciton transport over distances longer than 50 nm according to calculations . the intersystem crossing rate from the singlet to the triplet state is usually slower than the radiative and nonradiative decay of singlet excitons ; therefore , the triplet exciton yield is usually small . triplet exciton transport now attracts substantial interest when used in conjunction with singlet fission , where each singlet exciton can split into two triplet excitons and so can potentially double the charge carrier yield and photocurrent . singlet fission can be efficient in molecules with a triplet exciton energy less than half the energy of the singlet exciton and is an attractive way to utilize high - energy photons of the solar spectrum . the most researched material is polycrystalline pentacene , which shows very fast singlet fission in about 80 fs . planar heterojunction organic solar cells have been reported with a peak external quantum efficiency of up to 126% in a small portion of the visible spectrum using pentacene , demonstrating the generation of more than one electron per incident photon . determined the triplet exciton diffusion length in polycrystalline pentacene to be about 40 nm by modeling the photovoltaic spectral response . these studies suggest that harvesting of triplet excitons is a bottleneck for charge carrier generation in bilayer solar cells using polycrystalline pentacene , where the triplet exciton lifetime is on the order of 5 ns . much longer lifetimes of triplet excitons are observed in single pentacene crystals ( on the order of 0.42 s ) , so it may be possible to increase the triplet lifetime and diffusion length in thin films by increasing crystallinity . it has been shown that triplet exciton lifetime in other materials can be increased by controlling intermolecular interactions , so there might be an opportunity to increase triplet diffusion length by molecular engineering . measuring triplet exciton transport is also challenging because only some materials are phosphorescent . an interesting technique to study triplet diffusion was suggested by mikhnenko et al . , who used a thin layer of a metal ligand charge transfer complex with very fast intersystem crossing rate to generate triplet excitons and inject them into the studied material . a phosphorescent detector layer was deposited on the other side of the studied material to detect the transported triplet excitons . to demonstrate the technique , they measured the triplet exciton diffusion length in films of n , n-di-[(1-naphthyl)-n , n - diphenyl]-1,10-biphenyl-4,40-diamine ( npd ) and found a value of 87 nm . because charge transfer between electron donor and acceptor is a short - range process , initially an exciton is split into a bound geminate electron hole pair . this charge pair then has to overcome recombination and dissociate into free carriers in order to contribute to photocurrent , and this process usually limits the device performance . in this section , we briefly discuss the main experimental findings and concepts suggested to explain free carrier generation in bulk and planar heterojunctions . for a more detailed description of the theoretical models , we refer the reader to recently published perspectives . the most common technique is ultrafast transient absorption spectroscopy , where the formation of characteristic absorption bands of radical cations in an electron donor and radical anions in an electron acceptor can be observed . on the basis of experimental findings , several different mechanisms of free carrier generation have been proposed and they are shown schematically in figure 16 . it has been suggested that ballistic charge separation can occur in higher - energy ( hot ) charge transfer ( ct ) states if it can outcompete the electronic relaxation in the manifold of ct states and vibrational cooling . for example , extremely fast charge generation within 50 fs has been reported by grancini et al . in a photovoltaic blend of the conjugated polymer pcpdtbt and the soluble fullerene pc61bm . they observed very different transient absorption spectra when exciting the polymer with an excess photon energy of 1 ev above the optical band gap as compared to excitations with lower - energy photons and suggested that higher - energy exciton states convert almost resonantly into hot ct states that dissociate into free charge carriers in less than 200 fs by ballistic charge transport . this study suggests that electron transfer can be faster than electronic and vibrational relaxation of the exciton . faster electron transfer from hot vibronic states is also supported by theoretical studies . even the lowest - energy exciton in the donor or acceptor material , which is marked s1 in figure 16 , can couple almost resonantly to a hot ct state , where one of the carriers is delocalized and it can experience a short - distance ballistic transport before it forms a polaron . such a process can generate a loosely bound charge pair with an effective separation distance of several nanometers , which then dissociates into free carriers by one carrier hopping away from its counter charge . studied the relaxation of hot ct states generated in planar heterojunctions of copper phthalocyanine and electron acceptors c60 and c70 using transient two - photon photoemission spectroscopy . in this technique , a pump pulse generated ct states by splitting excitons at the heterojunction , whereas a probe pulse was incident upon the same spot after a variable time delay and led to ionization of the sample . they analyzed the kinetic energy of the photoelectrons emitted from the sample , identified hot ct states at 0.3 ev higher energy than the relaxed ct states , and observed relaxation of hot ct states within 1 ps . this study suggests that in order to assist free carrier generation , the dissociation of hot ct states has to be faster than 1 ps . hypothetical potential energy surfaces along the charge separation coordinate illustrating different free carrier generation mechanisms proposed in the literature . vertical arrows represent light absorption in the ground state ( g.s . ) to generate exciton states s1 ... sn or charge transfer ( ct ) states . the straight horizontal arrows illustrate ballistic transport of delocalized carriers in hot ct states suggested by some studies . the bent arrows indicate incoherent carrier hopping between sites on donor or acceptor , with red bent arrows representing thermally activated hopping . however , several studies have shown that the internal quantum efficiency ( the ratio of collected charges and absorbed photons at the short circuit condition ) in high - performance bulk heterojunctions is independent of the energy of absorbed photons , including a direct excitation of the relaxed intermolecular ct states formed between donor and acceptor with very low photon energy ( indicated by a dashed line in figure 16 ) . the fraction of light absorbed by these ct states was determined using electroluminescence measurements and was found to be lower by 4 orders of magnitude as compared to intramolecular absorption by the donor and acceptor . acceptor pair with a separation distance of less than 1 nm . the high efficiency of photocurrent generation observed by direct excitation of the relaxed ct states indicates that a charge pair in this state is loosely bound and can dissociate into free carriers with the help of a rather weak built - in electric field . ballistic charge transport is unlikely in the relaxed ct state ; thus , dissociation is expected to occur by incoherent carrier hopping , and some of its steps have to be thermally activated . these findings are consistent with observations of rather slow charge generation in efficient photovoltaic blends of poly(3-hexylthiophene ) ( p3ht ) with fullerene acceptors prepared by thermal annealing , where the majority of charge pairs were found to be generated on a picosecond time scale , presumably limited by exciton diffusion to a heterojunction . annealing has been shown to triple the power conversion efficiency of p3ht : fullerene bulk - heterojunction solar cells to 4% , suggesting that slower charge generation in these materials is not an obstacle to device efficiency . slower charge generation in annealed blends suggests larger domain sizes of donor and acceptor , which help dissociation of bound charge pairs into free carriers , as discussed in section 4.2.2 . in very efficient photovoltaic blends of the electron - donating polymer ptb7 with the acceptor pc71bm these results suggest that the rate of electron transfer at the heterojunction does not have to be ultrafast to achieve high device efficiencies in organic photovoltaics . in the next section , we discuss recent findings on how the rate depends on the energy offset between donor and acceptor . the difference of the gibbs free energy between the relaxed neutral excited state ( exciton ) and the relaxed ct state ( electron hole pair ) is known as the driving force for electron transfer . in organic photovoltaics , it is desirable to have the smallest driving force necessary to generate free charge carriers , as any excess will be converted into heat and will reduce the photovoltage ( and consequently the power conversion efficiency ) . a recent study by coffey et al . showed the existence of an optimal driving force for the highest yield of mobile charge carriers that was measured by microwave conductivity ( figure 17 ) . they observed a decrease of the mobile carrier yield when the optimal driving force was exceeded and explained their results using the marcus theory of electron transfer . yield of mobile charge carriers ( a ) and the rate constant for electron transfer ( b ) plotted against the driving force g . points 57 in the bottom panel have arrows denoting that they are lower limits . measured electron transfer rates from thermally relaxed excitons in the conjugated polymer ptb7 to different electron acceptors in the blends using time - resolved fluorescence quenching . measurements with very low and very high loadings of acceptor were used to separate electron transfer at a short distance to an acceptor ( < 1 nm ) from exciton diffusion in the ptb7 phase . they found very fast electron transfer in < 2 ps for values of the driving force between 0.2 and 0.6 ev . higher and lower driving forces outside this range gave slower rates of electron transfer , as expected from the marcus theory of electron transfer . these results show that electron transfer is very fast and efficient when the driving force equals the reorganization energy . fitting to the marcus model gave a reorganization energy of 0.4 ev . acceptor interface can be reduced by reducing the reorganization energy and maintaining the optimum offset of electron affinities or ionization potentials . although the driving force influences the rate of electron transfer from donor to acceptor , its effect on dissociation of photogenerated charge pairs into free carriers is not significant . this is indicated by the high photocurrent efficiency observed after direct excitation of the relaxed ct states and dissociation of bound charge pairs on a picosecond time scale that is much slower than the cooling of hot ct states . these findings imply that it should be possible to design efficient photovoltaic materials with small reorganization energies that would give high open circuit voltages without a trade - off in photocurrent . excitons can split into charge pairs in the bulk of an organic semiconductor , albeit with a lower efficiency than at heterojunctions . for example , charge pair generation with 1520% efficiency has been observed in films of conjugated polymers that are used as electron donors , and neat films of fullerene derivatives , which are commonly used as acceptors . generation of bound charge pairs inside donor or acceptor domains can be detrimental to device performance because these pairs often recombine before they can be spatially separated into donor and acceptor phases . to avoid recombination losses , the morphology of bulk heterojunction has to support fast extraction of holes and electrons into the donor and acceptor domains , respectively , where they can be transported to the correct electrodes , and this is discussed in section 4.2 of this review . charge pair dissociation into free carriers is a crucial step in the operation of organic solar cells ; therefore , its understanding is very important for the development of efficient heterojunctions . the observations of efficient dissociation of relaxed charge transfer states in high - performance bulk heterojunctions suggest that hot ct states are not required to achieve charge pair dissociation . the free carrier yield is independent of the internal electric field in efficient bulk heterojunctions or shows very weak dependence , indicating that the potential barrier for charge pair dissociation is very small . have estimated an activation energy of around 9 mev for charge pair dissociation in optimized p3ht : pc61bm bulk heterojunctions . several studies suggested that coulomb attraction is compensated by the free energy cascade from interfacial ct states to spatially separated charged states in the bulk of donor and acceptor domains . acceptor phases in the boundaries between pure domains in bulk heterojunctions and findings that the difference in ionization potentials and electron affinities is favorable for directional charge transport from a mixed phase into pure donor and acceptor domains . higher entropy of spatially separated charges as compared to ct states can also contribute to the free energy gain . have estimated that the entropy contribution is sufficient to diminish the activation energy needed for pair dissociation when it is spaced by just 4 nm , assuming uninterrupted three - dimensional charge transport at 300 k and a dielectric constant of 3.5 . in that case , charge pairs separated by > 4 nm can be considered unbound and can dissociate into free carriers provided there are continuous donor and acceptor domains to support charge transport . it has been suggested that long - range charge - separated states ( up to 4 nm separation ) can be accessed through ballistic transport of delocalized carriers in hot ct states or very fast initial carrier hopping . the dissociation efficiency has been shown to increase with increasing electric field up to 0.050.5 mv cm and to saturate above these values . the dissociation efficiency was found to saturate at lower field values when polymers with longer conjugation length were used as electron donors . this suggests that an increase of hole delocalization along the conjugated chain decreases the binding energy of the geminate electron pair and helps its dissociation into free carriers . in summary , the rate of charge generation has been shown to depend on the driving force for electron transfer , and in some cases , charge generation was found to be faster than electronic and vibrational relaxation . however , in high - performance photovoltaic blends , the free carrier yield was found to be independent of photon energy and electric field , indicating that hot charge transfer states do not play a major role . free carrier generation in these blends occurs through relaxed charge transfer states and is influenced by blend morphology , energetics of mixed and pure donor and acceptor phases , charge delocalization , and carrier mobility . free carrier generation in planar heterojunctions has been found to depend on applied electric field and conjugation length of the electron donor , indicating that the mechanism could be different from that in the bulk heterojunction . it is possible that there is no universal mechanism and more than one mechanism is involved in some cases . so far we have recounted that absorption of light by donor and acceptor materials in a bulk heterojunction readily occurs ; however , harvesting that light requires , among other things , getting the formed exciton to the donor : acceptor interface via exciton diffusion or fret and getting free charges through the active layer to their respective electrodes to be extracted , as discussed above . consequently , it is clear that the structural arrangement of the donor and acceptor in the blend if the donor and acceptor are too far apart , then excitons will not be able to traverse the distance to enable them to be split into charge pairs , and if the distance is too short , then while excitons will easily dissociate , sufficiently separate pathways of material will not be available to suppress recombination of the readily formed charge pairs . the optimal distance between the two materials is on the order of 1020 nm , approximately the exciton diffusion length . measuring the morphology on such length scales is very challenging ; the donor and acceptor are typically both carbon - based materials , soft in nature , with similar physical properties . in this section , we will review the principal methods of measuring the morphology of bhj blends , discuss some of the key results published to date and their implications for light harvesting in organic photovoltaics , and highlight the challenges that remain . this is not meant to be an exhaustive treatment of morphology in organic photovoltaic blends , which more detailed singular reviews already cover in great detail , but rather an overview of how it links to light harvesting for organic photovoltaics . the length scale of merit for bhjs as discussed above is 1020 nm , this is much smaller than the optical diffraction limit , and thus , alternative techniques to classical optical microscopy have been used to measure the blend morphology . a key technique to measure nanoscale morphology is atomic force microscopy ( afm ) and variants thereof . here a cantilevered tip honed to a 1020 nm point is scanned across the sample . atomic repulsion forces can be used to keep the tip a constant distance from the actual sample ( giving topographical information ) , and the frequencies of an oscillating tip can be used to derive more information on surface force response , revealing finer details ( so - called phase images ) . in the context of organic photovoltaics , afm can give information on the surface and subsurface topography of the film . conventional topography images simply show the surface of the film and can give information on the organization of the two materials in the blend only when they fully phase separate into clear separate domains , e.g. , in a polymer polymer blend , as shown in figure 18a . rarely is this the case in good opv blends , and thus phase images instead can be used to reveal more subtle details , as shown in figure 18b , c . ( a ) afm topography of a blend film of pfo and f8bt , showing clear phase separation . ( b ) topography and ( c ) phase image of the same area of a p3ht : pc61bm blend film , indicating the finer details that the phase measurement reveals . the image in panel a is reprinted with permission from ref ( 196 ) . the images in panels b and c are reprinted with permission from ref ( 197 ) . unfortunately , frequently in high - performance opv blends the blend morphology is such that there are subtle degrees of mixing between the two materials , leading overall to a relatively featureless image in afm topography and phase . here instead an extension of the afm concept can be used , wherein a metal - coated afm tip is used as a conductivity probe as it is scanned across the sample surface , giving rise to the technique of conductive afm ( c - afm ) . material composition can be derived by exploiting the clear conductivity differences between p - type electron donors and n - type electron acceptors used in the bhj blend . given the small contact area between the sample and the afm tip ( of diameter 40 nm ) , the measured currents tend to be very small ( pico or nanoamps ) , and significant biases ( a few volts ) are typically used to ensure clear current maps that have distinguishable features measurable above the noise floor . an extension of the conductivity map is to measure photoconductivity with an afm ( pc - afm ) . here light is shone onto the sample , creating excitons that dissociate in the blend into charge pairs . extraction of the charge pairs by the metal - coated afm tip under bias with respect to the sample substrate contact leads to a measured photocurrent , which then will vary depending upon blend composition as the tip is scanned across the sample , as depicted in figure 19 . this enables a photocurrent map to be built that can be qualitatively analogous to the donor : acceptor concentration . illumination of a sample leads to the generation of excitons that dissociate into charges . these charges are then extracted by the metal afm tip , which is under bias with respect to the sample substrate . spatial scanning of the sample ( or the tip ) allows a 2d photocurrent map to be recorded . the length scale of interest , 1050 nm , as indicated above , is well below the diffraction limit enabled with conventional confocal optical microscopy ; however , this is just within the capabilities of another optical microscopy technique , scanning near - field optical microscopy ( snom ) . here an afm is used , either with a tip that has a 50 nm aperture in it that light is coupled into or with a solid tip that light is scattered off of . in both cases , the evanescent wave of light from the end of the tip is coupled into the sample below , leading to a spatial resolution below the diffraction limit . transmission and reflection of the excitation light can be collected , as well as fluorescence from the sample under investigation . additionally , raman spectroscopy of the scattered incident excitation light can be performed . all of these modes can contribute to discriminating the blend morphology in a bulk heterojunction . despite the promising path for snom in helping with bhj morphology , this can be attributed to the advanced nature of the suite of snom techniques , along with the challenges in reaching the headline spatial resolution of < 50 nm . additionally , mixing between the donor and acceptor tends to blur the snom images , making high resolutions difficult to achieve . nevertheless , successful investigations of organic semiconductors using snom techniques have been made . in figure 20 , a blend of poly(9,9-dioctylfluorene ) and poly(9,9-dioctylfluorene - alt - benzothiadiazole ) is investigated with snom . in this system , the two polymers show clear phase separation , and this allows nanoscale mapping of the fluorescence from each of them to be made . opv blends were investigated by schubert et al . , who looked at the absorption of light by the donor and acceptor to build up maps of the two materials . shown in figure 20 are maps of the donor p3ht absorption in blends cast from different solvents , indicating both the changes in nanomorphology and the mixing enhancements that lead to loss of contrast with the acceptor . exploring the raman properties of materials , a recent paper has investigated the nanoscale composition of materials , as also shown in figure 20 , finding that materials can be distinguished by their vibrational responses . scanning near - field optical microscopy of the blend of the conjugated polymers pfo and f8bt showing the afm topography ( a ) and f8bt near - field pl ( b ) ; arrow 1 indicates regions of f8bt , while arrow 2 shows pfo . ( c and d ) optical density ( absorption ) measurements of p3ht blended with a naphthalene - polymer - based acceptor . casting from p - xylene ( c ) gives large - scale phase separation , while casting from a 1:1 mixture of p - xylene and chloronapthalene ( d ) gives a well - mixed blend . ( e and f ) block - co - polymer of polystyrene and poly(methyl methacrylate ) measured with afm phase ( e ) and near - field raman imaging of the 1735 cm carbonyl mode ( f ) . reprinted with permission from ref copyright 2014 nature publishing group . at first glance , the most practical route to achieving the high spatial resolution required for observing bhj the de broglie wavelength of accelerated electrons can reach the picometer length scale , and while other practicalities of electron microscopy restrict the spatial resolution to the angstrom scale , this is still well within the requirements for measuring the length scales of interest in opv blends . the qualifier on this promising route is that both the donor and acceptor in opv blends are primarily carbon - based , and thus , the two materials have a very similar response to electrons , thus measuring contrast can be challenging and frequently requires the application of specific blends with favorable contrast and advanced measurement techniques or sample preparation . in classical bright - field transmission electron microscopy ( tem ) , an electron gun is imaged onto a sample , and the transmitted intensity is measured on a phosphor screen . the whole thickness of the sample is imaged , and thus , absorption of electrons through the sample volume will lead to a reduced signal in the image plane , while transmission of electrons will lead to a higher signal . typically , for all - organic bhj films very limited contrast exists , and thus , only specific blends with fortuitous compositions can readily be imaged with bright - field conventional tem , such as choosing a blend with a high purity of phases , as demonstrated in figure 21a . ( a ) bright - field transmission electron microscopy of mdmo - ppv : pc61bm blend . strong demixing leads to easily measurable phase separation , with fullerene regions appearing dark . ( b ) tem tomography reconstruction of p3ht : zno blend , with regions of zno being yellow , p3ht appearing transparent , and the gray region on top being the aluminum top contact . reprinted with permission from ref ( 209 ) . conventional tem images are simple projections of the sample volume onto a plane ; however , 3d information can be derived if multiple projections are made at different sample angles with respect to the electron beam plane , enabling computer - aided tem tomography to be performed . high contrast between blend components is required as a prerequisite before attempting tomography , as the reconstruction process is challenging ; however , as shown in figure 21b , when a suitable system is measured , the results can lead to detailed and valuable information on the full three - dimensional morphology of the blend , leading to a large amount of secondary information , such as mean free paths of material and connectivity analysis . the stunning image shown illustrates the remarkable level of detail that can be achieved in a best - case situation . however , a hybrid solar cell , i.e. , an organic donor with an inorganic acceptor , is a situation that gives strong contrast . for organic / organic bulk heterojunctions , a range of advanced tem techniques discussed below have proved useful to enhance contrast and/or give compositional information . in energy - filtered transmission electron microscopy ( eftem ) , such inelastic scattering can occur with different amounts of energy loss for different elements , e.g. , due to different energies of core electron transitions . selective transmission of specific scattered electron energies , e.g. , carbon k edge , sulfur , or fluorine , can be made and imaged , leading to elemental maps . this can be used to distinguish between the components of a photovoltaic blend , for while carbon is generally a constituent in both blend materials , the polymer will typically include sulfur or fluorine atoms that can be used to give contrast . in high - resolution transmission electron microscopy ( hrtem ) , the highest spatial resolution is achieved , at the potential cost of not having discriminatory compositional information . this technique is thus most useful for resolving crystalline regions of material , commonly associated with opv blend materials , such as p3ht , that show high degrees of local ordering . advanced defocusing techniques may be used to enhance the resolved image ; however , caution has to be applied when doing this , as the dangers of misinterpretation are significant . shown in figure 22 is a hrtem image of a blend of the small molecule donor material p - dts(fbtth2)2 with pc61bm . highly ordered regions are observed , indicating both the lattice spacing and the domain boundaries of the crystallite . as noted above , compositional information is lacking ; thus , it is not known if the crystallite is set within the amorphous material of the donor itself or of the acceptor . high - resolution transmission electron microscopy of a blend film of p - dts(fbtth2)2 with pc61bm when processed with 0.4% diiodooctane . to probe the local morphology at the smallest length scales , angstroms to nanometers , there exists the possibility to use the time - dependent light emission or absorption from the organic materials in the blend themselves as nanoscale probes of the local composition . this has the added advantage of determining the exciton harvesting of the blend itself directly , with the population of excitons formed from light absorption being measured and the degree of quenching by the other component of the blend quantified . with this technique , transient photoluminescence ( tpl ) or transient absorption ( ta ) can be used to determine certain geometric shapes , if the exciton diffusion coefficient is known . for example , as shown schematically in figure 23 , if the exciton diffusion coefficient of a donor material ( blue ) is known , and it is surrounded by an acceptor material ( red ) , then with sufficient time resolution , the smoluchowski equation can be used to determine the radius , r , of the donor domain . this method relies on a presumed knowledge of the geometry of the donor domain ( spherical , cylindrical , oblate spheroid , etc . ) and for easiest modeling an analytical solution to the smoluchowski equation for that geometry . schematic of time - resolved pl derived morphology with donor material ( blue region in center ) surrounded by an acceptor ( red region ) . an exciton generated in the center of the donor region has an exciton diffusion radius ( ld ) denoted by the dashed line , which should correspond to the natural unquenched pl lifetime . measured shorter pl lifetimes can enable ( presuming a spherical geometry ) the radius to the acceptor ( r ) to be determined . the main advantage of this technique is that it is sensitive on the very smallest scale in the immediate vicinity surrounding the polymer or fullerene . a disadvantage is that the blend chosen for this technique has to be amenable to analysis , with relatively pure phases of material present to enable modeling of the diffusion to a quenching surface ; if the quencher is too dispersed in the blend , i.e. , if the blend has a significant mixed phase , then excitons are quenched without having to diffuse to the donor : acceptor interface . the blend ratios used in devices are typically on the order of 1:1 and have significant mixed phase components , thus leading to kinetics that may be too complicated to model and understand correctly using accessible analytical fitting methods . however , in systems amenable to analysis , the technique can give important information . shown in figure 24 is the derived domain size distribution in a blend of two polymers , pfb and f8bt , when the ratio between the two components is modified . one can immediately grasp why this technique has powerful conclusions when applied correctly , as such small length scale domain distributions would be very difficult to observe with the multitude of morphology techniques discussed above in the previous sections . derived probability density of exciton diffusion length as a function of distance for different blends of pfb : f8bt . mass ratios of pfb : f8bt are as follows : squares are 90:10 , circles are 75:25 , upward triangles are 50:50 , and downward triangles are 20:80 . the solid line indicates the theoretical exciton diffusion length distribution for a sphere of 2 nm radius and the dashed line for a 5 nm sphere radius . reprinted with permission from ref ( 211 ) . copyright 2008 american physical society . with an understanding of the key techniques to measure morphology , we now turn our attention to study the key donor : acceptor blend morphologies that have been reported over the last 15 years . in discussing the morphology here , we are particularly thinking of its role in light harvesting and the operation of the device : as already explained , in order to harvest light in a bulk heterojunction organic photovoltaic cell , the donor and acceptor domains need to be within an exciton diffusion length ( typically 10 nm ) of where the light is absorbed . in looking at the morphology of opv blends , we aim to examine why systems do and do not work and any common themes that we can deduce between different blends . the number of donor : acceptor combinations in the development of opvs is very large , so we focus on three key generations of opv donor polymer materials , namely , poly[2-methoxy-5-(3,7-dimethyloctyloxy)-1,4-phenylenevinylene ] ( mdmo - ppv ) , poly[3-hexylthiophene-2,5-diyl ] ( p3ht ) , and poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b]dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ] ] ( ptb7 ) . the chemical structures for these three key donor materials are shown in figure 25 . chemical structures of three donor materials ( mdmo - ppv , p3ht , ptb7 ) and two acceptor materials ( pc61bm , pc71bm ) that are examined in this section . each donor material represents a key generational point in the development of opvs , with mdmo - ppv blends reaching power conversion efficiencies of 13% , p3ht 35% , and ptb7 710% . here we will examine the efforts to understand the morphology of blends containing these donor materials to date . it is worth noting that recently small molecule donors have begun to match the performance of polymeric materials . the morphologies discussed in this review can be pertinent to small molecule bhjs , but we have chosen not to feature any specific study of a small molecule blend owing to the potentially different physics and chemistry at play in material organization ( e.g. , crystallinity , miscibility ) . a variety of acceptors have been used in fabricating bhj cells based on these donors , but generally , fullerene derivatives such as -phenyl - c61-butyric acid methyl ester ( pc61bm ) or -phenyl - c71-butyric acid methyl ester ( pc71bm ) , both chemical structures shown in figure 25 , have been used . mdmo - ppv was one of the earliest successful opv donor materials , giving reliable power conversion efficiencies in the region of 12% , with the highest efficiency of 3% being reached when blended with pc71bm . almost no spectral coverage in the red part of the visible spectrum and relatively low external quantum efficiencies are responsible for the overall power conversion efficiency of 2% . the completely amorphous nature of mdmo - ppv prevents the use of x - ray diffraction techniques to study the polymer or bulk heterojunction blends , so information on the structural arrangement of the materials relies solely on other techniques , as detailed below . the morphology of the mdmo - ppv : fullerene system has been extensively studied . the fullerene was found to mix intimately with mdmo - ppv up to 50% mass ratio . on the face of it , such fine mixing is ideal for light harvesting , as it will give very strong exciton dissociation and good charge - pair generation . unfortunately , while maximizing surface area for charge separation , the large surface area associated with such fine mixing also leads to strong charge recombination , as opposite charges can readily meet again . hence , charge extraction is very problematic is such a finely mixed structure . it was found that if the blend has more than 50% fullerene , then phase separation starts to occur , leading to the formation of pure unmixed domains comprised solely of pc61bm . the best device performance was obtained with 80% fullerene:20% mdmo - ppv by mass . at this blend ratio pure fullerene domains form that give rise to percolation pathways for electrons , thus spatially separating the charges and suppressing recombination . the phase separation is , however , relatively uncontrolled , and at the optimal fullerene loading ratio of 80% , large 60500 nm fullerene domains form , with the size dependent on the casting solvent used for the blend . these sizes and high degrees of purity enable observation with tem ( figure 26a , b ) , sem ( figure 26c , d ) , and afm ( figure 26e , f ) . an overall schematic of the morphology is shown in figure 27 , with charge transport and extraction to a tip contact indicated . fullerene domains that form near the top of the active layer promote good electron and hole extraction to their electrodes and could be thought of in effect as equivalent to not being too far away from ideal nanoscale - ordered heterojunctions . on the other hand , when the fullerene is buried inside the blend , then electrons have to transverse regions that only contain 50% fullerene ( meaning recombination with holes is likely ) , while , more critically , holes have to get through near - pure fullerene domains , an unlikely occurrence . these restrictions are partly responsible for the relatively low external quantum efficiency of this optimized blend and indicate how the morphology has an important role to play in determining the overall light harvesting and photovoltaic performance of organic bhj blends . microscopy images of mdmo - ppv : pc61bm blends spin - coated from toluene ( top row , a , c , e ) and chlorobenzene ( bottom row , b , d , f ) when measured with bright - field tem ( a , b ) , cross - section imaging with sem ( c , d ) , and afm topography ( e , f ) . large pure fullerene domains form when spin - coated from toluene , which get smaller but are still distinct from mdmo - ppv when spin - coated from chlorobenzene . images in panels c f are reprinted with permission from ref ( 227 ) . schematic of the morphology in mdmo - ppv : pc61bm blends when large pure fullerene domains are formed , indicating relative charge transport and extraction to an afm tip . the large size of the pure fullerene domains does have the distinct advantage of making them easier to characterize , and in particular , the work of coffey et al . in measuring the electrical characteristics on and off the domains with pc - afm enabled novel nanoscale electrical behavior to be explored . positioning the pc - afm tip on different regions of the blend enables local i v curves to be constructed , as shown in figure 28 . one can thus correlate regions of high and low current at different biases with the afm topography and other morphological information to understand how the electrical behavior of the blend is related to the physical organization of the material . ( c ) photocurrent map of the same region as in panel b , at 0 v bias , showing regions high and low in current . ( d ) i v curves for specific points as indicated by the symbols on panels b and c. reprinted with permission from ref ( 228 ) . finally , it is worth noting that the overall morphological structure of mdmo - ppv : pc61bm described here has become a recurring theme in a number of opv blends with higher than 50% fullerene weighting and has helped in understanding high - performance blends such as ptb7:pc71bm ( vide infra ) a number of years after the original work was reported . this indicates that knowledge derived for one blend , while specific to that blend , can also contain general information or indicators for others . p3ht was the prototypical opv donor polymer for a significant number of years and continues to be important as a benchmark system to study and understand . power conversion efficiencies range between 3 and 5% for p3ht blended with pc61bm . in contrast to the completely amorphous polymer mdmo - ppv discussed above , p3ht is characterized by its semicrystalline nature , where regions of p3ht are amorphous and small regions , 10 10 20 nm are ordered into crystallites . this semicrystalline nature of the polymer is important to the photovoltaic performance of the material and has major effects on the morphology and light harvesting in the blend . these effects are both positive ( crystalline structures lead to higher mobility and enable diffractive experimental techniques to be used to characterize the morphology ) and negative ( the details of the crystalline and mixed phases are complicated , with unknowns in both the spatial and size distributions along with complications in varieties of crystalline ordering controlled by chemical structure variations ) . consequently , while the p3ht : pc61bm blend has been very heavily studied and is an important blend to understand , a straightforward conclusive morphological narrative for it has failed to gain acceptance . the aim here is to highlight some informative studies that have been completed on the p3ht - based blends to date and attempt to build an overall sense of the likely morphology . it should be noted that some studies that will be covered here do not use pc61bm as the acceptor but alternatives that can help to provide better contrast for measures of morphology . starting with an examination of the crystallinity aspects of the polymer , p3ht itself is comprised of repeat units of a thiophene monomer with a hexyl side chain at the 3-position . the monomers can be arranged such that the hexyl side chain is at the same position on every ring ( regioregular , rr ) . this gives high crystallinity , as the side chains of the polymer can interleave , as shown in figure 29 , to form a periodic structure . -stacking of the thiophene backbones can also occur to give large three - dimensional aligned structures , which one can call crystallites and , as noted above , can grow to 10 10 20 nm in size . crystalline organization of p3ht chains , which can form edge - on ( a ) or face - on ( b ) aggregates to the substrate . reprinted with permission from ref ( 229 ) . as might be anticipated , these periodic regions of p3ht are important in the photovoltaic performance of the material . the hole mobility in crystalline p3ht can be close to double that of amorphous p3ht . the ability for charges to move with high mobilities in p3ht is an obvious advantage when one aims to get holes to the electrode and extract them quickly before they have a chance to meet an electron and recombine . this aim is also enhanced by the crystallinity in a second sense in that crystalline regions do not by definition contain any pc61bm molecules . thus , recombination of holes with electrons on fullerene molecules is suppressed inside the crystallite , allowing the hole to move freely without being lost . crystalline p3ht thus seems like an ideal candidate for use in photovoltaic blends ; there are , however , many challenges in bringing together each of the advantageous properties in the correct way to enable the optimal light harvesting . for example , holes may well have high mobilities and low recombination probabilities inside p3ht crystallites , but unless that crystallite connects to the anode , the holes will have to transverse other more disordered regions to be extracted . the morphology of the entire blend , ordered and disordered polymer and fullerene , thus determines the overall light - harvesting and photovoltaic performance of the system . where does one begin with such a complex question as to what is the morphology in p3ht - based blends ? compositionally , large - scale phase separation does not occur in blends of p3ht : pc61bm , and this makes the determination of compositional maps challenging . initial bright - field tem studies were able to discern some features , but the low contrast hampered strong interpretation of the data . a strategy of creating contrast by shrewd choice of blend components thus arose as a method for understanding the morphology . in work by roehling et al . , an endohedral fullerene was used as the acceptor . here a heavy - metal lutetium - based trimetallic nitride , lu3n , is contained within a solubilized c80 fullerene cage . blends of p3ht with lu3n pc81bh produce power conversion efficiencies of up to 4% , with advantageous open circuit voltages of 0.8 v , and so are a valid blend composition for investigation of morphology . the heavy metal contained within the fullerene affords strong contrast with the p3ht in the high - angle annular dark - field scanning transmission electron microscopy tomography experiments that the authors report . examination of the three phases ( p3ht , fullerene - rich , and mixed ) in as - cast films and annealed films that give optimal power conversion efficiencies show strong differences that explain the differing light harvesting . in the as - cast films , the p3ht and fullerene are separated into distinct , small domains surrounded by a large mixed - phase component ( figure 30 , left side ) . the role of thermal annealing is clear in the morphological evolution of the blend ( figure 30 , right side ) , as fullerene - rich and p3ht domains have become larger and connected together to form networks . crucially , the tomography technique enables the morphology in the z direction ( i.e. , perpendicular to the substrate ) to be determined . for the annealed blend , one can see in cross - sectional reconstructions ( figure 30 , bottom row ) that the p3ht and fullerene - rich domains have extended in the z direction as well as the x and y directions . this is important , for charges are flowing in primarily the z direction , and one can see that clear continuous phases of p3ht and fullerene exist throughout this direction , enabling charges to be extracted with low recombination probabilities . tem tomography reconstructions of as cast ( left column ) and thermally annealed ( right column ) p3ht : lu - pc61bm blend films . shown are identified p3ht regions ( first row ) , fullerene - rich regions ( second row ) , mixed - phase regions ( third row ) , and cross - sectional composites of all three ( fourth row ) . scale bars are 100 nm . reprinted with permission from ref ( 233 ) . the large endohedral fullerene molecules are not frequently used , so there is a desire for determination of the three - dimensional morphology of more commonly studied blends . to this end , another way of obtaining high contrast was to study the hybrid inorganic blend of p3ht with zinc oxide . power conversion efficiencies in these devices can reach 2% , and it is an interesting candidate for photovoltaic cells , as the inorganic component carries distinct advantages ( high dielectric constant , high charge mobility ) , so the formed morphology is important . the organic : inorganic blend gives rise to high contrast in transmission electron microscopy , enabling tem tomography to be performed . we chose to highlight this tomography study owing to both the exceptional contrast that is garnered from the blend components and the analysis that the authors have provided , which goes beyond simple pictorial representations of the morphology into a more detailed quantitative investigation . three thicknesses of p3ht : zno films were investigated , 57 , 100 , and 167 nm thick , and the tomography of each was measured , as shown in figure 31a c , where zno is highlighted in yellow and p3ht is transparent . it can be seen that the thinnest film contains a great degree of phase separation , with large areas of p3ht surrounded by small clusters of zno . when the films are made thicker , the zno becomes more distributed inside the film volume and the p3ht domains are substantially reduced in size . the work does not just end at the 3d reconstruction of the morphology , however , but applies the now known 3d organization of the materials to look at the efficiency of quenching of p3ht excitons by applying a diffusion model with the known exciton lifetime and diffusion coefficient . this enables a 3d model of the exciton quenching to be generated , as shown in figure 31d f . these results are now determining quantitative information on how the blend behaves in a photovoltaic context at harvesting light and are a gold standard for others to aim at in quantifying light harvesting of a blend as opposed to simply showing images . morphology of p3ht : zno blend films of three thicknesses ( 57 nm , first column ; 100 nm , second column ; and 157 nm , third column ) . panels a c show the reconstructed tem tomography for the films , with zno yellow and p3ht transparent . panels d f show modeled exciton harvesting for each determined morphology when using the known p3ht exciton diffusion coefficient , with red regions corresponding to near - unity harvesting and blue / black regions to almost none . the modeled exciton quenching reveals that large areas of the 57 nm thick film have no or very little light harvesting at all . doubling of the thickness to 100 nm shows a dramatic improvement , with only a very small region of the film not able to contribute to light harvesting . finally , at a film thickness of 167 nm , there are no regions of the film that do not contribute , and the worst areas are only a few small domains that have only 50% probability of light harvesting . it is clear from this study that the most powerful way to examine light harvesting in bulk heterojunction organic photovoltaic blends is with the ability to combine detailed morphological information with photophysical knowledge . the best examination of the morphology and light - harvesting properties of p3ht would be with pc61bm , as this is the most common blend combination and is the most well understood blend in terms of other electrical and photophysical studies . the challenge , however , as already noted , is that poor contrast exists between the materials to enable discrimination of the composition . the most promising study made use of a variant of energy - filtered tem and significant computational image analysis to enable the production of material maps looking through the film ( figure 32 ) . here p3ht and pc61bm domains could be mapped , along with a mixed phase of the two materials , showing that isolated domains of both materials are found embedded in a mixed - phase matrix . the compositional maps of as - cast films of the blend were compared with those of thermally annealed films . eftem of p3ht : pc61bm blend as - cast ( a ) and after thermal annealing ( b ) . green areas show p3ht , red areas pc61bm , and yellow areas a mixture of the two . thermal annealing is a postdeposition processing step commonly used to greatly enhance device efficiency . in thermal annealing , the film is heated , leading to it softening and the possibility of both the polymer and fullerene rearranging their packing in the film . in particular , when the film is heated to a temperature above the glass transition temperature ( tg ) of the polymer , the polymer enters a rubbery phase that enables the chains to move , bend , and reshape themselves to enable crystalline order between or among the chains to be formed . alternatively , solvent vapor annealing ( sva ) can be used to enhance crystallinity . here a film is exposed to solvent vapor , which causes it to swell , thereby allowing rearrangement of the polymer chains ( and fullerene ) and enabling crystallinity to increase . upon thermal annealing , the domains of p3ht and pc61bm are found to grow , joining together to form in - plane bicontinuous networks , with a feature size of 14100 nm found by analyzing the spectral power density of the image in figure 32b . this result is consistent with the endohedral fullerene stem tomography measurements already presented above and gives good confidence that thermal annealing consistently increases domain size and purity to enhance charge transport networks in the active layer . this result is a good starting point for understanding p3ht : pc61bm light - harvesting morphologies , but there are many unknowns remaining , as primarily the plane parallel to the substrate was mapped , with the compositional information integrated along the z direction , as is commonly the case with tem - based techniques . extracting z information is possible with spectroscopic ellipsometry , but at the cost of not having lateral information ( x and y directions ) on the same sample volume . ideally , one would like full three - dimensional information . two ways to obtain such information have developed a novel energy filtering of secondary electrons in scanning electron microscopy for use with organic bhj blends . the advantage of this is that only the top few ( 13 ) nanometers are able to contribute to the efsem signal , ensuring the observations give only xy plane morphology rather than the full - film thickness z - integrated morphology that was measured above with eftem . if one were to selectively remove layers of the sample with etching , then 3d information could be derived . the efsem measurements were as much a proof - of - principle on using the technique with all - organic bhj blends as a detailed examination of morphology , but the authors did choose to investigate p3ht : pc61bm to validate their methods . with confidence that only the surface of the film is being imaged , the authors first find a p3ht skin layer on top of the blend that they need to remove with plasma treatment . once that has been removed , they were able to use energy filtering to directly observe p3ht - rich , mixed , and pc61bm - rich phases before and after thermal annealing ( figure 33 ) , finding typical phase sizes of 16 and 28 nm . such phase sizes are indicative of why p3ht : pc61bm works well as a bhj opv blend , as they are on the correct order for ensuring that excitons created are able to diffuse to the interface and be separated into charge pairs , as already discussed in this review . consequently , with such phase sizes , good light harvesting can be achieved . these results corroborate what has been observed previously , but do so with the distinction that they are definitively only looking at a very thin plane of the morphology rather than z - integrated results . novel energy - filtered sem image of p3ht : pc61bm blends as cast ( a ) and after thermal annealing ( b ) , with red indicating p3ht - rich areas and blue pc61bm rich areas , while gray denotes mixed regions . reprinted from ref ( 255 ) . licensed under cc - by-4.0 . the second way to address the problem of getting z information for p3ht : pc61bm is the use of computer aided tem tomography as already discussed , but this is extremely challenging with the limited contrast available in conventional bright - field tem . recent pioneering studies have utilized energy - filtered tem tomography and energy - filtered scanning electron microscopy on a different opv blend , indicating that such techniques are possible . consequently , for p3ht : pc61bm - based blends some alternative strategies have been used to enable the generation of contrast . in work in 2009 , van bavel et al . undertook tem tomography on p3ht : pc61bm blend films to determine the three - dimensional organization of the blend . using relatively low molecular weight p3ht ( 19.4 kda ) that preferentially forms highly crystalline nanorods , contrast was enabled for tomography . the work enabled full 3d reconstructions of the blend morphology in as - cast , thermally annealed , and solvent - annealed samples . in the as - cast film , few features were seen , owing to the primarily amorphous nature of the p3ht being indistinguishable from the pc61bm . however , in the two annealed samples , strong p3ht nanorod crystallization was induced and strong contrast was formed . the 3d reconstruction allows information on the composition perpendicular to the substrate ( the z direction ) to be deduced , as shown in figure 34 . the proportion of crystalline p3ht nanorods as a function of depth through the film can be measured and shows that the majority lie close to the bottom of the film . this is an important result and is consistent with good device performance , as hole extraction is through the ito anode at the bottom of the film . tem tomography slices of a 100200 nm thick p3ht : pc61bm blend film , with p3ht indicated by the color yellow . slices are taken toward the bottom of the film ( a ) and toward the top of the film ( b ) , indicating differences in the amount of p3ht . this can enable the p3ht volume percentage as a function of z - position in the film , as shown in panel c , to be measured . the disadvantages of the study , as the authors concede , is that contrast is only provided with the highly crystalline p3ht nanorods ; thus , no information is deduced on the amorphous p3ht . in addition , the relatively low molecular weight of the p3ht used is a little different from the values typically used in devices , where such nanorods are not commonly observed . however , these limitations must be viewed in the context of the aim of truly observing the three - dimensional nanomorphology of the blend , which was achieved . the second part of the p3ht : pc61bm light - harvesting morphology narrative , as discussed at the beginning of this section , is the crystallinity of the p3ht . this is a difficult and challenging set of variables to measure , but numerous diffraction techniques can aid in understanding . given the number of studies that have been undertaken on p3ht , we have selected only a few that offer the most comprehensive overall impression of crystallinity in the polymer as it pertains to opv blends . to examine the blend crystallinity it is pertinent to begin by just looking at the organization in films of p3ht on its own . a comprehensive investigation by duong et al . using grazing - incidence x - ray diffraction ( gixrd ) was able to quantify the orientation , size , and spatial placement of crystalline p3ht regions within neat films . by analyzing the intensities of edge - on and face - on diffraction peaks in situ during film formation , along with optical absorption spectra , a picture is able to be drawn of the overall evolution of film crystallinity formation and orientations , as shown in figure 35 . initial edge - on aggregates align on the substrate ( a ) , which grow into crystallites ( b ) , followed by the formation of face - on aggregates higher up in the film ( c ) , which grow into crystallites ( d ) before all the elements finally form a tightly packed structure ( e ) . reprinted with permission from ref ( 229 ) . the term aggregate was used to describe one - dimensional -stacking , while crystallite was used to describe lamellae of those aggregates giving two - dimensional ordering . it was found that edge - on p3ht crystallites , 20 10 10 nm in size dominate at the substrate film boundary , before giving way to face - on orientated crystallites 6 8 3 nm in size further inside the film . the ratio of the overall edge- and face - on orientations was dependent on the casting solvent used , with chloroform giving predominantly face - on aggregates in the bulk of the film , while dichlorobenzene gave primarily edge - on . for light harvesting , the structure of the p3ht : pc61bm bhj blends is important , and it is found that some of the same observations hold true . in the as - cast blend , p3ht crystallites are primarily edge - on to the substrate , while after thermal annealing it was found that these reorientate to face - on . the size of the crystallites increases upon annealing , and in another study , it was found that the inclusion of pc61bm somewhat restricts the size that the p3ht crystallites can grow to upon annealing when compared to films of neat p3ht . a detailed discussion of the complex results that can be obtained from diffraction techniques is outside the scope of this work , but we note that a number of studies have investigated polymer : fullerene blends ( typically p3ht : pc61bm ) and attempted to place those results within the context of other morphological , electrical , and photophysical knowledge on the blend , and a comprehensive review of the topic has also been published by rivnay et al . one final application of diffraction techniques that we do wish to highlight is their use for in situ studies of the evolution of order in thin films as they are annealed . this can also be in combination with other photophysical measurements , such as variable - angle spectroscopic ellipsometry ( vase ) or field - effect mobility measurements to give a greater understanding of the annealing process . observing the evolution of the material crystallinity during annealing can give new insights into how the crystalline regions form and grow under the influence of heat , as shown for p3ht : pc61bm films in figure 36 . here grazing - incidence x - ray diffraction measurements have been made continuously while the blend film has been annealed , and the out - of - plane crystallite domain size has been calculated from the scattering peak size with the scherrer equation . it can be seen that as soon as heat is applied to the film , the domain size increases and is then followed by a slower period of growth lasting for 10 min of annealing . annealing longer than 10 min leads to a subtle loss of domain size over time , consistent with the optimal annealing time in devices being in the 1015 min range . when the film is cooled , a clear drop in the p3ht domain size is observed and may be linked to some disorder being reintroduced now that less thermal energy is available . from these results , one can see that dynamical experiments to study annealing can give unique information on the morphology of bhj blends , and a number of important studies have been reported that indicate the power of this methodology . time evolution of out - of - plane ( oop ) domain sizes for p3ht calculated from scherrer s equation on the recorded grazing incidence x - ray diffraction peaks . the sample is heated ( the temperature is indicated by the black line , right - hand axis ) and the domain size is monitored for different weight percentages of p3ht : pc61bm blend films . overall , the domain size increases when the film is heated for 15 min . a small reduction in domain sizes occurs when the film is cooled , but the overall sizes are still substantially larger than before annealing . copyright 2011 american chemical society . finally , to draw all of the knowledge presented on the morphology of p3ht : pc61bm together , we present an overall schematic of the as - cast organization of the material and how that is altered by thermal annealing , as depicted in figure 37 . overall p3ht : pc61bm morphology , in - plane ( a ) and laterally ( b ) of as - cast blend films ( left column ) and after thermal annealing ( right column ) , with p3ht - rich shown as shades of blue and pc61bm - rich as shades of red . initially , small spatially isolated domains of both materials form , which after thermal annealing join up to make contiguous domains in three dimensions . thermal annealing promoted the formation of pure crystalline domains of p3ht , which are shown as the darkest blue regions in the annealed sample . the impact of the morphology on the charge recombination in transient absorption is shown in panel c , and here the as - cast film is the black open squares and the black line and shows fast geminate recombination owing to a higher degree of mixing , while the annealed sample ( red open circles , red line ) has markedly less geminate recombination . pc61bm solution by spin - coating we have a primarily mixed blend of the two materials , with phase separation promoted on small length scales . small p3ht crystallites align edge - on to the substrate , but overall , light harvesting in an opv configuration is hindered owing to the small isolated domains of both materials that will enhance exciton dissociation but strongly hamper charge transport and extraction . thermal annealing of p3ht : pc61bm blends has been shown to triple the power conversion efficiency , and morphology studies can explain why . in this blend , the light harvesting is optimized owing to the larger phase separation that leads to bicontinuous pathways of polymer and fullerene that form in - plane and out - of - plane . a mixed phase of the two materials still exists , but it constitutes a smaller fraction of the overall film after annealing . these results are consistent with transient absorption studies that showed that charge generation is much slower in thermally annealed p3ht : pc61bm blends than in as - cast blends and presumably is limited by exciton diffusion to a heterojunction . the charge recombination mechanism also changes upon thermal annealing of p3ht : pc61bm blends : while as - cast blends show substantial geminate recombination , in annealed blends , recombination is predominantly bimolecular , indicating that charge pairs dissociate more efficiently into free carriers in annealed blends . geminate recombination is suppressed by thermal annealing , but the network is still spatially fine enough to ensure that all excitons reach heterojunction by diffusion and give efficient charge generation . the final material that we will examine in detail for how light harvesting is dependent on optimization of the morphology is the high - performance polymer ptb7 . with optimized electrodes and interlayers , ptb7-based blends are capable of reaching power conversion efficiencies in the range of 910% . the material has proven important in propelling organic photovoltaic devices to and across the 10% efficiency barrier , an important milestone not just psychologically , but also to enable opvs to be potentially competitive with amorphous silicon pv as a low - cost power generation technology . nearly always , the highest efficiencies are achieved with the use of the high boiling point additive 1,8-diiodooctane ( dio ) . dio is added at 3% by volume to the solution prior to spin - coating and is found to double the efficiency . current voltage curves and external quantum efficiency spectra for as - cast and additive - treated ptb7:pc71bm devices are shown in figure 38 , with efficiencies slightly lower than in optimum devices owing to nonoptimized contacts and lack of interlayers , but the blends have active layer deposition conditions ( blend ratio , concentration , spin speed , etc . ) the doubling of the efficiency with the use of dio is a good question to investigate in the context of the interplay between the blend morphology and its effect on light harvesting , for such dramatic device performance improvements are at the heart of understanding why bulk heterojunctions work well and how the can be fully optimized . i v curves ( a ) and external quantum efficiencies ( b ) for ptb7:pc71bm devices from chlorobenzene as cast ( black lines ) and with 3% diiodooctane ( dio ) added to the casting solvent ( red lines ) . licensed under cc - by-3.0 . without the use of the additive dio , the blend films of ptb7 with pc71bm at a ratio of 1:1.5 show large quasi - periodic round features 150200 nm in diameter that are clearly visible in afm and tem images ( figure 39 ) , not dissimilar to the observed morphology of mdmo - ppv : pc61bm ( vide supra ) . afm topography ( a ) and bright - field tem ( b ) of ptb7:pc71bm blend films from chlorobenzene as - cast . copyright 2010 john wiley and sons . for such strong tem contrast there must be a high degree of material purity between the ptb7 and pc71bm regions , and the fullerene can be identified as the main constituent of the large 150200 nm domains from its interactions with electrons that form the tem image . in opv blends containing pc71bm , the fullerene contributes significantly ( up to half ) of the photocurrent , absorbing strongly in the blue and green . with large pure fullerene domains this should lead to a low degree of light harvesting from the fullerene , for excitons that form inside such a large volume will only be able to diffuse 3 nm before decaying to the ground state . it is thus surprising when time - resolved photoluminescence of the fullerene was measured in the blend and showed a very fast decay , with an average lifetime of 67 ps , indicating that there was a significant amount of quencher ( i.e. , ptb7 ) in the immediate vicinity of the fullerene molecules . this is in contrast to the afm and tem images , which show homogeneous , presumed pure , domains of pc71bm . the average lifetime is in fact comprised of a number of components , including a fast 300 fs decay that is consistent with fullerene exciton dissociation due to hole transfer to ptb7 that has to be right next to the fullerene molecules . slower picosecond decays represent exciton diffusion within regions of pc71bm before quenching due to hole transfer to ptb7 . as the diffusion coefficient of pc71bm has been determined , the morphology can be derived from the time - resolved pl decays . a best - fit is found for a region of pure fullerene material , spherical in shape with a diameter of 60 nm , as shown in figure 40a , well below the observed fullerene domain size of 150200 nm . this led to a further examination of the morphology , and upon closer investigation , a skin layer of ptb7 was observed to be covering the active layer ( figure 40b ) , similar to some mdmo - ppv and p3ht blends , as briefly mentioned in their respective sections above . this skin layer is polymer - rich in character , blankets the top of the film , and therefore obscures the structural organization of the material beneath it when probed with surface characterization techniques such as afm . by removing this skin layer with plasma etching , the true morphology of the as - cast blend could then be observed with afm ( figure 40c ) , and the blend displays a remarkable ordered morphology , with the large fullerene domains appearing to actually comprise numerous small fullerene spheres 2060 nm in size , surrounded by a ptb7-rich matrix . ( a ) results of time - resolved pl decay modeling , using the pc71bm exciton diffusion coefficient to determine that pure fullerene spheres 60 nm in size exist , much smaller than what is observed from the initial morphology measurements . ( b ) sem cross - sectional image of the blend , showing the skin on top of the blend layer that was obscuring the true morphology . ( c ) after removal of the skin with plasma ashing , afm topography shows that the large domains actually consist of a large number of small fullerene domains , 2060 nm in size , agreeing well with the time - resolved pl data in panel a. reprinted from ref ( 77 ) . this work is a good example of utilizing time - resolve photophysics to derive nanoscale morphology in an opv blend , for with a cursory glance the fullerene domains would be presumed to be 200 nm in diameter , whereas in fact they are 60 nm . the small pure - fullerene spheres sit in a ptb7-rich matrix , which studies have shown to be 70% ptb7 and 30% pc71bm . the matrix and skin layer are presumed to be a quintessentially mixed phase , with polymer and fullerene completely mixed together at the smallest length scales with no discernible phase separation . an overall picture of the morphology without additive is thus able to be built and explains the low power conversion efficiencies that are obtained . absorption of light and dissociation of excitons into charge pairs by the polymer and fullerene are both good , but charge separation , transport , and extraction are made difficult owing to the morphology . the small pure - fullerene spheres lead to regions where there is a large surface area for dissociation but no continuous network for electron transport . furthermore , the intimately mixed - phase regions will promote charge recombination , for there are no separated domains to keep charges apart . finally , the polymer - rich skin at the top of the film acts as a partial electron - blocking layer , which is not the most advantageous arrangement next to the electron - extracting cathode in the device stack . consequently , this morphology tells us that even when one does have good exciton dissociation and clear domains of material , light harvesting may not be possible owing to the fact that all parts of the photovoltaic process need to be satisfied to turn absorbed photons into extracted charges . with the use of a solvent additive , the device performance of ptb7:pc71bm solar cells doubles . as the morphology of blends without additive was found to be particularly unattractive for good devices , it was speculated that this is where major improvements must occur . the use of solvent additives with opvs has occurred for a number of different blends , generally to control the evaporation rates of the host solvent and to enable the correct amount of crystallization or phase separation to occur . the type of additive , its boiling point , and the volume percentage used are typically determined by trial and error in order to achieve the optimal device performance . for ptb7:pc71bm , the additive found to work best is dio , and the volume added is 3% into chlorobenzene for an overall 25 mg ml solute concentration ( 10 mg of ptb7 and 15 mg of pc71bm ) . afm or tem studies of the optimized blend with dio show a uniformly mixed blend ( figure 41a , b ) with no discernible features . this is consistent with the time - resolved pl from the blend , which shows a very fast predominantly 100 fs decay of fullerene excitons . using x - ray microscopy , the miscibility of the fullerene into ptb7 is measured and found to be 60% , with variations existing between 52 and 68% , creating regions that are slightly polymer - rich and regions that are fullerene - rich ; however , the spatial resolution of the technique does not allow any quantifiable information on that spatial concentration variability . afm topography ( a ) and bright - field tem ( b ) of ptb7:pc71bm blend cast from chlorobenzene with 3% dio added to the solution prior to spin - coating . as shown , afm and tem do not have the ability to discriminate between the two materials with such a high degree of mixing between them . the greatest success was seen with photoconductive afm ( figure 42 ) , where remarkable contrast in photocurrent was observed , with feature sizes down to a few tens of nanometers . the pc - afm photocurrent map shows swirling linear features of high and low photocurrent . light for the generation of photocurrent is provided by the afm tip laser at 670 nm and thus is primarily absorbed in regions that are rich in ptb7 , leading to high photocurrent , while low photocurrent is due to regions rich in pc71bm . while the observed morphology is in the substrate plane , and current flow is perpendicular to that , what is observed is still relevant , as charges may have to move laterally before finding vertical pathways . if a closer look is taken at the morphology ( figure 42b ) , then one can see that the ptb7-rich and pc71bm - rich domains form extended elongated shapes , with transitionary photocurrent measured between the domains ( i.e. , the photocurrent transition follows a gradual linear dependence rather than a sharp step function between the domains ) . ( a ) photocurrent map of ptb7:pc71bm blends spin - coated from chlorobenzene solution with 3% dio . measurements were made by photoconductive afm at a bias of 3 v and with photoexcitation at 670 nm . ptb7-rich regions are shown in blue , mixed - phase regions in green , and pc71bm - rich regions in red . in panel b is shown a close - up of the boxed region in panel a , indicating that the two materials form elongated fiberlike domains with respect to each other . examination has also been made of the crystallinity of ptb7 by x - ray diffraction experiments . in work by hammond et al . , ptb7 was measured in blends with and without dio , and when combined with spectroscopic ellipsometry , information on the alignment of the polymer chains and the degree of crystallinity can be obtained . what was found was that ptb7 was not highly crystalline ; indeed , by one measure only 21% of the polymer chains were aligned in blend films . however , this does not mean that the majority of ptb7 is amorphous , as the authors comment that paracrystallinity is an important consideration in the blend films , i.e. , partial local ordering aided by lattice strain . in addition , there is some evidence from other work that polymers very similar to ptb7 readily aggregate , and while this is not crystalline order , it is not what one would class as a truly amorphous polymer either . hammond et al . also found that dio plays no role in enhancing the crystallinity in the blend films , with xrd being almost identical in blends with and without dio . the crystals that form are small by the standards of p3ht discussed above , with typical dimensions of 1.5 5 nm ( i.e. , ca . thus , overall the morphology generated with 3% dio is highly advantageous for light harvesting in a photovoltaic cell , creating a blend with large surface areas between donor and acceptor for efficient exciton dissociation into charge pairs and quite small , interconnected , pure domains of both materials for charge separation and transport with minimal recombination losses . it is clear that in ptb7:pc71bm the morphology plays a very important role in light harvesting , for if the morphology is as it is when no dio additive is used , solar cell efficiency is halved . the role of dio in the blend is to enable the highly advantageous morphology for opv performance ; however , why dio has that role is still not fully understood . the common function of dio as a high boiling point solvent to slow the evaporation process to enhance crystallinity is not relevant here , where neither material is highly crystalline . instead , dio seems to help mixing and miscibility of ptb7 and pc71bm , with fullerene miscibility into the polymer enhanced from 30 to 60% when dio is used . however , the improved miscibility not only leads to well - mixed materials but also to an optimal fiberlike nanoscale organized morphology , thereby enabling not only efficient light harvesting to generate charge , but also extraction of those charges . although the highest efficiencies with ptb7 are obtained with pc71bm , bhjs can also be made with pc61bm , with power conversion efficiencies in the region of 34% . as a testament to how complex bhj morphology can be , the organization of the two materials in the pc61bm blend is completely different from the pc71bm one discussed above . the higher miscibility of pc61bm than pc71bm leads to a higher degree of mixing and a more complex nanoscale morphology . small crystallites a few nanometers in size of polymer and fullerene form near to each other , embedded in a more disordered matrix of both materials . a subtle large - scale phase separation on the order of 150 nm exists , but is not as well - defined and impactful as in the pc71bm blends . overall the role of dio in the pc61bm blends is less important , with the additive enhancing mixing when chlorobenzene is used as the casting solvent but otherwise not changing measured parameters drastically . this is borne out by the overall device efficiency , which only increases by a third with the use of dio , compared to the doubling of device efficiency for pc71bm - based blends with chlorobenzene . to conclude this section , we can see that the morphology the spatial organization of the donor and acceptor materials in the blend is vitally important to successful harvesting of sunlight in photovoltaic devices . the optimal morphology for each blend combination frequently reduces to a compromise of competing photophysical and electrical requirements for light absorption , charge generation , transport , and extraction . if the donor and acceptor are too spatially disconnected into phase - separated domains , then exciton diffusion is unable to ensure that absorbed light can be converted into charge pairs . conversely , if the two materials are too well mixed , then charge pairs form easily but can not be readily separated or kept apart for long enough to enable extraction . if crystallinity in some of the opv materials is not great enough , then the charge mobility is too low ; if the crystallinity is too high , then charge - pair generation is restricted . the morphology in opv blends is a delicate balancing act , the formed morphology has to be just right , as otherwise losses in device performance are incurred . almost always this just right morphology is found by trial - and - error device fabrication runs , combining different solvents , additives , deposition methods , and postdeposition treatments ( solvent vapor annealing , thermal annealing , etc . ) until an optimum is obtained . material scientists then explore these morphologies with a suite of techniques , as detailed in this section to determine what this optimal scientifically this is not the most efficient way of improving device performance and finding the best deposition methodologies . it is thus a pressing problem to attempt to understand the actual nanoscale organizational physics of how these materials interact and blend with each other and then determine / predict what the resultant morphology would be . computationally and in terms of physics understanding this is obviously a grand challenge , all the more challenging because each blend combination of donor and acceptor forms a different morphology . the combination of donor and acceptor materials in terms of synthetic chemistry capabilities is near infinite ; thus , exploring these via trial - and - error device fabrication with all the possible solvents , additives , etc . is simply impossible . significant improvements have been realized , as this section demonstrates , with approximate doubling of device efficiencies at each node on the path of progress , from 1 to 2% efficiencies with mdmo - ppv , through 34% with p3ht and now 710% with ptb7 . to continue this progress , new materials with advantageous photovoltaic properties will need to be created and optimal morphologies for them determined , preferably predictively rather than retrospectively , to enable the best light harvesting . in this review , we have provided a detailed picture of the main processes and variables that govern light harvesting in organic photovoltaic cells . the great strength of organic materials for photovoltaic applications is the strong absorption of light that enables the use of thin films on the order of 100 nm from nontoxic , relatively stable , plastic materials . the materials can be deposited from solution by simple coating processes , and they offer the prospect of simple fabrication of large areas of solar cells at low cost and with low energy of manufacture . as the primary photoexcitations are tightly bound frenkel excitons , a key step in light harvesting is splitting them into free charges which is achieved using a charge - accepting material ( normally an electron acceptor , if as is most often the case the absorbing material is an electron donor ) . a combination of exciton diffusion and fret leads to exciton transport to the acceptor . since excitons in most materials diffuse < 20 nm , planar heterojunctions are limited to be thin organic layers that do not fully absorb the incident light . this is overcome by creating a bulk heterojunction throughout the full 100 nm thickness of the film , with separation between the electron donor and acceptor on the order of 20 nm . the bulk heterojunction is used out of necessity rather than desire , as it creates a wide - range of complications in designing materials for efficient harvesting of light . the efficient operation of a bulk heterojunction requires a particular arrangement of the donors and acceptors ( morphology ) that facilitates excitons reaching the heterojunction and also enables charge separation and extraction . at present it is not known in advance if a new material will be able to make a suitable morphology , and progress in the field is slowed by needing to explore a wide range of processing conditions with no guarantee of success . the first is by making so - called ordered heterojunctions in which either donor or acceptor are patterned and the other material added to make the desired morphology . the length scales involved are demanding , and so far efficiencies using this approach have been limited . nevertheless , progress in self - assembly and in nanoimprint lithography could help to achieve this . the second main approach is to enhance the effective exciton diffusion length ; a factor of 5 increase would enable planar heterojunctions to harvest almost all of the incident sunlight . this would simplify fabrication , avoid the time - consuming optimization of donor acceptor morphology , and enable materials to be used that do not form appropriate bhj morphologies . the suggested increase in the exciton diffusion length is clearly very challenging , but advances in materials , processing , and possibly device structure combined with the advanced experimental techniques and synthetic chemistry available today could enable it to be realized . the third approach is further improvement of the bulk heterojunction by moving from an empirical to a predictive approach . understanding of exciton diffusion and dissociation and charge separation , transport , and extraction in bulk heterojunctions all convolved with a complex nanoscale morphological arrangement of the materials has reached a remarkable degree of maturity , given the complexity of the blends . moving from a regime of reactive understanding of the systems created to predictive control of such systems with the use of advanced electrical , photophysical , morphological , theoretical , and synthetic chemical scientific techniques is a prospect with serious merit and one that could transform the relatively trial - and - error approach that has been used thus far into a powerful predictive method for improving or optimizing solar cell efficiencies . finally , it is worth pausing to consider the remarkable progress that has been made in organic photovoltaics , with power conversion efficiencies of opvs increasing by a factor of 5 in the last 20 years , with lab - based devices now regularly exceeding 10% . in this review we have described the key processes that all have to be present in order for the successful harvesting of light in an organic solar cell , and have detailed how understanding of these processes has contributed to such performance increases . with good prospects for further understanding and optimization , future improvements in device efficiency are anticipated . the external quantum efficiencies at short - circuit conditions now reach 7080% in record - efficiency organic photovoltaic cells over spectral regions of strong light absorption . however , the open - circuit voltage from single - junction cells is only about 0.8 v or lower and potentially can be increased . there are several factors that make the open - circuit voltage much lower than the energy of the absorbed photon could allow . first , the higher - energy exciton states usually relax to the lowest - energy exciton before splitting into an electron hole pair . these energy losses can be avoided by developing tandem cells or multijunction cells that use a combination of different absorbers . in such cells , higher energy photons are absorbed and converted in a subcell with higher band gap materials . in february 2016 , the german company heliatek demonstrated a record power conversion efficiency of 13.2% in small organic solar cells using this approach with their proprietary materials . a second source of energy loss is the energy offset between exciton states and charge transfer states , which is the driving force for charge pair generation . we have discussed recent findings that the optimum driving force for charge separation equals the reorganization energy in sections 3.1 and 3.2 . this implies that photovoltaic materials with small reorganization energies and a small driving force can minimize the loss of open - circuit voltage at a heterojunction without a trade - off of photocurrent . a further loss of open - circuit voltage of typically 0.5 v is associated with charge extraction . multiple factors were suggested to contribute here , including energetic disorder for charge transport , the nature of the electrical contacts , and carrier recombination . the main findings on this topic have been reviewed several times . overcoming limitations in the open - circuit voltage outlined above can be expected to lead to further progress in the power conversion efficiency of organic photovoltaic devices .

the field of organic photovoltaics has developed rapidly over the last 2 decades , and small solar cells with power conversion efficiencies of 13% have been demonstrated . light absorbed in the organic layers forms tightly bound excitons that are split into free electrons and holes using heterojunctions of electron donor and acceptor materials , which are then extracted at electrodes to give useful electrical power . this review gives a concise description of the fundamental processes in photovoltaic devices , with the main emphasis on the characterization of energy transfer and its role in dictating device architecture , including multilayer planar heterojunctions , and on the factors that impact free carrier generation from dissociated excitons . we briefly discuss harvesting of triplet excitons , which now attracts substantial interest when used in conjunction with singlet fission . finally , we introduce the techniques used by researchers for characterization and engineering of bulk heterojunctions to realize large photocurrents , and examine the formed morphology in three prototypical blends .

Introduction Excitation Energy Transfer to Heterojunction Free Carrier Generation Optimum Morphologies of Bulk Heterojunctions Summary and Outlook

consequently , harvesting of sunlight for the generation of electrical energy is highly desirable , and great developments in the conversion efficiency of photovoltaic cells have been made over the last 50 years . light absorbed in the organic layer forms tightly bound excitons that with clever choices of materials and device architectures are split into free electrons and holes , which are then extracted at electrodes to give useful electrical power . organic photovoltaic materials and devices for solar energy conversion have been researched extensively in the last 2 decades , and remarkable progress has been achieved , with recorded power conversion efficiencies over 10% for organic solar cells . in order to split an exciton into a charge pair , a heterojunction of electron donor and acceptor materials is used in which the homo and lumo energies of the acceptor are lower than those of the donor ( figure 1b , c ) . for simplicity in organic photovoltaics schematic illustration of charge generation in organic photovoltaic materials which involves ( a ) light absorption and the generation of a singlet exciton with opposite up and down spins , followed by energy transfer to a type ii heterojunction of an electron donor and acceptor and then ( b ) electron or ( c ) hole transfer at the heterojunction . excitons can be transported to a heterojunction by frster resonance energy transfer ( fret ) between the electron donor and electron acceptor and by exciton diffusion in the donor or acceptor materials . in the first , shown on the left in figure 2 , the donor and acceptor materials are deposited as a simple bilayer structure , while on the right , the two are mixed throughout the active layer to form a bulk heterojunction . because of a limited width of the absorption spectra of organic materials , for the best coverage of the solar spectrum , one has to use donor and acceptor materials with distinct absorption spectra . power conversion efficiencies ( pce ) of around 6% have been achieved in research solar cells with 1 cm active area using organic bilayers . this has been successfully implemented by blending electron donor and acceptor materials , where spontaneous separation of donor and acceptor phases forms a nanostructure that is known as a bulk heterojunction . the small length scale of the bulk heterojunction phase separation enables good harvesting of excitons , with only a few example tracks for the drawn excitons in figure 2 being lost , with most harvested ( some easily and some only just , depending on the initial position and the path followed ) . recent studies of bulk heterojunctions have shown that the blend morphology has to be just right for the charge pair to dissociate into free carriers , and we discuss this in more detail in section 4.2 . the free carrier generation and their transport to the correct electrodes in bulk heterojunctions relies on continuous and uninterrupted donor and acceptor phases . in a heterojunction with small donor and acceptor domains and very high interface area , a charge is more likely to encounter the interface with the other component of the blend and hence more likely to recombine with its opposite charge . this complex series of steps involved in light harvesting has led to intensive research efforts on bulk heterojunction solar cells , and recently , multiple 1 cm active area research cells have been demonstrated with pce above 10% . the progress was achieved by rational design of donor and acceptor materials with efficient light absorption and good charge transport , morphology optimization of the active layers , and development of new charge transporting layers . in summation , the key steps of converting light into electrical power in organic photovoltaics are ( a ) light absorption , ( b ) energy transfer ( by exciton diffusion and/or fret ) to a heterojunction , ( c ) exciton splitting into an electron hole pair , ( d ) dissociation of bound charge pairs into free carriers , and ( e ) charge extraction to the electrodes . we present an overview of energy transfer mechanisms , methods to measure exciton transport , and recent efforts to improve light harvesting in planar heterojunctions by enhancing the exciton diffusion length , by encouraging interlayer fret , and by directing exciton diffusion . we briefly discuss harvesting of triplet excitons , which now attracts substantial interest when used in conjunction with singlet fission , where one high - energy singlet exciton can generate two triplet excitons and so double the charge carrier yield . in particular , we discuss the role of the morphology of bulk heterojunctions , which has to be just right for the best solar conversion efficiency . we introduce the main techniques for measuring the morphology of bulk heterojunctions and discuss optimized morphologies for photovoltaic performance . in this section we discuss the mechanisms of energy transfer , the techniques to measure exciton diffusion , and recent efforts to enhance the exciton harvesting range by increasing their diffusion length and by directing exciton transport . in the weak coupling limit the rate of fret can be calculated using the fermi golden rule2where v is the electronic coupling energy between the exciton and energy - accepting chromophore ; s is the dielectric screening of the interaction by the surrounding medium ; j is the spectral overlap between the homogeneous spectral profiles of exciton luminescence f(e ) and absorption of energy - accepting chromophore a(e ) , which have each been normalized to unit area on an energy scale , ; and emax is the upper energy of the luminescence and absorption spectra . when the emitting and absorbing dipoles can be approximated as point dipoles , the energy transfer rate has a simple expression3where is the luminescence lifetime of the excited chromophore in the absence of energy transfer , r is the distance between the exciton emission dipole and the energy - accepting dipole , and r0 is the frster radius , which depends on the photoluminescence quantum yield of the excited chromophore , the angle between the donor emission and acceptor absorption transition dipole moments , and the spectral overlap j , which was defined above . as efficient fret requires strong spectral overlap of absorption and emission , fret from the triplet to singlet state is inefficient , and triplet the dominant transport mechanism of triplet excitons is by electron exchange , which is often referred to as dexter transfer . it is assumed that excitons move by a random walk , and the results can then be modeled using a one - dimensional diffusion equation4where g(x , t ) is the exciton generation rate , d is the exciton diffusivity in the direction perpendicular to the quenching interface , kq(x ) is the rate constant of surface quenching , which depends on the distance to the quenching interface , and ks is the rate constant of spontaneous exciton decay measured in the reference film of the same thickness but on a nonquenching surface ( for example , fused silica ) . in the simplest analysis and estimation of the exciton diffusion length , it is usually assumed that exciton diffusivity is time - independent ; however , different models have been proposed to describe time - dependent exciton diffusivity in materials with significant energetic disorder , and we discuss this in more detail in section 2.3 . it was assumed that all excitons excited within a diffusion length of the interface are ionized at the interface , lead to separated charges , and are then extracted . to absorb most of the incident light , the thickness of donor and acceptor layers has to be at least 100 nm ; therefore , there is a strong drive to increase the exciton diffusion length in order to improve light harvesting in planar heterojunctions . using this approach , they made planar - heterojunction solar cells with the fullerene c60 as an electron acceptor and obtained a power conversion efficiency of 4.4% , which is 30% higher than that of the control cell made using undiluted subphthalocyanine molecules . in contrast to exciton diffusion , fret between electron donor and acceptor materials is directional and can bring excitons directly to a heterojunction . the general equation to describe the rate of fret from a point donor to a slab of acceptor molecules of thickness is10where z is the distance between the excited donor and the acceptor film , is the pl decay time of the donor in the absence of energy transfer , and d0 is the critical transfer distance ( figure 14 ) . triplet exciton transport now attracts substantial interest when used in conjunction with singlet fission , where each singlet exciton can split into two triplet excitons and so can potentially double the charge carrier yield and photocurrent . these studies suggest that harvesting of triplet excitons is a bottleneck for charge carrier generation in bilayer solar cells using polycrystalline pentacene , where the triplet exciton lifetime is on the order of 5 ns . because charge transfer between electron donor and acceptor is a short - range process , initially an exciton is split into a bound geminate electron hole pair . in this section , we briefly discuss the main experimental findings and concepts suggested to explain free carrier generation in bulk and planar heterojunctions . on the basis of experimental findings , several different mechanisms of free carrier generation have been proposed and they are shown schematically in figure 16 . however , several studies have shown that the internal quantum efficiency ( the ratio of collected charges and absorbed photons at the short circuit condition ) in high - performance bulk heterojunctions is independent of the energy of absorbed photons , including a direct excitation of the relaxed intermolecular ct states formed between donor and acceptor with very low photon energy ( indicated by a dashed line in figure 16 ) . slower charge generation in annealed blends suggests larger domain sizes of donor and acceptor , which help dissociation of bound charge pairs into free carriers , as discussed in section 4.2.2 . in very efficient photovoltaic blends of the electron - donating polymer ptb7 with the acceptor pc71bm these results suggest that the rate of electron transfer at the heterojunction does not have to be ultrafast to achieve high device efficiencies in organic photovoltaics . in the next section , we discuss recent findings on how the rate depends on the energy offset between donor and acceptor . for example , charge pair generation with 1520% efficiency has been observed in films of conjugated polymers that are used as electron donors , and neat films of fullerene derivatives , which are commonly used as acceptors . to avoid recombination losses , the morphology of bulk heterojunction has to support fast extraction of holes and electrons into the donor and acceptor domains , respectively , where they can be transported to the correct electrodes , and this is discussed in section 4.2 of this review . charge pair dissociation into free carriers is a crucial step in the operation of organic solar cells ; therefore , its understanding is very important for the development of efficient heterojunctions . free carrier generation in these blends occurs through relaxed charge transfer states and is influenced by blend morphology , energetics of mixed and pure donor and acceptor phases , charge delocalization , and carrier mobility . free carrier generation in planar heterojunctions has been found to depend on applied electric field and conjugation length of the electron donor , indicating that the mechanism could be different from that in the bulk heterojunction . so far we have recounted that absorption of light by donor and acceptor materials in a bulk heterojunction readily occurs ; however , harvesting that light requires , among other things , getting the formed exciton to the donor : acceptor interface via exciton diffusion or fret and getting free charges through the active layer to their respective electrodes to be extracted , as discussed above . consequently , it is clear that the structural arrangement of the donor and acceptor in the blend if the donor and acceptor are too far apart , then excitons will not be able to traverse the distance to enable them to be split into charge pairs , and if the distance is too short , then while excitons will easily dissociate , sufficiently separate pathways of material will not be available to suppress recombination of the readily formed charge pairs . measuring the morphology on such length scales is very challenging ; the donor and acceptor are typically both carbon - based materials , soft in nature , with similar physical properties . in this section , we will review the principal methods of measuring the morphology of bhj blends , discuss some of the key results published to date and their implications for light harvesting in organic photovoltaics , and highlight the challenges that remain . in the context of organic photovoltaics , afm can give information on the surface and subsurface topography of the film . this has the added advantage of determining the exciton harvesting of the blend itself directly , with the population of excitons formed from light absorption being measured and the degree of quenching by the other component of the blend quantified . with an understanding of the key techniques to measure morphology , we now turn our attention to study the key donor : acceptor blend morphologies that have been reported over the last 15 years . in discussing the morphology here , we are particularly thinking of its role in light harvesting and the operation of the device : as already explained , in order to harvest light in a bulk heterojunction organic photovoltaic cell , the donor and acceptor domains need to be within an exciton diffusion length ( typically 10 nm ) of where the light is absorbed . each donor material represents a key generational point in the development of opvs , with mdmo - ppv blends reaching power conversion efficiencies of 13% , p3ht 35% , and ptb7 710% . mdmo - ppv was one of the earliest successful opv donor materials , giving reliable power conversion efficiencies in the region of 12% , with the highest efficiency of 3% being reached when blended with pc71bm . the phase separation is , however , relatively uncontrolled , and at the optimal fullerene loading ratio of 80% , large 60500 nm fullerene domains form , with the size dependent on the casting solvent used for the blend . blends of p3ht with lu3n pc81bh produce power conversion efficiencies of up to 4% , with advantageous open circuit voltages of 0.8 v , and so are a valid blend composition for investigation of morphology . examination of the three phases ( p3ht , fullerene - rich , and mixed ) in as - cast films and annealed films that give optimal power conversion efficiencies show strong differences that explain the differing light harvesting . power conversion efficiencies in these devices can reach 2% , and it is an interesting candidate for photovoltaic cells , as the inorganic component carries distinct advantages ( high dielectric constant , high charge mobility ) , so the formed morphology is important . the ratio of the overall edge- and face - on orientations was dependent on the casting solvent used , with chloroform giving predominantly face - on aggregates in the bulk of the film , while dichlorobenzene gave primarily edge - on . finally , to draw all of the knowledge presented on the morphology of p3ht : pc61bm together , we present an overall schematic of the as - cast organization of the material and how that is altered by thermal annealing , as depicted in figure 37 . the doubling of the efficiency with the use of dio is a good question to investigate in the context of the interplay between the blend morphology and its effect on light harvesting , for such dramatic device performance improvements are at the heart of understanding why bulk heterojunctions work well and how the can be fully optimized . by removing this skin layer with plasma etching , the true morphology of the as - cast blend could then be observed with afm ( figure 40c ) , and the blend displays a remarkable ordered morphology , with the large fullerene domains appearing to actually comprise numerous small fullerene spheres 2060 nm in size , surrounded by a ptb7-rich matrix . finally , the polymer - rich skin at the top of the film acts as a partial electron - blocking layer , which is not the most advantageous arrangement next to the electron - extracting cathode in the device stack . although the highest efficiencies with ptb7 are obtained with pc71bm , bhjs can also be made with pc61bm , with power conversion efficiencies in the region of 34% . to conclude this section , we can see that the morphology the spatial organization of the donor and acceptor materials in the blend is vitally important to successful harvesting of sunlight in photovoltaic devices . in this review , we have provided a detailed picture of the main processes and variables that govern light harvesting in organic photovoltaic cells . as the primary photoexcitations are tightly bound frenkel excitons , a key step in light harvesting is splitting them into free charges which is achieved using a charge - accepting material ( normally an electron acceptor , if as is most often the case the absorbing material is an electron donor ) . this is overcome by creating a bulk heterojunction throughout the full 100 nm thickness of the film , with separation between the electron donor and acceptor on the order of 20 nm . finally , it is worth pausing to consider the remarkable progress that has been made in organic photovoltaics , with power conversion efficiencies of opvs increasing by a factor of 5 in the last 20 years , with lab - based devices now regularly exceeding 10% .

spondylotic lesions that cause cervical cord compression require surgical decompression either through anterior cervical discectomy / corpectomy with fusion or laminectomy / laminoplasty when they result in progressive neurological deficits2,3,13,20 ) . these procedures have been regarded as a gold - standard technique for decompression and stabilization . however , fusion - related problems such as loss of the cervical range of motion , non - union , instrumentation failure , and graft extrusion are still the subject of debates7,20 ) . the anterior cervical microforaminotomy ( acmf ) technique involves not only the direct removal of the compressive abnormality but also the preservation of the motion segments without bone fusion or post - operative immobilization9,11,15 ) . but , it naturally advanced to spinal cord decompression with extended technique ( extended anterior cervical foraminotomy , eacf ) . although many previous studies showed favorable clinical and radiological results , it has been used only for nerve root decompression . here , twenty - two of the patients who showed cervical cord compression on their pre - operative magnetic resonance scan images with radicular and/or myelopathic symptoms were enrolled in this study . the preoperative instability on cervical dynamic x - ray and already pre - existing narrowing of cervical spinal canal ( that is , developmental stenosis ) excluded in this study . the detailed surgical technique of acmf for cord compression has been reported by jho8 ) . a brief summary of our procedure from skin incision to exposure of the anterior column of the cervical spine is similar to other anterior approach to the cervical spine . an anterior cervical discectomy retractor system is applied to expose the ipsilateral longus colli muscle rather than the midline anterior disc surface . the lateral portion of the longus colli muscle is excised to expose the medial parts of the transverse processes of the upper and lower vertebrae . once the medial portion of the transverse processes of the upper and lower vertebrae is identified , the ipsilateral uncovertebral joint between them can be seen ; however , in advanced spondylosis , anatomical landmarks of the uncovertebral joint and transverse processes can not be delineated . the uncovertebral joint is removed between the transverse processes using a high - speed microsurgical drill attached to an angled hand piece . to prevent injury to the vertebral artery ( va ) , a thin layer of cortical bone is left attached to the ligamentous tissue covering the medial portion of this artery . as drilling advances posteriorly , the direction of the drill is gently inclined medially . when the posterior longitudinal ligament is exposed , a piece of thin cortical bone is left attached laterally to the periosteal and ligamentous tissue covering the va . this lateral remnant of the uncinate process is dissected from the ligamentous tissue and fractured at the base of the uncinate process . it is further dissected from the surrounding soft tissue and removed , which enables identification of the va by its pulsation between the transverse processes of the vertebrae . it is necessary to proceed cautiously with drilling at the base of the uncinate process because the nerve root lies just adjacent to it . after the uncinate process becomes loosened at its base , it is safer to remove the thin layer of remaining bone of the uncinate process by fracturing it rather than by continued drilling . the posterior osteophytes , herniated part of disc , some parts of the upper and lower endplates are removed . the procedure crossed over the midline diagonally toward the opposite margin of the spinal dura mater . the posterior longitudinal ligament is incised and resected to achieve adequate decompression of the ipsilateral nerve root and spinal cord . sometimes the beginning of the contralateral nerve root is identified for adequate decompression of the spinal canal in the transverse axis . the spinal cord canal is enlarged in the longitudinal axis by removing the posterior portion of the vertebral bodies with kerrison rongeurs and a long - armed up - biting curette . all the patients took pre - operative plain radiography of cervical spine , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and dynamic plain radiography of cervical spine in six weeks after their operation . analyses of the pre- and post - operative dynamic plain radiographs were done to evaluate postoperative instability . we measured five radiological data for evaluate to presence of postoperative instability and effectiveness of cervical cord decompression : the disc space height ( dsh ) , sagittal plane displacement ( translation ) , cobb angle , vertebral body resection rate and degree of cervical cord decompression . the disc space height was measured from the mid - point of the upper vertebral body to the mid - point of the lower vertebral body on the sagittal plane . to allow different magnification factors on different films , the measurements were compared with the height of the third cervical vertebrae ( c3)18 ) . the sagittal plane displacement was measured on the cervical flexion - extension radiographs as the linear distance in millimeter scale from the posterior - inferior corner of the superior vertebra to the posterior - superior corner of the inferior vertebra body . a distance greater than 3.5 mm defined instability19 ) . the sagittal plane alignment was defined on the lateral neutral radiographs in relation to the line that joined the postero - inferior edge of c2 to the postero - inferior edge of c7 . when all the intervening vertebral bodies were found anterior to this line , the alignment was defined as a lordotic curvature . the sagittal angulation , which is the angle between a line on the superior end plate of the vertebral body above and a line on the inferior border of the body below the levels operated 1 ) , was measured on the neutral lateral view at the operated level , using cobb 's method . the vertebral body resection rate was measured on pre- and post - cervical 3-dimensional ct scan . all scans were performed on a 64-slice multi - detector computed tomography scanner ( toshiba aquilion , tokyo , japan ) . the scanning parameters were as follows : voltage of 120 kv , current 150 mas , field - of - view 147 mm and slice thickness of 3 mm ( fig . the degree of cervical cord decompression was measured antero - posterior diameter and transverse area of the spinal cord at the site of maximal compression . the data from preoperative and postoperative t2-weighted axial mri analyzed using aquarius inutrition edition ver4.4.6 . the post - operative pain improvement was measured using the visual analogue score ( vas ) . the clinical outcome was graded as " excellent " when the patient showed complete resolution of all symptoms , " good " when the patient experienced relief of radiculopathy but still experienced occasional minimal / mild residual non - radicular discomfort , " fair " when the patient showed mild residual symptoms of radiculopathy with or without mild / moderated residual non - radicular discomfort , and " poor " when the patient continued to show significant radicular symptoms with or without non - radicular discomfort . we were analyzed to the degree of cervical cord decompression , disc space height loss , translation , cobb angle and vertebral body resection rate using paired t - test . a 52-year - old woman was referred to our hospital with a 6-month history of posterior neck pain , right shoulder pain and progressive myelopathy that both upper and lower extremity numbness ( right > left ) . on neurologic examination , she had grade 4-/5 weakness of her right elbow flexion and extension . mri scans demonstrated bulging disc c4 - 5 , c5 - 6 with focal ossification posterior longitudinal ligament and showed cervical cord compression . the patient underwent spinal cord decompression via right - sided microsurgical anterior foraminotomy holes at c4 - 5 , c5 - 6 . , she showed no instability on a flexion - extension dynamic roentgenogram of the cervical spine ( fig . twenty - two of the patients who showed cervical cord compression on their pre - operative magnetic resonance scan images with radicular and/or myelopathic symptoms were enrolled in this study . the preoperative instability on cervical dynamic x - ray and already pre - existing narrowing of cervical spinal canal ( that is , developmental stenosis ) excluded in this study . the detailed surgical technique of acmf for cord compression has been reported by jho8 ) . a brief summary of our procedure from skin incision to exposure of the anterior column of the cervical spine is similar to other anterior approach to the cervical spine . an anterior cervical discectomy retractor system is applied to expose the ipsilateral longus colli muscle rather than the midline anterior disc surface . the lateral portion of the longus colli muscle is excised to expose the medial parts of the transverse processes of the upper and lower vertebrae . once the medial portion of the transverse processes of the upper and lower vertebrae is identified , the ipsilateral uncovertebral joint between them can be seen ; however , in advanced spondylosis , anatomical landmarks of the uncovertebral joint and transverse processes can not be delineated . the uncovertebral joint is removed between the transverse processes using a high - speed microsurgical drill attached to an angled hand piece . to prevent injury to the vertebral artery ( va ) , a thin layer of cortical bone is left attached to the ligamentous tissue covering the medial portion of this artery . as drilling advances posteriorly , the direction of the drill is gently inclined medially . when the posterior longitudinal ligament is exposed , a piece of thin cortical bone is left attached laterally to the periosteal and ligamentous tissue covering the va . this lateral remnant of the uncinate process is dissected from the ligamentous tissue and fractured at the base of the uncinate process . it is further dissected from the surrounding soft tissue and removed , which enables identification of the va by its pulsation between the transverse processes of the vertebrae . it is necessary to proceed cautiously with drilling at the base of the uncinate process because the nerve root lies just adjacent to it . after the uncinate process becomes loosened at its base , it is safer to remove the thin layer of remaining bone of the uncinate process by fracturing it rather than by continued drilling . the posterior osteophytes , herniated part of disc , some parts of the upper and lower endplates are removed . the procedure crossed over the midline diagonally toward the opposite margin of the spinal dura mater . the posterior longitudinal ligament is incised and resected to achieve adequate decompression of the ipsilateral nerve root and spinal cord . sometimes the beginning of the contralateral nerve root is identified for adequate decompression of the spinal canal in the transverse axis . using the holes of anterior foraminotomies , the spinal cord canal is enlarged in the longitudinal axis by removing the posterior portion of the vertebral bodies with kerrison rongeurs and a long - armed up - biting curette . all the patients took pre - operative plain radiography of cervical spine , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and dynamic plain radiography of cervical spine in six weeks after their operation . analyses of the pre- and post - operative dynamic plain radiographs were done to evaluate postoperative instability . we measured five radiological data for evaluate to presence of postoperative instability and effectiveness of cervical cord decompression : the disc space height ( dsh ) , sagittal plane displacement ( translation ) , cobb angle , vertebral body resection rate and degree of cervical cord decompression . the disc space height was measured from the mid - point of the upper vertebral body to the mid - point of the lower vertebral body on the sagittal plane . to allow different magnification factors on different films , the measurements were compared with the height of the third cervical vertebrae ( c3)18 ) . the sagittal plane displacement was measured on the cervical flexion - extension radiographs as the linear distance in millimeter scale from the posterior - inferior corner of the superior vertebra to the posterior - superior corner of the inferior vertebra body . a distance greater than 3.5 mm defined instability19 ) . the sagittal plane alignment was defined on the lateral neutral radiographs in relation to the line that joined the postero - inferior edge of c2 to the postero - inferior edge of c7 . when all the intervening vertebral bodies were found anterior to this line , the alignment was defined as a lordotic curvature . the sagittal angulation , which is the angle between a line on the superior end plate of the vertebral body above and a line on the inferior border of the body below the levels operated 1 ) , was measured on the neutral lateral view at the operated level , using cobb 's method . the vertebral body resection rate was measured on pre- and post - cervical 3-dimensional ct scan . all scans were performed on a 64-slice multi - detector computed tomography scanner ( toshiba aquilion , tokyo , japan ) . the scanning parameters were as follows : voltage of 120 kv , current 150 mas , field - of - view 147 mm and slice thickness of 3 mm ( fig . the degree of cervical cord decompression was measured antero - posterior diameter and transverse area of the spinal cord at the site of maximal compression . the data from preoperative and postoperative t2-weighted axial mri analyzed using aquarius inutrition edition ver4.4.6 . the post - operative pain improvement was measured using the visual analogue score ( vas ) . the clinical outcome was graded as " excellent " when the patient showed complete resolution of all symptoms , " good " when the patient experienced relief of radiculopathy but still experienced occasional minimal / mild residual non - radicular discomfort , " fair " when the patient showed mild residual symptoms of radiculopathy with or without mild / moderated residual non - radicular discomfort , and " poor " when the patient continued to show significant radicular symptoms with or without non - radicular discomfort . we were analyzed to the degree of cervical cord decompression , disc space height loss , translation , cobb angle and vertebral body resection rate using paired t - test . a 52-year - old woman was referred to our hospital with a 6-month history of posterior neck pain , right shoulder pain and progressive myelopathy that both upper and lower extremity numbness ( right > left ) . on neurologic examination , she had grade 4-/5 weakness of her right elbow flexion and extension . ct and mri scans demonstrated bulging disc c4 - 5 , c5 - 6 with focal ossification posterior longitudinal ligament and showed cervical cord compression . the patient underwent spinal cord decompression via right - sided microsurgical anterior foraminotomy holes at c4 - 5 , c5 - 6 . follow up examination after 6 weeks , she showed no instability on a flexion - extension dynamic roentgenogram of the cervical spine ( fig . the average follow - up duration was 30.36 months ( ranged 9 - 57 months ) . the age of the patients ranged 29 - 75 years ( median 55 years ) . duration of the occurrence of the symptoms was four weeks to 10 years ( median 12 months ) . in addition to the symptoms and signs of myelopathy , 22 patients ( 100% ) had radicular symptoms and motor weakness , 18 patients ( 82% ) had a sensory deficit , four patients ( 18% ) had gait disturbance , and one patient ( 5% ) had a bladder symptom ( table 1 ) . single - level operations were performed on five patients , and two - level operations were done on 17 patients . the operative levels were c4 - 5 for 10 patients , c5 - 6 for 20 patients and c6 - 7 for nine patients . fourteen patients ( 64% ) showed excellent results ; six patients ( 27% ) , good results ; and one patient ( 4% ) , a fair result . however , one patient ( 5% ) had a poor result , and he was re - operated on due to post - operative instability ( table 2 ) . the mean pre - operative vas score was 7.5 ( 1.01 ) , the post - operative vas score was 1.8 ( 0.85 ) , and the vas score difference was 5.7 . the mean preoperative disc space height ( dsh ) was 6.36 ( 2 - 8.4 ) mm , and the post - operative dsh was 5.5 ( 2 - 8 ) mm . the mean pre - operative translation ( retrolisthesis ) was 0.13 ( 0 - 1.5 ) mm , and the post - operative translation was 0.49 ( 0 - 2.51 ) mm . the mean pre - operative cobb angle in the neutral position was 7.67 ( -6.8-19 ) , and the mean post - operative cobb angle was 4.21 ( -10.2-20 ) . the change in the cobb angle decreased by 3.46 and showed slight kyphosis ( p<0.166 ) . the average vertebral body resection rate was 11.47% ( 8.10 - 16.14% , p<0.001 ) ( table 3 ) . the mean preoperative ap diameter at the site of maximal com - pression improved from 5.430.93 mm preoperatively to 6.35 1.10 mm , postoperatively ( p<0.001 ) . and the mean preoperative transverse area at the site of maximal compression improved from 63.589.47 mm preoperatively to 73.3410.85 mm , postoperatively ( p<0.001 ) ( table 4 ) . no procedure - related complications occurred such as vertebral artery injury , horner 's syndrome , or hoarseness . fourteen patients ( 64% ) showed excellent results ; six patients ( 27% ) , good results ; and one patient ( 4% ) , a fair result . however , one patient ( 5% ) had a poor result , and he was re - operated on due to post - operative instability ( table 2 ) . the mean pre - operative vas score was 7.5 ( 1.01 ) , the post - operative vas score was 1.8 ( 0.85 ) , and the vas score difference was 5.7 . the mean preoperative disc space height ( dsh ) was 6.36 ( 2 - 8.4 ) mm , and the post - operative dsh was 5.5 ( 2 - 8 ) mm . the mean pre - operative translation ( retrolisthesis ) was 0.13 ( 0 - 1.5 ) mm , and the post - operative translation was 0.49 ( 0 - 2.51 ) mm . the mean pre - operative cobb angle in the neutral position was 7.67 ( -6.8-19 ) , and the mean post - operative cobb angle was 4.21 ( -10.2-20 ) . the change in the cobb angle decreased by 3.46 and showed slight kyphosis ( p<0.166 ) . the average vertebral body resection rate was 11.47% ( 8.10 - 16.14% , p<0.001 ) ( table 3 ) . the mean preoperative ap diameter at the site of maximal com - pression improved from 5.430.93 mm preoperatively to 6.35 1.10 mm , postoperatively ( p<0.001 ) . and the mean preoperative transverse area at the site of maximal compression improved from 63.589.47 mm preoperatively to 73.3410.85 mm , postoperatively ( p<0.001 ) ( table 4 ) . no procedure - related complications occurred such as vertebral artery injury , horner 's syndrome , or hoarseness . but some patients presented only radicular symptoms as current clinical manifestations . even in patients who had radicular symptoms , we frequently observed spinal cord compression in mri and ct scan . in the literature , seventy - five percent of patients with myelopathy deteriorate in a stepwise fashion , 20% deteriorate slowly and steadily , and 5% have a rapid onset of symptoms with a stable plateau of dysfunction5 ) . most patients require surgical decompression when their condition results in progressive neurological deficit and functional declination . traditionally , surgical decompression has been performed to arrest neurological deterioration and prevent further disability . however , recent studies have shown improved clinical outcomes . at present , surgical treatment of cervical myelopathy can be broadly divided into anterior and posterior approaches . the anterior approaches include anterior cervical discectomy and corpectomy , while the posterior approaches involve laminectomy with or without fusion , and laminoplasty12,13,17,21 ) . the choice between an anterior or posterior approach depends on the location , extent and type of the compressive pathology , the curvature of the spine , and the presence of instability . like this , various techniques can be used presently , but the main goal of surgical intervention is spinal cord decompression . optimal treatment of csm is still controversial , and fusion - related complications such as loss of the range of motion , non - union , instrumentation failure and graft extrusion , and the adjacent - segment syndrome were exist20,22 ) . in our study , although all patients are not enough to criteria of cervical spondylotic myelopathy , main pathologic finding is cervical cord compression with degenerative change such as stenosis , opll . through this study , we evaluate the effectiveness of eacf technique to csm patients . we propose eacf as an alternative minimally invasive non - fusion technique for cervical cord decompression . the ecaf technique is originally based on acmf ; actually it 's one of technical variation . most surgeons have accepted the acmf as a surgical procedure for cervical nerve root decompression . so , we suggest changing the name of this technical variation into extended technique in descriptive terminology . some authors reported anterolateral technique to decompress the cervical cord , but these procedures were targeted for vertebral body not to the foramen where nerve root trespassing2,3 ) . in the extended acf , decompression started form the exit of the intervertebral foramen and proceeded into more anterolateral portion of the spinal canal . with extended resection from the pathologic part of the foramen to the anterolateral portion of the cord , a funnel shaped widened area can be obtained . this space contained a decompressed nerve root and spinal cord without a large area of bony resection . so this terminology , extended anterior cervical foraminotomy technique , is more reasonable to describe this procedure for cord decompression . acmf was developed by jho et al.10 ) under the concept of " functional spine surgery , " which directly eliminates the compressive pathological factor while preserving the functional anatomic features . this minimally invasive surgical procedure is widely used in cervical radiculopathy and has shown favorable outcomes in many studies . this technique was also used in cervical myelopathy in 2002 and had good surgical results . in his study , 40 patients were treated with acmf . eighty percent of them showed good to excellent surgical results , with minimal morbidities , six weeks after their operation , and 90% of them showed the same results at the long - term follow - up . a total of 91% patients had an excellent or good outcome and no procedure related complication occurred . , we measured five radiologic outcomes . and except the vertebral body resection rate and degree of cervical cord decompression , radiologic outcome was similar to other studies that were acmf for radiculopathy patients11,14,15 ) . therefore , our study results support the extended acf for cervical cord decompression is safety and effectiveness . the main advantages of this procedure are the quick patient recovery without the need for bone fusion , and the preservation of the motion segments . in generally , the anterior cervical discectomy and fusion ( acdf ) procedure provides good access to the spinal canal , but not to the neural foramen . however , this procedure enables foraminal decompression through the same hole for patients with mixed myelopathy and radiculopathy symptoms . the overall incidence of complications is known to be 7%10 ) to 22%,6 ) with horner 's syndrome ( 5.3% ) , hoarseness ( 4.8 - 5.9% ) , va injury ( 1% ) , recurrence ( 1 - 17% ) and discitis ( 1% ) reported . in our study , some studies had postulated that uncovertebral joint resection with a degenerative disc leads to hypermobility , which results in a painful joint and post - operative instability . however , resection of the uncovertebral joint does not correctly describe anterior cervical foraminotomy . to maintain spinal stability and cervical spine motion , it was developed by placing the foraminotomy hole more laterally and making it progressively smaller . recently , kim14 ) reported the surgical long - term outcome of acmf for cervical degenerative disease . he reported that the disc space height decreased more when the acmf diameter was more than 4.7 mm , and that the ideal range of the acmf diameter may be from 2.6 mm to 4.7 mm . concerning the biomechanical effect , chen et al.4 ) studied the relative biomechanical contributions of various structures in seven human cadaver c4-t1 specimens . the various destructive steps , which included discectomy , unilateral uncinate process removal , bilateral uncinate process removal , and posterior longitudinal ligament transection , it was reported that the preservation of much of the anterior structures , including the uncinated process , achieved better cervical stability when cervical fusion was not performed . however , these biomechanical tests do not accurately represent the actual methods currently being used . unlike with the actual anterior cervical microforaminotomy , these results were obtained after removing the uncinate process or much of the surrounding structures . thus , these tests are inaccurate . also , unlike previous assumptions that the disc space height will decrease and the retrolisthesis and sagittal rotation will increase and add to the instability due to the degeneration of the uncovertebral joint long after the surgery , there are cases in which the uncovertebral joint is fused as the foraminotomy hole this is similar to the reported bone reformation , a long time after posterior decompression , as in lumbar laminectomy and also in cases when the uncovertebral joint is retained without being fused but instead , filled up11 ) . there are many studies on the instability that follows after acmf surgery in radiculopathy patients . for this reason , the vertebral body resection rate in our study would be different from previous studies . but , one patient developed instability after surgery despite of mean vertebral body resection rate was 9% . the patient was young and had a soft disc , flexible neck without spondylotic change and a unilateral approach was used to resect both lesions . conclusionally , the patient was not truly cervical spondylotic myelopathy and all these factors could be attributed to postoperative instability . eventually , a dynamic stenosis may occur after decompression only surgery for cervical spondylotic myelopathy patients . but , our study did not show any symptoms related to restenosis during the follow up period . concerning to this fact , our follow up periods seemed to be relatively short . in long - term follow up , some patients will develop restenosis and may need further surgery according to its pathological conditions . nevertheless , we emphasized to preservation of motion segment as a primary advantage . and this procedure will contribute to decrease the risk of developing adjacent segment disease as compared with fusion surgery . also , cervical spondylotic myelopathy is a degenerative disease that occurs mostly in older patients . therefore , a better surgical strategy is to preserve the motion unit rather than the bone fusion . first , it is a retrospective study with the subjects being mostly cervical cord decompression patients rather than symptomatic myelopathy only patients . as a result a decompression and fusion surgery has been considered as a standard surgical treatment for cervical cord compressing lesions . but , in our study , a decompression without fusion procedure was successfully performed using eacf technique . this procedure achieves adequate anterior decompression of the spinal cord with preservation of the functional motion , and avoids fusion - related complications . with our clinical and radiological results , authors conclude that eacf is a possible and applicable surgical option for the spondylotic patients who need cervical cord decompression . in selecting patients , the pre - operative clinical status should be carefully considered to achieve an excellent outcome . long - term follow - up and a further study in a larger series are needed .

objectiveat present , gold - standard technique of cervical cord decompression is surgical decompression and fusion . but , many complications related cervical fusion have been reported . we adopted an extended anterior cervical foraminotomy ( eacf ) technique to decompress the anterolateral portion of cervical cord and report clinical results and effectiveness of this procedure.methodsfifty-three patients were operated consecutively using eacf from 2008 to 2013 . all of them were operated by a single surgeon via the unilateral approach . twenty - two patients who exhibited radicular and/or myelopathic symptoms were enrolled in this study . all of them showed cervical cord compression in their preoperative magnetic resonance scan images.resultsin surgical outcomes , 14 patients ( 64% ) were classified as excellent and six ( 27% ) , as good . the mean difference of cervical cord anterior - posterior diameter after surgery was 0.92 mm ( p<0.01 ) and transverse area was 9.77 mm2 ( p<0.01 ) . the dynamic radiological study showed that the average post - operative translation ( retrolisthesis ) was 0.36 mm and the disc height loss at the operated level was 0.81 mm . the change in the cobb angle decreased to 3.46 , and showed slight kyphosis . the average vertebral body resection rate was 11.47% . no procedure - related complications occurred . only one patient who had two - level decompression needed anterior fusion at one level as a secondary surgery due to postoperative instability.conclusionscervical cord decompression was successfully performed using eacf technique . this procedure will be an alternative surgical option for treating cord compressing lesions . long - term follow - up and a further study in larger series will be needed .

INTRODUCTION MATERIALS AND METHODS Patients Operative procedure Radiological assessment Clinical outcome assessment Statistical analysis Illustrative cases RESULTS Clinical outcomes Radiologic outcomes Complications DISCUSSION CONCLUSION

spondylotic lesions that cause cervical cord compression require surgical decompression either through anterior cervical discectomy / corpectomy with fusion or laminectomy / laminoplasty when they result in progressive neurological deficits2,3,13,20 ) . these procedures have been regarded as a gold - standard technique for decompression and stabilization . however , fusion - related problems such as loss of the cervical range of motion , non - union , instrumentation failure , and graft extrusion are still the subject of debates7,20 ) . the anterior cervical microforaminotomy ( acmf ) technique involves not only the direct removal of the compressive abnormality but also the preservation of the motion segments without bone fusion or post - operative immobilization9,11,15 ) . but , it naturally advanced to spinal cord decompression with extended technique ( extended anterior cervical foraminotomy , eacf ) . here , twenty - two of the patients who showed cervical cord compression on their pre - operative magnetic resonance scan images with radicular and/or myelopathic symptoms were enrolled in this study . the preoperative instability on cervical dynamic x - ray and already pre - existing narrowing of cervical spinal canal ( that is , developmental stenosis ) excluded in this study . the detailed surgical technique of acmf for cord compression has been reported by jho8 ) . an anterior cervical discectomy retractor system is applied to expose the ipsilateral longus colli muscle rather than the midline anterior disc surface . the lateral portion of the longus colli muscle is excised to expose the medial parts of the transverse processes of the upper and lower vertebrae . once the medial portion of the transverse processes of the upper and lower vertebrae is identified , the ipsilateral uncovertebral joint between them can be seen ; however , in advanced spondylosis , anatomical landmarks of the uncovertebral joint and transverse processes can not be delineated . to prevent injury to the vertebral artery ( va ) , a thin layer of cortical bone is left attached to the ligamentous tissue covering the medial portion of this artery . sometimes the beginning of the contralateral nerve root is identified for adequate decompression of the spinal canal in the transverse axis . the spinal cord canal is enlarged in the longitudinal axis by removing the posterior portion of the vertebral bodies with kerrison rongeurs and a long - armed up - biting curette . all the patients took pre - operative plain radiography of cervical spine , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and dynamic plain radiography of cervical spine in six weeks after their operation . analyses of the pre- and post - operative dynamic plain radiographs were done to evaluate postoperative instability . we measured five radiological data for evaluate to presence of postoperative instability and effectiveness of cervical cord decompression : the disc space height ( dsh ) , sagittal plane displacement ( translation ) , cobb angle , vertebral body resection rate and degree of cervical cord decompression . the disc space height was measured from the mid - point of the upper vertebral body to the mid - point of the lower vertebral body on the sagittal plane . when all the intervening vertebral bodies were found anterior to this line , the alignment was defined as a lordotic curvature . the sagittal angulation , which is the angle between a line on the superior end plate of the vertebral body above and a line on the inferior border of the body below the levels operated 1 ) , was measured on the neutral lateral view at the operated level , using cobb 's method . the vertebral body resection rate was measured on pre- and post - cervical 3-dimensional ct scan . the scanning parameters were as follows : voltage of 120 kv , current 150 mas , field - of - view 147 mm and slice thickness of 3 mm ( fig . the degree of cervical cord decompression was measured antero - posterior diameter and transverse area of the spinal cord at the site of maximal compression . the post - operative pain improvement was measured using the visual analogue score ( vas ) . the clinical outcome was graded as " excellent " when the patient showed complete resolution of all symptoms , " good " when the patient experienced relief of radiculopathy but still experienced occasional minimal / mild residual non - radicular discomfort , " fair " when the patient showed mild residual symptoms of radiculopathy with or without mild / moderated residual non - radicular discomfort , and " poor " when the patient continued to show significant radicular symptoms with or without non - radicular discomfort . we were analyzed to the degree of cervical cord decompression , disc space height loss , translation , cobb angle and vertebral body resection rate using paired t - test . mri scans demonstrated bulging disc c4 - 5 , c5 - 6 with focal ossification posterior longitudinal ligament and showed cervical cord compression . the patient underwent spinal cord decompression via right - sided microsurgical anterior foraminotomy holes at c4 - 5 , c5 - 6 . twenty - two of the patients who showed cervical cord compression on their pre - operative magnetic resonance scan images with radicular and/or myelopathic symptoms were enrolled in this study . the preoperative instability on cervical dynamic x - ray and already pre - existing narrowing of cervical spinal canal ( that is , developmental stenosis ) excluded in this study . the detailed surgical technique of acmf for cord compression has been reported by jho8 ) . once the medial portion of the transverse processes of the upper and lower vertebrae is identified , the ipsilateral uncovertebral joint between them can be seen ; however , in advanced spondylosis , anatomical landmarks of the uncovertebral joint and transverse processes can not be delineated . to prevent injury to the vertebral artery ( va ) , a thin layer of cortical bone is left attached to the ligamentous tissue covering the medial portion of this artery . it is necessary to proceed cautiously with drilling at the base of the uncinate process because the nerve root lies just adjacent to it . using the holes of anterior foraminotomies , the spinal cord canal is enlarged in the longitudinal axis by removing the posterior portion of the vertebral bodies with kerrison rongeurs and a long - armed up - biting curette . all the patients took pre - operative plain radiography of cervical spine , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and dynamic plain radiography of cervical spine in six weeks after their operation . analyses of the pre- and post - operative dynamic plain radiographs were done to evaluate postoperative instability . we measured five radiological data for evaluate to presence of postoperative instability and effectiveness of cervical cord decompression : the disc space height ( dsh ) , sagittal plane displacement ( translation ) , cobb angle , vertebral body resection rate and degree of cervical cord decompression . the disc space height was measured from the mid - point of the upper vertebral body to the mid - point of the lower vertebral body on the sagittal plane . the sagittal angulation , which is the angle between a line on the superior end plate of the vertebral body above and a line on the inferior border of the body below the levels operated 1 ) , was measured on the neutral lateral view at the operated level , using cobb 's method . the vertebral body resection rate was measured on pre- and post - cervical 3-dimensional ct scan . the scanning parameters were as follows : voltage of 120 kv , current 150 mas , field - of - view 147 mm and slice thickness of 3 mm ( fig . the degree of cervical cord decompression was measured antero - posterior diameter and transverse area of the spinal cord at the site of maximal compression . the post - operative pain improvement was measured using the visual analogue score ( vas ) . the clinical outcome was graded as " excellent " when the patient showed complete resolution of all symptoms , " good " when the patient experienced relief of radiculopathy but still experienced occasional minimal / mild residual non - radicular discomfort , " fair " when the patient showed mild residual symptoms of radiculopathy with or without mild / moderated residual non - radicular discomfort , and " poor " when the patient continued to show significant radicular symptoms with or without non - radicular discomfort . we were analyzed to the degree of cervical cord decompression , disc space height loss , translation , cobb angle and vertebral body resection rate using paired t - test . ct and mri scans demonstrated bulging disc c4 - 5 , c5 - 6 with focal ossification posterior longitudinal ligament and showed cervical cord compression . the average follow - up duration was 30.36 months ( ranged 9 - 57 months ) . in addition to the symptoms and signs of myelopathy , 22 patients ( 100% ) had radicular symptoms and motor weakness , 18 patients ( 82% ) had a sensory deficit , four patients ( 18% ) had gait disturbance , and one patient ( 5% ) had a bladder symptom ( table 1 ) . single - level operations were performed on five patients , and two - level operations were done on 17 patients . fourteen patients ( 64% ) showed excellent results ; six patients ( 27% ) , good results ; and one patient ( 4% ) , a fair result . however , one patient ( 5% ) had a poor result , and he was re - operated on due to post - operative instability ( table 2 ) . the mean pre - operative vas score was 7.5 ( 1.01 ) , the post - operative vas score was 1.8 ( 0.85 ) , and the vas score difference was 5.7 . the mean preoperative disc space height ( dsh ) was 6.36 ( 2 - 8.4 ) mm , and the post - operative dsh was 5.5 ( 2 - 8 ) mm . the mean pre - operative translation ( retrolisthesis ) was 0.13 ( 0 - 1.5 ) mm , and the post - operative translation was 0.49 ( 0 - 2.51 ) mm . the mean pre - operative cobb angle in the neutral position was 7.67 ( -6.8-19 ) , and the mean post - operative cobb angle was 4.21 ( -10.2-20 ) . the change in the cobb angle decreased by 3.46 and showed slight kyphosis ( p<0.166 ) . the average vertebral body resection rate was 11.47% ( 8.10 - 16.14% , p<0.001 ) ( table 3 ) . the mean preoperative ap diameter at the site of maximal com - pression improved from 5.430.93 mm preoperatively to 6.35 1.10 mm , postoperatively ( p<0.001 ) . and the mean preoperative transverse area at the site of maximal compression improved from 63.589.47 mm preoperatively to 73.3410.85 mm , postoperatively ( p<0.001 ) ( table 4 ) . no procedure - related complications occurred such as vertebral artery injury , horner 's syndrome , or hoarseness . fourteen patients ( 64% ) showed excellent results ; six patients ( 27% ) , good results ; and one patient ( 4% ) , a fair result . however , one patient ( 5% ) had a poor result , and he was re - operated on due to post - operative instability ( table 2 ) . the mean pre - operative vas score was 7.5 ( 1.01 ) , the post - operative vas score was 1.8 ( 0.85 ) , and the vas score difference was 5.7 . the mean preoperative disc space height ( dsh ) was 6.36 ( 2 - 8.4 ) mm , and the post - operative dsh was 5.5 ( 2 - 8 ) mm . the mean pre - operative translation ( retrolisthesis ) was 0.13 ( 0 - 1.5 ) mm , and the post - operative translation was 0.49 ( 0 - 2.51 ) mm . the mean pre - operative cobb angle in the neutral position was 7.67 ( -6.8-19 ) , and the mean post - operative cobb angle was 4.21 ( -10.2-20 ) . the change in the cobb angle decreased by 3.46 and showed slight kyphosis ( p<0.166 ) . the average vertebral body resection rate was 11.47% ( 8.10 - 16.14% , p<0.001 ) ( table 3 ) . the mean preoperative ap diameter at the site of maximal com - pression improved from 5.430.93 mm preoperatively to 6.35 1.10 mm , postoperatively ( p<0.001 ) . and the mean preoperative transverse area at the site of maximal compression improved from 63.589.47 mm preoperatively to 73.3410.85 mm , postoperatively ( p<0.001 ) ( table 4 ) . no procedure - related complications occurred such as vertebral artery injury , horner 's syndrome , or hoarseness . even in patients who had radicular symptoms , we frequently observed spinal cord compression in mri and ct scan . in the literature , seventy - five percent of patients with myelopathy deteriorate in a stepwise fashion , 20% deteriorate slowly and steadily , and 5% have a rapid onset of symptoms with a stable plateau of dysfunction5 ) . most patients require surgical decompression when their condition results in progressive neurological deficit and functional declination . at present , surgical treatment of cervical myelopathy can be broadly divided into anterior and posterior approaches . the anterior approaches include anterior cervical discectomy and corpectomy , while the posterior approaches involve laminectomy with or without fusion , and laminoplasty12,13,17,21 ) . the choice between an anterior or posterior approach depends on the location , extent and type of the compressive pathology , the curvature of the spine , and the presence of instability . optimal treatment of csm is still controversial , and fusion - related complications such as loss of the range of motion , non - union , instrumentation failure and graft extrusion , and the adjacent - segment syndrome were exist20,22 ) . in our study , although all patients are not enough to criteria of cervical spondylotic myelopathy , main pathologic finding is cervical cord compression with degenerative change such as stenosis , opll . through this study , we evaluate the effectiveness of eacf technique to csm patients . we propose eacf as an alternative minimally invasive non - fusion technique for cervical cord decompression . most surgeons have accepted the acmf as a surgical procedure for cervical nerve root decompression . some authors reported anterolateral technique to decompress the cervical cord , but these procedures were targeted for vertebral body not to the foramen where nerve root trespassing2,3 ) . in the extended acf , decompression started form the exit of the intervertebral foramen and proceeded into more anterolateral portion of the spinal canal . with extended resection from the pathologic part of the foramen to the anterolateral portion of the cord , a funnel shaped widened area can be obtained . so this terminology , extended anterior cervical foraminotomy technique , is more reasonable to describe this procedure for cord decompression . in his study , 40 patients were treated with acmf . eighty percent of them showed good to excellent surgical results , with minimal morbidities , six weeks after their operation , and 90% of them showed the same results at the long - term follow - up . a total of 91% patients had an excellent or good outcome and no procedure related complication occurred . and except the vertebral body resection rate and degree of cervical cord decompression , radiologic outcome was similar to other studies that were acmf for radiculopathy patients11,14,15 ) . therefore , our study results support the extended acf for cervical cord decompression is safety and effectiveness . the main advantages of this procedure are the quick patient recovery without the need for bone fusion , and the preservation of the motion segments . in generally , the anterior cervical discectomy and fusion ( acdf ) procedure provides good access to the spinal canal , but not to the neural foramen . the overall incidence of complications is known to be 7%10 ) to 22%,6 ) with horner 's syndrome ( 5.3% ) , hoarseness ( 4.8 - 5.9% ) , va injury ( 1% ) , recurrence ( 1 - 17% ) and discitis ( 1% ) reported . in our study , some studies had postulated that uncovertebral joint resection with a degenerative disc leads to hypermobility , which results in a painful joint and post - operative instability . however , resection of the uncovertebral joint does not correctly describe anterior cervical foraminotomy . recently , kim14 ) reported the surgical long - term outcome of acmf for cervical degenerative disease . he reported that the disc space height decreased more when the acmf diameter was more than 4.7 mm , and that the ideal range of the acmf diameter may be from 2.6 mm to 4.7 mm . the various destructive steps , which included discectomy , unilateral uncinate process removal , bilateral uncinate process removal , and posterior longitudinal ligament transection , it was reported that the preservation of much of the anterior structures , including the uncinated process , achieved better cervical stability when cervical fusion was not performed . also , unlike previous assumptions that the disc space height will decrease and the retrolisthesis and sagittal rotation will increase and add to the instability due to the degeneration of the uncovertebral joint long after the surgery , there are cases in which the uncovertebral joint is fused as the foraminotomy hole this is similar to the reported bone reformation , a long time after posterior decompression , as in lumbar laminectomy and also in cases when the uncovertebral joint is retained without being fused but instead , filled up11 ) . for this reason , the vertebral body resection rate in our study would be different from previous studies . but , one patient developed instability after surgery despite of mean vertebral body resection rate was 9% . the patient was young and had a soft disc , flexible neck without spondylotic change and a unilateral approach was used to resect both lesions . conclusionally , the patient was not truly cervical spondylotic myelopathy and all these factors could be attributed to postoperative instability . in long - term follow up , some patients will develop restenosis and may need further surgery according to its pathological conditions . nevertheless , we emphasized to preservation of motion segment as a primary advantage . and this procedure will contribute to decrease the risk of developing adjacent segment disease as compared with fusion surgery . first , it is a retrospective study with the subjects being mostly cervical cord decompression patients rather than symptomatic myelopathy only patients . as a result a decompression and fusion surgery has been considered as a standard surgical treatment for cervical cord compressing lesions . but , in our study , a decompression without fusion procedure was successfully performed using eacf technique . this procedure achieves adequate anterior decompression of the spinal cord with preservation of the functional motion , and avoids fusion - related complications . with our clinical and radiological results , authors conclude that eacf is a possible and applicable surgical option for the spondylotic patients who need cervical cord decompression . long - term follow - up and a further study in a larger series are needed .

metabolic syndrome is a cluster of conditions abdominal obesity , dyslipidemia , elevated fasting glucose , and hypertension with serious consequences for physical health , such as increased risk of atherosclerotic cardiovascular disease , type 2 diabetes , and allcause mortality.1 , 2 , 3 , 4 metabolic syndrome and its components are also associated with a range of psychological characteristics , including depression , anxiety , anger , hostility , and impaired cognitive functioning.5 , 6 , 7 , 8 these associations may in part explain why people with psychopathology are at high risk of developing a range of chronic illnesses.9 metabolic syndrome has been defined as a discrete , binary entity ; however , previous research indicates that some components are more strongly associated with psychopathology than others , particularly obesity and dyslipdemia.10 , 11 , 12 given these discrepancies between components in the context of psychiatric illness , using a dichotomous definition of metabolic syndrome may not be the strongest way of identifying the nature of associations between psychopathology and metabolic dysregulation . rather , describing components on their own is warranted to investigate uniformities across the components . although there is much evidence for crosssectional associations between metabolic syndrome dysregulations and psychopathology , few longitudinal studies assess temporal relationships , with even fewer examining both directions of the relationship simultaneously.6 furthermore , most of the current evidence involves depressive symptoms , whereas associations for anxiety symptoms or diagnosis of depression and anxiety have been infrequently reported , and results have been inconsistent.13 , 14 , 15 consequently , it remains unclear as to whether , over time , psychopathology impairs components of the metabolic syndrome , or vice versa . each direction is biologically plausible , since the development and progression of both psychopathology and metabolic dysregulation are associated with a range of detrimental biological processes , including altered autonomic and neuroendocrine stress functioning , low grade inflammation , cellular aging , and oxidative and nitrosative damage.16 , 17 , 18 thus , the pathophysiology of psychopathology may increase the risk of metabolic dysregulation , and vice versa . the unhealthy lifestyle of people with psychopathology and metabolic dysregulation may also contribute , since smoking , physical inactivity and alcohol use are frequently observed in people with psychopathology19 , 20 and may also drive impairments in metabolic indicators , particularly obesity.21 , 22 independent of psychiatric disorder , antidepressants may also directly influence metabolic syndrome components . the use of tricyclic antidepressants ( tca ) has been associated with metabolic dysregulation , particularly abdominal obesity.10 , 23 however , the effects of selective serotonin reuptake inhibitors ( ssris ) on metabolic syndrome components are less clear.24 , 25 , 26 for instance , ssri use has been associated with weight gain , loss and no change , as well as both impaired and improved glucose and lipid profiles.23 , 26 ssri use is also associated with positive impacts on biological correlates of metabolic dysregulation , such as inflammation,27 , 28 which may in part explain why their metabolic effects are diverse . thus it is uncertain , particularly in the longitudinal context , the effect of antidepressants on metabolic syndrome components independent of psychopathology . the current study contributes to the understanding of three issues that remain largely unclear regarding associations between psychopathology and metabolic dysregulation : ( 1 ) is psychopathology associated across all the metabolic syndrome components ? ( 2 ) how are psychopathology indicators and metabolic syndrome components associated longitudinally ? ( 3 ) are antidepressants independently associated with metabolic dysregulation over time ? we addressed these issues using baseline , 2 and 6year data from a largescale , longitudinal cohort study of depression and anxiety . first , we examined the consistency of associations of five metabolic syndrome components with depression , anxiety and antidepressant use across the three data waves . second , to investigate prospective associations we examined whether psychopathology at one assessment predicted metabolic dysregulation at the next , and vice versa . participants were drawn from the netherlands study of depression and anxiety ( nesda ) , an ongoing prospective cohort study of 2,981 adults with and without depression and anxiety ( 1865 years ) . detailed study rationale , design , and methods have been published elsewhere.29 briefly , between september 2004 and february 2007 , participants were recruited from the community , primary care , and specialized mental health care . exclusion criteria were a primary diagnosis of psychosis , obsessive compulsive disorder , bipolar disorder or severe addiction disorder or a lack of fluency in dutch . informed consent was obtained after the nature of the procedures was explained and all procedures were approved by institutional ethical review boards . participants completed a detailed interview , selfreport questionnaires , and a medical examination involving physical assessments and fasting blood draw . metabolic syndrome components and psychopathology were assessed at baseline , 2 and 6year followup assessments . retention of participants across followup assessments was high ( 87% of baseline sample at 2year followup and 76% of baseline sample at 6year followup ) . participants who did not contribute to the 2 or 6year followup were relatively similar to participants in demographic characteristics and antidepressant use , although they had higher depression and anxiety symptoms at baseline as well as poorer metabolic syndrome components ( including lower hdlc and greater waist circumference , glucose , and triglycerides and number of abnormalities ) , compared to participants . participants were included in analyses when they had data for at least one metabolic syndrome component , psychopathology , and covariates for at least one assessment ( baseline assessment : n = 2,776 , 93% of available sample ; 2year assessment : n = 2,203 , 85% of available sample ; 6year assessment : n = 1,899 , 84% of available sample ) . psychopathology indicators , metabolic syndrome components , and covariates were assessed at baseline , 2 and 6year assessment waves . participants completed the world health organization ( who ) composite international diagnostic interview ( cidi , version 2.1 ) to derive lifetime and 6month dsmiv diagnoses of major depressive disorder , dysthymia , social phobia , panic disorder , agoraphobia , and generalized anxiety disorder . from these diagnoses , participants were classified at each wave as having : ( 1 ) any current depressive or anxiety disorder in the previous 6 months ; ( 2 ) remitted depression and/or anxiety disorder ( disorder > 6 months prior ) ; or ( 3 ) no lifetime history of depression or anxiety . participants also completed validated selfreport measures for the severity of psychopathology symptoms : the 30item inventory of depressive symptomatology ( ids)30 , 31 for depressive symptoms , the 21item beck anxiety inventory ( bai)32 for measuring the psychological and arousal components of anxiety , and the 24item fear questionnaire ( fq)33 for measuring fear avoidance symptoms . medication use was derived from medication container inspection and interview , and coded with the who anatomical therapeutic chemical ( atc ) classification system . antidepressant use was defined as frequent use ( 50% of the time ) of a tca ( n06aa ) , ssri ( n06ab ) , or other antidepressant ( remaining na06 ) in the previous month . waist circumference was measured with a measuring tape at the central point between the lowest front rib and highest point of the pelvis over light clothing . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . supine resting systolic blood pressure ( sbp ) was taken using omron m4 intellisense digital blood pressure monitor , as an average of two rightarm measurements . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.03 mmol / l in men , < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . for analyses using continuous values of metabolic syndrome components , raw values were adjusted for use of medication as previously described.14 , 18 , 35 specifically , for hdlc 0.10 mmol / l was subtracted for use of fibrates ( c10ab ) ; for triglycerides 0.67 mmol / l was added for use of fibrates ; for sbp 10 mmhg was added for use of antihypertensive medication ( c02 , c03 , c0709 ) ; and for glucose a value of 7 mmol / l was given for those on antidiabetic medication ( a10 ) with glucose level below 7 mmol / l . covariates were selfreported age , sex , years of education , smoking status ( current , not current ) , alcohol use , and physical activity . categories of alcohol use were derived from responses on the audit36 by calculating the average number of drinks per week : no use ( 0 ) , moderate use ( 121 for males , 114 for females ) , and heavy use ( > 21 for males , > 14 for females ) . physical activity was calculated from response to the international physical activity questionnaire ( ipaq ) , which uses the weekly duration of selfreported vigorous , moderate , walking , and sitting activity to calculate metabolic equivalent total ( met ) minutes of activity per week ( met level minutes of activity events per week).37 , 38 analyses were conducted using ibm spss statistics version 20.0 ( ibm corp . descriptive statistics were calculated at baseline , 2 and 6year assessments . to examine the consistency of the associations between psychopathology or antidepressant use and metabolic dysregulation across the baseline , 2 and 6year followup assessments , generalized estimating equations ( gee ) time ( 0 , 2 , 6 ) and psychopathology ( diagnosis or symptom severity ) or antidepressant use ( none , tcas , ssris , other antidepressants ) were entered as main effects , with metabolic syndrome components as the outcome ( normal distribution model for continuous outcomes and poisson distribution for count outcomes ) . time was entered as a continuous variable for ease of reporting , since results were identical when time was treated categorically . covariates of age and sex were held at baseline values , whereas years of education , smoking status , alcohol use , and physical activity could vary over time . whether the effects of psychopathology on metabolic syndrome components were the same across the three waves , analyses were repeated including an interaction term between psychopathology indicators and time . to further examine longitudinal relationships , autoregression models were tested , which essentially remove the crosssectional components of the relationship . autoregression gee models were applied to examine whether levels of psychopathology symptom severity or antidepressant use at one time point ( t ) predicted levels of metabolic syndrome components at the next time point ( t + 1 ) , controlling for covariates and metabolic syndrome components at t. thus , there were two comparisons made : between baseline and 2year followup assessment , and between 2 and 6year followup assessment . given the differential time gap between the two assessments ( 2 vs. 4 years ) , a binary variable coding the assessment comparison group was tested as an independent variable and also in interaction with psychopathology . most of these interactions were significant , indicating that the effect of psychopathology on subsequent metabolic syndrome components differed in the two assessment comparisons . analyses were repeated with metabolic syndrome components as predictor and psychopathology as outcome . in these autoregression analyses , since the pattern across antidepressant classes were relatively consistent and to retain sufficient group size , antidepressant use was coded as a binary variable ( use vs. no use ) . participants were drawn from the netherlands study of depression and anxiety ( nesda ) , an ongoing prospective cohort study of 2,981 adults with and without depression and anxiety ( 1865 years ) . detailed study rationale , design , and methods have been published elsewhere.29 briefly , between september 2004 and february 2007 , participants were recruited from the community , primary care , and specialized mental health care . exclusion criteria were a primary diagnosis of psychosis , obsessive compulsive disorder , bipolar disorder or severe addiction disorder or a lack of fluency in dutch . informed consent was obtained after the nature of the procedures was explained and all procedures were approved by institutional ethical review boards . participants completed a detailed interview , selfreport questionnaires , and a medical examination involving physical assessments and fasting blood draw . metabolic syndrome components and psychopathology were assessed at baseline , 2 and 6year followup assessments . retention of participants across followup assessments was high ( 87% of baseline sample at 2year followup and 76% of baseline sample at 6year followup ) . participants who did not contribute to the 2 or 6year followup were relatively similar to participants in demographic characteristics and antidepressant use , although they had higher depression and anxiety symptoms at baseline as well as poorer metabolic syndrome components ( including lower hdlc and greater waist circumference , glucose , and triglycerides and number of abnormalities ) , compared to participants . participants were included in analyses when they had data for at least one metabolic syndrome component , psychopathology , and covariates for at least one assessment ( baseline assessment : n = 2,776 , 93% of available sample ; 2year assessment : n = 2,203 , 85% of available sample ; 6year assessment : n = 1,899 , 84% of available sample ) . psychopathology indicators , metabolic syndrome components , and covariates were assessed at baseline , 2 and 6year assessment waves . participants completed the world health organization ( who ) composite international diagnostic interview ( cidi , version 2.1 ) to derive lifetime and 6month dsmiv diagnoses of major depressive disorder , dysthymia , social phobia , panic disorder , agoraphobia , and generalized anxiety disorder . from these diagnoses , participants were classified at each wave as having : ( 1 ) any current depressive or anxiety disorder in the previous 6 months ; ( 2 ) remitted depression and/or anxiety disorder ( disorder > 6 months prior ) ; or ( 3 ) no lifetime history of depression or anxiety . participants also completed validated selfreport measures for the severity of psychopathology symptoms : the 30item inventory of depressive symptomatology ( ids)30 , 31 for depressive symptoms , the 21item beck anxiety inventory ( bai)32 for measuring the psychological and arousal components of anxiety , and the 24item fear questionnaire ( fq)33 for measuring fear avoidance symptoms . medication use was derived from medication container inspection and interview , and coded with the who anatomical therapeutic chemical ( atc ) classification system . antidepressant use was defined as frequent use ( 50% of the time ) of a tca ( n06aa ) , ssri ( n06ab ) , or other antidepressant ( remaining na06 ) in the previous month . waist circumference was measured with a measuring tape at the central point between the lowest front rib and highest point of the pelvis over light clothing . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . supine resting systolic blood pressure ( sbp ) was taken using omron m4 intellisense digital blood pressure monitor , as an average of two rightarm measurements . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.03 mmol / l in men , < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . for analyses using continuous values of metabolic syndrome components , raw values were adjusted for use of medication as previously described.14 , 18 , 35 specifically , for hdlc 0.10 mmol / l was subtracted for use of fibrates ( c10ab ) ; for triglycerides 0.67 mmol / l was added for use of fibrates ; for sbp 10 mmhg was added for use of antihypertensive medication ( c02 , c03 , c0709 ) ; and for glucose a value of 7 mmol / l was given for those on antidiabetic medication ( a10 ) with glucose level below 7 mmol / l . covariates were selfreported age , sex , years of education , smoking status ( current , not current ) , alcohol use , and physical activity . categories of alcohol use were derived from responses on the audit36 by calculating the average number of drinks per week : no use ( 0 ) , moderate use ( 121 for males , 114 for females ) , and heavy use ( > 21 for males , > 14 for females ) . physical activity was calculated from response to the international physical activity questionnaire ( ipaq ) , which uses the weekly duration of selfreported vigorous , moderate , walking , and sitting activity to calculate metabolic equivalent total ( met ) minutes of activity per week ( met level minutes of activity events per week).37 , 38 participants completed the world health organization ( who ) composite international diagnostic interview ( cidi , version 2.1 ) to derive lifetime and 6month dsmiv diagnoses of major depressive disorder , dysthymia , social phobia , panic disorder , agoraphobia , and generalized anxiety disorder . from these diagnoses , participants were classified at each wave as having : ( 1 ) any current depressive or anxiety disorder in the previous 6 months ; ( 2 ) remitted depression and/or anxiety disorder ( disorder > 6 months prior ) ; or ( 3 ) no lifetime history of depression or anxiety . participants also completed validated selfreport measures for the severity of psychopathology symptoms : the 30item inventory of depressive symptomatology ( ids)30 , 31 for depressive symptoms , the 21item beck anxiety inventory ( bai)32 for measuring the psychological and arousal components of anxiety , and the 24item fear questionnaire ( fq)33 for measuring fear avoidance symptoms . medication use was derived from medication container inspection and interview , and coded with the who anatomical therapeutic chemical ( atc ) classification system . antidepressant use was defined as frequent use ( 50% of the time ) of a tca ( n06aa ) , ssri ( n06ab ) , or other antidepressant ( remaining na06 ) in the previous month . waist circumference was measured with a measuring tape at the central point between the lowest front rib and highest point of the pelvis over light clothing . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . supine resting systolic blood pressure ( sbp ) was taken using omron m4 intellisense digital blood pressure monitor , as an average of two rightarm measurements . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . for analyses using continuous values of metabolic syndrome components , raw values were adjusted for use of medication as previously described.14 , 18 , 35 specifically , for hdlc 0.10 mmol / l was subtracted for use of fibrates ( c10ab ) ; for triglycerides 0.67 mmol / l was added for use of fibrates ; for sbp 10 mmhg was added for use of antihypertensive medication ( c02 , c03 , c0709 ) ; and for glucose a value of 7 mmol / l was given for those on antidiabetic medication ( a10 ) with glucose level below 7 mmol / l . covariates were selfreported age , sex , years of education , smoking status ( current , not current ) , alcohol use , and physical activity . categories of alcohol use were derived from responses on the audit36 by calculating the average number of drinks per week : no use ( 0 ) , moderate use ( 121 for males , 114 for females ) , and heavy use ( > 21 for males , > 14 for females ) . physical activity was calculated from response to the international physical activity questionnaire ( ipaq ) , which uses the weekly duration of selfreported vigorous , moderate , walking , and sitting activity to calculate metabolic equivalent total ( met ) minutes of activity per week ( met level minutes of activity events per week).37 , 38 descriptive statistics were calculated at baseline , 2 and 6year assessments . to examine the consistency of the associations between psychopathology or antidepressant use and metabolic dysregulation across the baseline , 2 and 6year followup assessments , generalized estimating equations ( gee ) time ( 0 , 2 , 6 ) and psychopathology ( diagnosis or symptom severity ) or antidepressant use ( none , tcas , ssris , other antidepressants ) were entered as main effects , with metabolic syndrome components as the outcome ( normal distribution model for continuous outcomes and poisson distribution for count outcomes ) . time was entered as a continuous variable for ease of reporting , since results were identical when time was treated categorically . covariates of age and sex were held at baseline values , whereas years of education , smoking status , alcohol use , and physical activity could vary over time . missing values were considered missing completely at random . to examine whether the effects of psychopathology on metabolic syndrome components were the same across the three waves , analyses were repeated including an interaction term between psychopathology indicators and time . to further examine longitudinal relationships , autoregression models were tested , which essentially remove the crosssectional components of the relationship . autoregression gee models were applied to examine whether levels of psychopathology symptom severity or antidepressant use at one time point ( t ) predicted levels of metabolic syndrome components at the next time point ( t + 1 ) , controlling for covariates and metabolic syndrome components at t. thus , there were two comparisons made : between baseline and 2year followup assessment , and between 2 and 6year followup assessment . given the differential time gap between the two assessments ( 2 vs. 4 years ) , a binary variable coding the assessment comparison group was tested as an independent variable and also in interaction with psychopathology . most of these interactions were significant , indicating that the effect of psychopathology on subsequent metabolic syndrome components differed in the two assessment comparisons . analyses were repeated with metabolic syndrome components as predictor and psychopathology as outcome . in these autoregression analyses , since the pattern across antidepressant classes were relatively consistent and to retain sufficient group size , antidepressant use was coded as a binary variable ( use vs. no use ) . 66% of the participants were female and were on average 42 years old with 12 years of education . physical activity was relatively stable across the three assessment waves , whereas smoking rates and the proportion of heavy drinkers and nondrinkers declined . depression and anxiety symptoms and the frequency of current diagnoses significantly decreased over time , whereas components of metabolic syndrome generally worsened over time . baseline , 2 and 6year characteristics of the sample adjusted for use of medications with metabolic effects . table 2 shows the main effects of time and psychopathology indicators on metabolic syndrome components . there were significant main effects of time on metabolic syndrome components , indicating that levels of hdlc decreased across assessment waves , whereas number of metabolic syndrome abnormalities , waist circumference , triglycerides , sbp , and fasting glucose increased ( table 2 ) . across the three assessments , current and remitted anxiety or depressive disorders were associated with lower sbp , although associations with other components were not significant . in contrast , the results for continuous symptom severity measures were much stronger . across all assessment waves , higher ids was associated with higher number of metabolic syndrome abnormalities , waist circumference , hdlc and triglycerides , and lower sbp . bai scores were significantly and strongly associated with higher values for number of metabolic syndrome components , waist circumference , triglycerides , and glucose , whereas fq scores were only weakly associated with higher hdlc . associations of psychopathology diagnosis , symptoms , and antidepressant use with metabolic syndrome components across baseline , 2 and 6 year assessments estimates were obtained from generalized estimating equations . analyses were adjusted for age , sex , education , smoking status , alcohol use , and physical activity . n ranges of cases : number of components : 6,6116,633 ; waist : 6,8246,847 ; hdl : 6,6116,633 ; triglycerides : 6,6146,636 ; sbp : 6,8496,872 ; glucose : 6,6076,629 . antidepressant use was also associated with metabolic syndrome components ( table 2 ) . compared with antidepressant nonusers , tca use was associated with lower hdlc and the use any type of antidepressant ( tca , ssri , or others ) was associated with higher waist circumference , triglycerides , and number of metabolic syndrome abnormalities . the effect sizes observed were somewhat stronger for tca use compared with use of ssris and other antidepressants , particularly for waist circumference . to see whether the effects of antidepressant use were independent of symptom severity , both the effects were largely the same magnitude as those reported in table 2 , indicating independent effects ( detailed in supporting informationtable s1 ) . we did not observe consistent interactions between psychopathology indicators and time , indicating that the relationship between psychopathology indicators and metabolic syndrome components did not differ over time ( data not shown ) . in the prospective analyses , several psychopathology indicators at baseline significantly predicted levels of metabolic syndrome components at 2year assessment , taking into account covariates and baseline metabolic syndrome component values ( table 3 ) . in contrast , there was little evidence that psychopathology indicators at the 2year assessment predicted levels of metabolic dysregulation at 6year assessment . higher ids or bai scores and antidepressant use at baseline were all associated with an increasing or a higher number of metabolic syndrome abnormalities at 2year followup . higher ids and bai scores at baseline were associated with increasing waist circumference by 2year followup . a contrasting pattern emerged for antidepressant use . antidepressant use at baseline was associated with increasing waist circumference by 2year followup , whereas antidepressant use at 2year followup was associated with decreasing waist circumference by 6year followup . to explore this effect , an additional withinsubject ancova was conducted on waist circumference change between 2 and 6years , comparing groups according to antidepressant use pattern , adjusting for baseline waist circumference and covariates . participants using antidepressants at both 2 and 6year assessments had similar waist gain ( marginal mean [ m ] = 2.2 cm , se = 0.4 ) to those who had no use ( m = 3.0 cm , se = 0.2 , pairwise contrast p = .108 ) or commenced use at the 6year assessment ( m = 2.6 cm , se = 0.8 , pairwise contrast p = .622 ) , whereas those who stopped using antidepressants at 6year assessment had , on average , a slight waist reduction ( m = 0.1 cm , se = 0.7 , pairwise contrast p = .006 ) . these differences were less pronounced between baseline and 2 years , rendering a discrepant average effect . these effects indicate that current antidepressant use has strongest influence on waist , which aligns with findings of table 2 . prospective association of baseline psychopathology ( t ) with metabolic syndrome components at the next assessment ( t + 1 ) estimates were obtained from generalized estimating equations . analyses were adjusted for baseline values of the outcome , age , sex , education , smoking , alcohol use , and physical activity . fewer consistent patterns across symptom severity and antidepressant use were observed for the other components . antidepressant use , but not severity measures , at baseline was also associated with higher fasting triglycerides and glucose by 2year followup . psychopathology indicators did not significantly predict subsequent levels of sbp . finally , we examined the opposite predictive relationship to see whether metabolic syndrome component values at t were associated with psychopathology scores at t + 1 , controlling for psychopathology at t and covariates ( table 4 ) . there was only one weak effect ( at p = .025 ) , suggesting that metabolic syndrome components do not consistently predict subsequent psychopathology in this sample . prospective association of baseline metabolic syndrome components ( t ) with psychopathology at the next assessment ( t + 1 ) estimates were obtained from generalized estimating equations . analyses were adjusted for baseline values of the outcome , age , sex , education , smoking , alcohol use , and physical activity . 66% of the participants were female and were on average 42 years old with 12 years of education . physical activity was relatively stable across the three assessment waves , whereas smoking rates and the proportion of heavy drinkers and nondrinkers declined . depression and anxiety symptoms and the frequency of current diagnoses significantly decreased over time , whereas components of metabolic syndrome generally worsened over time . baseline , 2 and 6year characteristics of the sample adjusted for use of medications with metabolic effects . table 2 shows the main effects of time and psychopathology indicators on metabolic syndrome components . there were significant main effects of time on metabolic syndrome components , indicating that levels of hdlc decreased across assessment waves , whereas number of metabolic syndrome abnormalities , waist circumference , triglycerides , sbp , and fasting glucose increased ( table 2 ) . across the three assessments , current and remitted anxiety or depressive disorders were associated with lower sbp , although associations with other components were not significant . in contrast , the results for continuous symptom severity measures were much stronger . across all assessment waves , higher ids was associated with higher number of metabolic syndrome abnormalities , waist circumference , hdlc and triglycerides , and lower sbp . bai scores were significantly and strongly associated with higher values for number of metabolic syndrome components , waist circumference , triglycerides , and glucose , whereas fq scores were only weakly associated with higher hdlc . associations of psychopathology diagnosis , symptoms , and antidepressant use with metabolic syndrome components across baseline , 2 and 6 year assessments estimates were obtained from generalized estimating equations . analyses were adjusted for age , sex , education , smoking status , alcohol use , and physical activity . n ranges of cases : number of components : 6,6116,633 ; waist : 6,8246,847 ; hdl : 6,6116,633 ; triglycerides : 6,6146,636 ; sbp : 6,8496,872 ; glucose : 6,6076,629 . antidepressant use was also associated with metabolic syndrome components ( table 2 ) . compared with antidepressant nonusers , tca use was associated with lower hdlc and the use any type of antidepressant ( tca , ssri , or others ) was associated with higher waist circumference , triglycerides , and number of metabolic syndrome abnormalities . the effect sizes observed were somewhat stronger for tca use compared with use of ssris and other antidepressants , particularly for waist circumference . to see whether the effects of antidepressant use were independent of symptom severity , both the effects were largely the same magnitude as those reported in table 2 , indicating independent effects ( detailed in supporting informationtable s1 ) . we did not observe consistent interactions between psychopathology indicators and time , indicating that the relationship between psychopathology indicators and metabolic syndrome components did not differ over time ( data not shown ) . in the prospective analyses , several psychopathology indicators at baseline significantly predicted levels of metabolic syndrome components at 2year assessment , taking into account covariates and baseline metabolic syndrome component values ( table 3 ) . in contrast , there was little evidence that psychopathology indicators at the 2year assessment predicted levels of metabolic dysregulation at 6year assessment . higher ids or bai scores and antidepressant use at baseline were all associated with an increasing or a higher number of metabolic syndrome abnormalities at 2year followup . higher ids and bai scores at baseline were associated with increasing waist circumference by 2year followup . a contrasting pattern emerged for antidepressant use . antidepressant use at baseline was associated with increasing waist circumference by 2year followup , whereas antidepressant use at 2year followup was associated with decreasing waist circumference by 6year followup . to explore this effect , an additional withinsubject ancova was conducted on waist circumference change between 2 and 6years , comparing groups according to antidepressant use pattern , adjusting for baseline waist circumference and covariates . participants using antidepressants at both 2 and 6year assessments had similar waist gain ( marginal mean [ m ] = 2.2 cm , se = 0.4 ) to those who had no use ( m = 3.0 cm , se = 0.2 , pairwise contrast p = .108 ) or commenced use at the 6year assessment ( m = 2.6 cm , se = 0.8 , pairwise contrast p = .622 ) , whereas those who stopped using antidepressants at 6year assessment had , on average , a slight waist reduction ( m = 0.1 cm , se = 0.7 , pairwise contrast p = .006 ) . these differences were less pronounced between baseline and 2 years , rendering a discrepant average effect . these effects indicate that current antidepressant use has strongest influence on waist , which aligns with findings of table 2 . prospective association of baseline psychopathology ( t ) with metabolic syndrome components at the next assessment ( t + 1 ) estimates were obtained from generalized estimating equations . analyses were adjusted for baseline values of the outcome , age , sex , education , smoking , alcohol use , and physical activity . fewer consistent patterns across symptom severity and antidepressant use were observed for the other components . antidepressant use , but not severity measures , at baseline was also associated with higher fasting triglycerides and glucose by 2year followup . psychopathology indicators did not significantly predict subsequent levels of sbp . finally , we examined the opposite predictive relationship to see whether metabolic syndrome component values at t were associated with psychopathology scores at t + 1 , controlling for psychopathology at t and covariates ( table 4 ) . there was only one weak effect ( at p = .025 ) , suggesting that metabolic syndrome components do not consistently predict subsequent psychopathology in this sample . prospective association of baseline metabolic syndrome components ( t ) with psychopathology at the next assessment ( t + 1 ) estimates were obtained from generalized estimating equations . analyses were adjusted for baseline values of the outcome , age , sex , education , smoking , alcohol use , and physical activity . elevated depressive or anxiety symptoms and antidepressant use were independently associated with generally poorer metabolic syndrome component outcomes , consistently across three assessment waves . the association was stronger for symptom severity and antidepressant use , as compared to the clinical diagnosis . although all antidepressant medication types were associated with negative outcomes , as expected the deleterious effects on metabolic syndrome components were stronger for tcas compared to other antidepressants . the differences between classes were observed after controlling for sociodemographic characteristics , health , and symptom severity . in terms of prospective relationships , there was some evidence that more severe depression and anxiety symptoms and antidepressant use were associated with poorer metabolic syndrome component outcomes at followup , at least in the shortterm , whereas there was no evidence that baseline metabolic syndrome components predicted psychopathology outcomes at followup . the crosssectional relationship between psychopathology and metabolic dysregulation has been relatively wellestablished , and the current study adds to this in highlighting the consistency of the relationship with repeated measurement over time . most of effects were in the expected direction , whereby psychopathology severity and antidepressant use were associated with poorer metabolic outcomes across the three assessment waves . however , in contrast to the other components , results for sbp and hdlc were unexpected , whereby greater depressive and anxiety symptom severity were associated with lower sbp and higher hdlc . although a relationship between depression and hypertension is often observed,39 , 40 a negative association between psychopathology and sbp has been reported previously in nesda and other cohorts.41 , 42 , 43 , 44 the effect does not seem to be driven by medication use . lowered blood pressure in psychopathology may be a consequence of shared risk factors , such as levels of neuropeptide y , which is associated with suppressed parasympathetic activity and stress , depression and anxiety.45 the reason for the positive association between symptom severity and hdlc is unclear , particularly since hdlc has antiinflammatory properties 46 and recent evidence suggests that use of cholesterol improving medication is associated with improved mental health.47 nevertheless , the findings are consistent with previous research demonstrating that depressive symptoms and suicidality are associated with lowered total cholesterol and higher hdlc,48 , 49 although heterogeneity between studies is high and the effect is not always observed.50 , 51 overall , people with psychopathology may have a more nuanced risk pattern across metabolic syndrome components , rather than increased risk of metabolic abnormalities across the board . we also observed contrasting effects for the two anxiety scales used in this study , which is important to note since anxiety symptoms are less frequently reported in relation to metabolic dysregulation than depressive symptoms . in the current study , the general anxiety arousal symptoms measured with the bai were significantly associated with higher waist circumference , glucose , triglycerides , and number of metabolic syndrome abnormalities , rather than phobic / avoidance symptoms measured in the fq , which was only weakly associated with higher hdlc . anxiety symptoms related to somatic arousal may be a stronger correlate of metabolic syndrome components than those related to fear.52 regarding prospective relationships , higher psychopathology severity and antidepressant use at baseline were associated with poorer metabolic syndrome component outcomes at 2year followup . this was particularly true for use of antidepressants , which compared with depressive and anxiety symptoms , negatively influenced a range of metabolic syndrome components after 2years . antidepressants have pleiotropic effects across biological systems , including effects on autonomic nervous system , immune and inflammatory processes , oxidation , and cellular aging.53 , 54 , 55 these systems are all associated with negative consequences for cardiometabolic health and the development and maintenance cardiometabolic diseases.56 , 57 , 58 the effects of antidepressants were largely independent of psychopathology , highlighting antidepressants as important risk factors in their own right . in an extended examination from 2 to 6year followup , indeed , one of the observed effects was that antidepressant users at 2 years had on average waist reduction at 6year followup ; an effect explained by waist reduction or a lack of gain in those who had stopped using antidepressants by the 6year assessment . around 30% of antidepressant users at 2 years were no longer users at 6 years , allowing a fouryear window to normalize waist after cessation of antidepressant use . the findings indicate that it is the current use of antidepressants that has the strongest influence on waist circumference . the limited evidence of prospective relationships in the longer term indicates that effects of psychopathology and antidepressants on metabolic dysregulation may be diluted over time , or better explained by lifestyle or another enduring behavioral or biological factor . several studies indicate that presence of depression or anxiety or more severe baseline levels of psychopathology are associated with poor metabolic outcomes over time , particularly obesity and dyslipidemia,6 , 14 and sometimes over extended periods , up to 15 years later.59 other studies indicate effects in the opposite direction.13 , 60 however , many of these previous studies by nature of their study design are unable to test both directions of the prospective relationship , or are unable to take into account current psychological functioning or confounding from unhealthy lifestyle as a possible explanations for effects . for instance , one explanation for significant prospective associations in previous studies may involve unhealthy lifestyle , in particular smoking and heavy alcohol use , which often remain present in people with depression and anxiety even though their other symptoms resolve.19 the current study controls for such factors . nevertheless , in the current study , longitudinal effects of psychopathology on metabolic syndrome components 2 years later were present after adjusting for several key lifestyle factors . for instance , higher baseline parasympathetic nervous system activity and shortened telomere length have been associated with worsening metabolic dysregulation after 2 years in nesda participants.18 , 61 we did not observe evidence supporting the opposite prospective relationship ; levels of metabolic syndrome components were not associated with psychopathology at the next assessment . thus , if there is a prospective relationship among people with psychopathology , the direction from psychopathology to metabolic dysregulation may be stronger . it is possible that the lack of association may be partly due to the overall good health of the sample , with previous findings demonstrating stronger associations between obesity and depression than overweight and depression.62 , 63 it may also be partly due to the sample composition , which at baseline largely consisted of people with high psychopathology symptoms who decrease in their symptoms over time as a consequence of treatment or naturalistic change . nevertheless , a large majority of participants enrolled at baseline did not have a current diagnosis ( see symptom levels in remitted and healthy controls in table 1 ) , and since the focus of analyses was on change in symptom severity , it is likely that there was sufficient range in psychopathology scores to detect change in psychopathology in either direction . the major limitation of this study is the lack of assessments at finer time gradations , which may have provided a better understanding of the reciprocal relationship between psychopathology and metabolic dysregulation . another limitation is that although many participants with severe psychopathology were retained in nesda , there is evidence that participants with higher psychopathology symptom severity and current diagnosis were more likely to drop out . maximum likelihood analyses showed largely similar patterns to those reported here , indicating low risk of selective bias . nevertheless , the findings may be less generalizable to more severe psychopathology cases and people with more severe metabolic dysregulation , and may underestimate the true effect . a final consideration is the influence of multiple testing on the interpretation of findings . numerous tests were conducted to examine the hypotheses , yet these analyses were expected to be largely correlated so pvalue adjustment methods that assume independence between tests would be too conservative . the results of this study were instead interpreted based on the consistency of patterns across the results , rather than statistical significance . however , calculating a benjamini yekutieli falsediscovery rate,64 which controls for false significance at = .05 when hypotheses are dependent , gives an adjusted pvalue of .01 . with this threshold , most results , particularly the crosssectional findings , remain statistically significant and so the primary conclusions of the study are not substantially altered . a substantial strength of the current study is the prospective design with measurement of predictors , outcomes , and confounders at three waves . the design of nesda also oversampled participants with depression and anxiety disorders , which is a strength over general communitybased cohorts in that there is a broader range of depression and anxiety symptoms than the typically positively skewed distribution . metabolic dysregulation is associated with depressive symptoms , anxiety symptoms , and antidepressant use . furthermore , psychological and pathophysiological perturbations in depression and anxiety and the pharmacological effects of antidepressants may have shortterm enduring effects on metabolic health . although in the longterm one does not appear to exacerbate the other , the consistent crosssectional associations and negative consequences of psychopathology on shortterm metabolic health are nonetheless concerning since depression and anxiety are chronic conditions and antidepressants are widely prescribed . the results of the current study highlight the ongoing need to assess metabolic health in people with psychopathology , particularly those on antidepressant medication . associations of both psychopathology indicators and antidepressants with metabolic syndrome components across baseline , 2 and 6year assessments click here for additional data file .

backgroundmetabolic syndrome components waist circumference , highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , systolic blood pressure and fasting glucose are crosssectionally associated with depression and anxiety with differing strength . few studies examine the relationships over time or whether antidepressants have independent effects.methodsparticipants were from the netherlands study of depression and anxiety ( nesda ; n = 2,776 ; 1865 years ; 66% female ) . at baseline , 2 and 6year followup , participants completed diagnostic interviews , depression and anxiety symptom inventories , antidepressant use assessment , and measurements of the five metabolic syndrome components . data were analyzed for the consistency of associations between psychopathology indicators and metabolic syndrome components across the three assessment waves , and whether psychopathology or antidepressant use at one assessment predicts metabolic dysregulation at the next and vice versa.resultsconsistently across waves , psychopathology was associated with generally poorer values of metabolic syndrome components , particularly waist circumference and triglycerides . stronger associations were observed for psychopathology symptom severity than diagnosis . antidepressant use was independently associated with higher waist circumference , triglycerides and number of metabolic syndrome abnormalities , and lower hdlc . symptom severity and antidepressant use were associated with subsequently increased number of abnormalities , waist circumference , and glucose after 2 but not 4 years . conversely , there was little evidence that metabolic syndrome components were associated with subsequent psychopathology outcomes.conclusionssymptom severity and antidepressant use were independently associated with metabolic dysregulation consistently over time and also had negative consequences for shortterm metabolic health . this is of concern given the chronicity of depression and anxiety and prevalence of antidepressant treatment .

INTRODUCTION MATERIALS AND METHODS PARTICIPANTS AND PROCEDURES MEASURES Psychopathology Metabolic Syndrome Components Covariates STATISTICAL ANALYSIS RESULTS SAMPLE CHARACTERISTICS CONSISTENCY OF ASSOCIATIONS ACROSS ASSESSMENT WAVES PROSPECTIVE, AUTOREGRESSIVE RELATIONSHIPS DISCUSSION CONCLUSIONS Conflict of interest Supporting information

metabolic syndrome is a cluster of conditions abdominal obesity , dyslipidemia , elevated fasting glucose , and hypertension with serious consequences for physical health , such as increased risk of atherosclerotic cardiovascular disease , type 2 diabetes , and allcause mortality.1 , 2 , 3 , 4 metabolic syndrome and its components are also associated with a range of psychological characteristics , including depression , anxiety , anger , hostility , and impaired cognitive functioning.5 , 6 , 7 , 8 these associations may in part explain why people with psychopathology are at high risk of developing a range of chronic illnesses.9 metabolic syndrome has been defined as a discrete , binary entity ; however , previous research indicates that some components are more strongly associated with psychopathology than others , particularly obesity and dyslipdemia.10 , 11 , 12 given these discrepancies between components in the context of psychiatric illness , using a dichotomous definition of metabolic syndrome may not be the strongest way of identifying the nature of associations between psychopathology and metabolic dysregulation . although there is much evidence for crosssectional associations between metabolic syndrome dysregulations and psychopathology , few longitudinal studies assess temporal relationships , with even fewer examining both directions of the relationship simultaneously.6 furthermore , most of the current evidence involves depressive symptoms , whereas associations for anxiety symptoms or diagnosis of depression and anxiety have been infrequently reported , and results have been inconsistent.13 , 14 , 15 consequently , it remains unclear as to whether , over time , psychopathology impairs components of the metabolic syndrome , or vice versa . each direction is biologically plausible , since the development and progression of both psychopathology and metabolic dysregulation are associated with a range of detrimental biological processes , including altered autonomic and neuroendocrine stress functioning , low grade inflammation , cellular aging , and oxidative and nitrosative damage.16 , 17 , 18 thus , the pathophysiology of psychopathology may increase the risk of metabolic dysregulation , and vice versa . the use of tricyclic antidepressants ( tca ) has been associated with metabolic dysregulation , particularly abdominal obesity.10 , 23 however , the effects of selective serotonin reuptake inhibitors ( ssris ) on metabolic syndrome components are less clear.24 , 25 , 26 for instance , ssri use has been associated with weight gain , loss and no change , as well as both impaired and improved glucose and lipid profiles.23 , 26 ssri use is also associated with positive impacts on biological correlates of metabolic dysregulation , such as inflammation,27 , 28 which may in part explain why their metabolic effects are diverse . the current study contributes to the understanding of three issues that remain largely unclear regarding associations between psychopathology and metabolic dysregulation : ( 1 ) is psychopathology associated across all the metabolic syndrome components ? we addressed these issues using baseline , 2 and 6year data from a largescale , longitudinal cohort study of depression and anxiety . first , we examined the consistency of associations of five metabolic syndrome components with depression , anxiety and antidepressant use across the three data waves . second , to investigate prospective associations we examined whether psychopathology at one assessment predicted metabolic dysregulation at the next , and vice versa . participants were drawn from the netherlands study of depression and anxiety ( nesda ) , an ongoing prospective cohort study of 2,981 adults with and without depression and anxiety ( 1865 years ) . metabolic syndrome components and psychopathology were assessed at baseline , 2 and 6year followup assessments . participants who did not contribute to the 2 or 6year followup were relatively similar to participants in demographic characteristics and antidepressant use , although they had higher depression and anxiety symptoms at baseline as well as poorer metabolic syndrome components ( including lower hdlc and greater waist circumference , glucose , and triglycerides and number of abnormalities ) , compared to participants . participants were included in analyses when they had data for at least one metabolic syndrome component , psychopathology , and covariates for at least one assessment ( baseline assessment : n = 2,776 , 93% of available sample ; 2year assessment : n = 2,203 , 85% of available sample ; 6year assessment : n = 1,899 , 84% of available sample ) . psychopathology indicators , metabolic syndrome components , and covariates were assessed at baseline , 2 and 6year assessment waves . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.03 mmol / l in men , < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . for analyses using continuous values of metabolic syndrome components , raw values were adjusted for use of medication as previously described.14 , 18 , 35 specifically , for hdlc 0.10 mmol / l was subtracted for use of fibrates ( c10ab ) ; for triglycerides 0.67 mmol / l was added for use of fibrates ; for sbp 10 mmhg was added for use of antihypertensive medication ( c02 , c03 , c0709 ) ; and for glucose a value of 7 mmol / l was given for those on antidiabetic medication ( a10 ) with glucose level below 7 mmol / l . to examine the consistency of the associations between psychopathology or antidepressant use and metabolic dysregulation across the baseline , 2 and 6year followup assessments , generalized estimating equations ( gee ) time ( 0 , 2 , 6 ) and psychopathology ( diagnosis or symptom severity ) or antidepressant use ( none , tcas , ssris , other antidepressants ) were entered as main effects , with metabolic syndrome components as the outcome ( normal distribution model for continuous outcomes and poisson distribution for count outcomes ) . whether the effects of psychopathology on metabolic syndrome components were the same across the three waves , analyses were repeated including an interaction term between psychopathology indicators and time . autoregression gee models were applied to examine whether levels of psychopathology symptom severity or antidepressant use at one time point ( t ) predicted levels of metabolic syndrome components at the next time point ( t + 1 ) , controlling for covariates and metabolic syndrome components at t. thus , there were two comparisons made : between baseline and 2year followup assessment , and between 2 and 6year followup assessment . participants were drawn from the netherlands study of depression and anxiety ( nesda ) , an ongoing prospective cohort study of 2,981 adults with and without depression and anxiety ( 1865 years ) . metabolic syndrome components and psychopathology were assessed at baseline , 2 and 6year followup assessments . participants who did not contribute to the 2 or 6year followup were relatively similar to participants in demographic characteristics and antidepressant use , although they had higher depression and anxiety symptoms at baseline as well as poorer metabolic syndrome components ( including lower hdlc and greater waist circumference , glucose , and triglycerides and number of abnormalities ) , compared to participants . participants were included in analyses when they had data for at least one metabolic syndrome component , psychopathology , and covariates for at least one assessment ( baseline assessment : n = 2,776 , 93% of available sample ; 2year assessment : n = 2,203 , 85% of available sample ; 6year assessment : n = 1,899 , 84% of available sample ) . psychopathology indicators , metabolic syndrome components , and covariates were assessed at baseline , 2 and 6year assessment waves . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.03 mmol / l in men , < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , and glucose levels were determined from fasting blood samples using routine methods . the number of metabolic syndrome abnormalities present was defined as a count of the number of atpiii criteria34 present : ( 1 ) abdominal obesity : waist circumference > 102 cm in men , > 88 cm in women ; ( 2 ) low hdlc : hdlc < 1.30 mmol / l in women ; ( 3 ) hypertriglyceridemia : triglycerides 1.7 mmol / l ; ( 4 ) hypertension : blood pressure 130/85 mmhg or using antihypertensive medication ; ( 5 ) hyperglycemia : fasting plasma glucose 6.1 mmol / l or using antidiabetic medication . physical activity was calculated from response to the international physical activity questionnaire ( ipaq ) , which uses the weekly duration of selfreported vigorous , moderate , walking , and sitting activity to calculate metabolic equivalent total ( met ) minutes of activity per week ( met level minutes of activity events per week).37 , 38 descriptive statistics were calculated at baseline , 2 and 6year assessments . to examine the consistency of the associations between psychopathology or antidepressant use and metabolic dysregulation across the baseline , 2 and 6year followup assessments , generalized estimating equations ( gee ) time ( 0 , 2 , 6 ) and psychopathology ( diagnosis or symptom severity ) or antidepressant use ( none , tcas , ssris , other antidepressants ) were entered as main effects , with metabolic syndrome components as the outcome ( normal distribution model for continuous outcomes and poisson distribution for count outcomes ) . to examine whether the effects of psychopathology on metabolic syndrome components were the same across the three waves , analyses were repeated including an interaction term between psychopathology indicators and time . autoregression gee models were applied to examine whether levels of psychopathology symptom severity or antidepressant use at one time point ( t ) predicted levels of metabolic syndrome components at the next time point ( t + 1 ) , controlling for covariates and metabolic syndrome components at t. thus , there were two comparisons made : between baseline and 2year followup assessment , and between 2 and 6year followup assessment . baseline , 2 and 6year characteristics of the sample adjusted for use of medications with metabolic effects . there were significant main effects of time on metabolic syndrome components , indicating that levels of hdlc decreased across assessment waves , whereas number of metabolic syndrome abnormalities , waist circumference , triglycerides , sbp , and fasting glucose increased ( table 2 ) . across the three assessments , current and remitted anxiety or depressive disorders were associated with lower sbp , although associations with other components were not significant . across all assessment waves , higher ids was associated with higher number of metabolic syndrome abnormalities , waist circumference , hdlc and triglycerides , and lower sbp . bai scores were significantly and strongly associated with higher values for number of metabolic syndrome components , waist circumference , triglycerides , and glucose , whereas fq scores were only weakly associated with higher hdlc . associations of psychopathology diagnosis , symptoms , and antidepressant use with metabolic syndrome components across baseline , 2 and 6 year assessments estimates were obtained from generalized estimating equations . antidepressant use was also associated with metabolic syndrome components ( table 2 ) . compared with antidepressant nonusers , tca use was associated with lower hdlc and the use any type of antidepressant ( tca , ssri , or others ) was associated with higher waist circumference , triglycerides , and number of metabolic syndrome abnormalities . we did not observe consistent interactions between psychopathology indicators and time , indicating that the relationship between psychopathology indicators and metabolic syndrome components did not differ over time ( data not shown ) . in the prospective analyses , several psychopathology indicators at baseline significantly predicted levels of metabolic syndrome components at 2year assessment , taking into account covariates and baseline metabolic syndrome component values ( table 3 ) . in contrast , there was little evidence that psychopathology indicators at the 2year assessment predicted levels of metabolic dysregulation at 6year assessment . higher ids or bai scores and antidepressant use at baseline were all associated with an increasing or a higher number of metabolic syndrome abnormalities at 2year followup . antidepressant use at baseline was associated with increasing waist circumference by 2year followup , whereas antidepressant use at 2year followup was associated with decreasing waist circumference by 6year followup . fewer consistent patterns across symptom severity and antidepressant use were observed for the other components . antidepressant use , but not severity measures , at baseline was also associated with higher fasting triglycerides and glucose by 2year followup . there was only one weak effect ( at p = .025 ) , suggesting that metabolic syndrome components do not consistently predict subsequent psychopathology in this sample . depression and anxiety symptoms and the frequency of current diagnoses significantly decreased over time , whereas components of metabolic syndrome generally worsened over time . baseline , 2 and 6year characteristics of the sample adjusted for use of medications with metabolic effects . there were significant main effects of time on metabolic syndrome components , indicating that levels of hdlc decreased across assessment waves , whereas number of metabolic syndrome abnormalities , waist circumference , triglycerides , sbp , and fasting glucose increased ( table 2 ) . across the three assessments , current and remitted anxiety or depressive disorders were associated with lower sbp , although associations with other components were not significant . across all assessment waves , higher ids was associated with higher number of metabolic syndrome abnormalities , waist circumference , hdlc and triglycerides , and lower sbp . bai scores were significantly and strongly associated with higher values for number of metabolic syndrome components , waist circumference , triglycerides , and glucose , whereas fq scores were only weakly associated with higher hdlc . associations of psychopathology diagnosis , symptoms , and antidepressant use with metabolic syndrome components across baseline , 2 and 6 year assessments estimates were obtained from generalized estimating equations . antidepressant use was also associated with metabolic syndrome components ( table 2 ) . compared with antidepressant nonusers , tca use was associated with lower hdlc and the use any type of antidepressant ( tca , ssri , or others ) was associated with higher waist circumference , triglycerides , and number of metabolic syndrome abnormalities . to see whether the effects of antidepressant use were independent of symptom severity , both the effects were largely the same magnitude as those reported in table 2 , indicating independent effects ( detailed in supporting informationtable s1 ) . we did not observe consistent interactions between psychopathology indicators and time , indicating that the relationship between psychopathology indicators and metabolic syndrome components did not differ over time ( data not shown ) . in the prospective analyses , several psychopathology indicators at baseline significantly predicted levels of metabolic syndrome components at 2year assessment , taking into account covariates and baseline metabolic syndrome component values ( table 3 ) . in contrast , there was little evidence that psychopathology indicators at the 2year assessment predicted levels of metabolic dysregulation at 6year assessment . higher ids or bai scores and antidepressant use at baseline were all associated with an increasing or a higher number of metabolic syndrome abnormalities at 2year followup . antidepressant use at baseline was associated with increasing waist circumference by 2year followup , whereas antidepressant use at 2year followup was associated with decreasing waist circumference by 6year followup . prospective association of baseline psychopathology ( t ) with metabolic syndrome components at the next assessment ( t + 1 ) estimates were obtained from generalized estimating equations . fewer consistent patterns across symptom severity and antidepressant use were observed for the other components . antidepressant use , but not severity measures , at baseline was also associated with higher fasting triglycerides and glucose by 2year followup . there was only one weak effect ( at p = .025 ) , suggesting that metabolic syndrome components do not consistently predict subsequent psychopathology in this sample . elevated depressive or anxiety symptoms and antidepressant use were independently associated with generally poorer metabolic syndrome component outcomes , consistently across three assessment waves . although all antidepressant medication types were associated with negative outcomes , as expected the deleterious effects on metabolic syndrome components were stronger for tcas compared to other antidepressants . in terms of prospective relationships , there was some evidence that more severe depression and anxiety symptoms and antidepressant use were associated with poorer metabolic syndrome component outcomes at followup , at least in the shortterm , whereas there was no evidence that baseline metabolic syndrome components predicted psychopathology outcomes at followup . the crosssectional relationship between psychopathology and metabolic dysregulation has been relatively wellestablished , and the current study adds to this in highlighting the consistency of the relationship with repeated measurement over time . most of effects were in the expected direction , whereby psychopathology severity and antidepressant use were associated with poorer metabolic outcomes across the three assessment waves . however , in contrast to the other components , results for sbp and hdlc were unexpected , whereby greater depressive and anxiety symptom severity were associated with lower sbp and higher hdlc . lowered blood pressure in psychopathology may be a consequence of shared risk factors , such as levels of neuropeptide y , which is associated with suppressed parasympathetic activity and stress , depression and anxiety.45 the reason for the positive association between symptom severity and hdlc is unclear , particularly since hdlc has antiinflammatory properties 46 and recent evidence suggests that use of cholesterol improving medication is associated with improved mental health.47 nevertheless , the findings are consistent with previous research demonstrating that depressive symptoms and suicidality are associated with lowered total cholesterol and higher hdlc,48 , 49 although heterogeneity between studies is high and the effect is not always observed.50 , 51 overall , people with psychopathology may have a more nuanced risk pattern across metabolic syndrome components , rather than increased risk of metabolic abnormalities across the board . in the current study , the general anxiety arousal symptoms measured with the bai were significantly associated with higher waist circumference , glucose , triglycerides , and number of metabolic syndrome abnormalities , rather than phobic / avoidance symptoms measured in the fq , which was only weakly associated with higher hdlc . anxiety symptoms related to somatic arousal may be a stronger correlate of metabolic syndrome components than those related to fear.52 regarding prospective relationships , higher psychopathology severity and antidepressant use at baseline were associated with poorer metabolic syndrome component outcomes at 2year followup . for instance , higher baseline parasympathetic nervous system activity and shortened telomere length have been associated with worsening metabolic dysregulation after 2 years in nesda participants.18 , 61 we did not observe evidence supporting the opposite prospective relationship ; levels of metabolic syndrome components were not associated with psychopathology at the next assessment . it is possible that the lack of association may be partly due to the overall good health of the sample , with previous findings demonstrating stronger associations between obesity and depression than overweight and depression.62 , 63 it may also be partly due to the sample composition , which at baseline largely consisted of people with high psychopathology symptoms who decrease in their symptoms over time as a consequence of treatment or naturalistic change . nevertheless , a large majority of participants enrolled at baseline did not have a current diagnosis ( see symptom levels in remitted and healthy controls in table 1 ) , and since the focus of analyses was on change in symptom severity , it is likely that there was sufficient range in psychopathology scores to detect change in psychopathology in either direction . another limitation is that although many participants with severe psychopathology were retained in nesda , there is evidence that participants with higher psychopathology symptom severity and current diagnosis were more likely to drop out . although in the longterm one does not appear to exacerbate the other , the consistent crosssectional associations and negative consequences of psychopathology on shortterm metabolic health are nonetheless concerning since depression and anxiety are chronic conditions and antidepressants are widely prescribed . associations of both psychopathology indicators and antidepressants with metabolic syndrome components across baseline , 2 and 6year assessments click here for additional data file .

osteonecrosis of the femoral head ( onfh ) , which is also called avascular necrosis of femoral head or aseptic necrosis of femoral head , is a progressive disease that can be divided into 2 categories . primary ( idiopathic ) osteonecrosis of hips is commonly associated with inherited hrombophilia or hypofibrinolysis or antiphospholipid antibodies syndrome , whereas onfh can also be secondary to multiple risk factors such as high - dose or long - term corticosteroids , alcoholism , decompression sickness , hip trauma including femoral neck fractures and hip dislocations , and so on . although the pathogenesis of this multifactorial disease has not been completely elucidated , a sequence for the development of onfh has been postulated by some scholars : osseous venous outflow obstruction is caused by venous thrombosis because of thrombophilia - hypofibrinolysis , circulating lipids , nitrogen bubble formation or direct interruption , leading to reduced arterial flow , ischemia , bone death , and collapse of the articular surface ( fig . 1 ) . and there were also some animal experiments or other experimental models of osteonecrosis confirming venous occlusion as a primary event . as this devastating disease mainly affects adults in their 3rd , 4th , or 5th decade of life , and often necessitates total hip arthroplasty ( tha ) at a young age , it is increasingly essential to find a feasible and less - invasive therapeutic regimen to save the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosis ( table 1 ) . various options for the management of onfh to prevent surgical procedures have been applied , including bisphosphonates , vasodilators , and biophysical modalities . recently , some scholars have investigated the effects of anticoagulants on relieving or reversing venous occlusion . anticoagulant drugs , which prevent blood coagulation by participating in physiological coagulation , could be classified into 4 kinds : indirect thrombin inhibitor ( heparin and enoxaparin , among others ) , direct thrombin inhibitor ( argatroban and hirudin , among others ) , vitamin k antagonists ( warfarin and dicoumaral , among others ) , and selective factor xa inhibitors such fondaparinux and ximelagatran . ficat and arlet classification system . given this process characterized by bone death and reduced local blood flow , we performed a study with a purpose of determining the efficacy of anticoagulants for preventing and treating the primary and secondary onfh on its precollapse stage to improve the hip prognosis . this meta - analysis was carried out in accordance with the preferred reporting items for systematic reviews and meta - analyses reporting guidelines for the meta - analysis of intervention trials . ethical approval for this study was unnecessary because it was a review of existing literature and did not involve any handling of individual patient data . three electronic databases ( pubmed , embase , and web of science ) were searched and we used terms and boolean operators as follows : ( anticoagulants or anticoagulant drugs ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . we did not limit the year of publication , publication status , or language , and there was also no limitation on any particular study design : randomized or nonrandomized clinical trials , cohort , and case - control studies . in addition , we also checked the references of the articles manually to identify other potentially relevant publications . the study selection was then independently performed by 2 of the authors , and any different opinions were resolved through discussion . studies were considered eligible if they met the following criteria : the study enrolled patients with onfh ; the intervention considered in the study was the use of anticoagulants saving the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosisresults ; the efficiency before and after the treatment were at least evaluated by ficat or arlet stage . articles that reported at least 1 outcome were included and those without the outcome measures of interest were excluded . we extracted the following details from each article : the first author 's name , publication year , study design , the type of onfh , study population ( hips / patients ) , patient 's sex and age , follow - up time , interventions , outcomes , and adverse effects . review manager software ( revman v5.3 ) was used to analyze experimental data from the included trials . heterogeneity among studies was estimated using the i statistics ; substantial heterogeneity was reflected by i > 50% . a fixed - effects model was used when i < 50% ; otherwise , the random - effects model was adopted . three electronic databases ( pubmed , embase , and web of science ) were searched and we used terms and boolean operators as follows : ( anticoagulants or anticoagulant drugs ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . we did not limit the year of publication , publication status , or language , and there was also no limitation on any particular study design : randomized or nonrandomized clinical trials , cohort , and case - control studies . in addition , we also checked the references of the articles manually to identify other potentially relevant publications . the study selection was then independently performed by 2 of the authors , and any different opinions were resolved through discussion . studies were considered eligible if they met the following criteria : the study enrolled patients with onfh ; the intervention considered in the study was the use of anticoagulants saving the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosisresults ; the efficiency before and after the treatment were at least evaluated by ficat or arlet stage . articles that reported at least 1 outcome were included and those without the outcome measures of interest were excluded . we extracted the following details from each article : the first author 's name , publication year , study design , the type of onfh , study population ( hips / patients ) , patient 's sex and age , follow - up time , interventions , outcomes , and adverse effects . review manager software ( revman v5.3 ) was used to analyze experimental data from the included trials . heterogeneity among studies was estimated using the i statistics ; substantial heterogeneity was reflected by i > 50% . a fixed - effects model was used when i < 50% ; otherwise , the random - effects model was adopted . a total of 75 articles were initially searched from the pubmed , embase , and web of science databases . after duplicating , title screening , and abstract or full - text screening , 4 articles with a total of 218 hips were included in this review.figure 2 showed the process and details of our searching work . tables 2 and 3 provided the basic information and outcomes of the 4 studies , which included 2 prospective noncontrolled studies conducted by the same group during different follow - up periods and 2 comparative studies . all of the studies evaluated the efficacy of anticoagulants according to ficat or arlet stage identified by x - ray or magnetic resonance imaging ( mri ) or both . two articles studied the primary onfh and 1 was performed on secondary onfh , whereas the rest one included both of the types . data could not be pooled because of the methodological heterogeneity and limited number of the available controlled studies . the selection process of this review . characteristics of studies of the prevention and treatment of anticoagulants for onfh . methods and outcomes of studies of the prevention and treatment of anticoagulants for onfh . glueck et al studied 6 patients ( four men and two women ( 9 hips , 8 ficat stage ii , 1 stage i ) with familial thrombophilia ( 5 factor v leiden heterozygotes , 1 with resistance to activated protein c [ rapc ] ) . they used enoxaparin ( 60 mg / day ) for 3 months for every subjects , in 4 patients , coumadin was used in long term with the international normalized ratio targeted between 2 and 3 , and 1 patient was given pradaxa 150 mg twice per day after 90 days of enoxaparin , whereas in 1 patient , coumadin was later supplanted by xarelto , 20 mg / day . they found that after the anticoagulants therapy , 9 hips in the 6 patients ( 8 originally ficat ii and 1 ficat i ) remained unchanged , contrasted to untreated onfh , ficat stage ii , wherein 50% to 80% of hips progress to collapse ( ficat stages iii - iv ) within 2 years after diagnosis . within 3 , 3 , 3 , 9 , and 16 months after starting anticoagulation , 5 patients became pain - free and remained asymptomatic throughout follow - up , the rest 1 patient required prcocet for pain . these authors confirmed that long - term anticoagulation started before segmental collapse of the head of the femur ( ficat stages iii ) , in patients with thrombophilic factor v leiden or rapc , would be expected to stop the progression of idiopathic osteonecrosis and relieve symptoms , thus preventing the need for total hip replacement . thirty - six patients ( 25 men and 11 women ) diagnosed of idiopathic osteonecrosis of the hips with 49 hips been in the precollapsed stage were randomized into 2 groups averagely ; the study group consisting of 26 hips had been administered with 6000 u of enoxaparin daily for 12 weeks , whereas 23 hips in the control group had not received additional treatment . all patients had been radiographically evaluated with x - ray of the studied hips at 3 , 6 , 12 , 18 , and 24 months . at 24 months follow - up , 15 hips ( 57.7% ) from the study group remained in precollapse stage , whereas only 5 hips ( 21.7% ) in the control group remained in precollapse stage ( p = .042 ) . in the study group , 10 of 11 hips ( 90.9% ) that were in the ficat & arlet stage 0 or i at the time of enrollment have not progressed beyond stage ii , whereas only 5 of 15 hips ( 33.3% ) that were initially in stage ii had remained unchanged , and only 1 patient from the study group developed transient hematuria , which spontaneously subsided . they concluded that enoxaparin administration for the idiopathic osteonecrosis of the hip in precollapse stage can significantly prevent the progression of the disease in 24 months of follow - up . glueck et al also performed a study including patients with primary and secondary onfh . in their study , 16 patients ( age 45 8 years , 5 women and 11 men ) had primary osteonecrosis ( 25 hips : 13 stage i and 12 stage ii ) ; they were treated with enoxaparin ( 60 mg / day in a preloaded syringe ) administered for 12 weeks , with serial hip radiographs taken every 36 weeks to 108 weeks . during the study , 1 patient was lost to follow - up at 36 weeks , and 1 patient , on worsening of left hip osteonecrosis , stopped follow - up at 36 weeks despite no change in his right hip , and another patient progressed to ficat stage iii and iv osteonecrosis and received tha at 36 weeks . at 108 weeks or more of follow - up ( mean , 161 weeks ; range , 108216 weeks ) in 13 patients ( 20 hips ) with primary osteonecrosis , 19 hips ( 95% ) had no change from ficat stages i and ii osteonecrosis . at 108 weeks follow - up in patients with primary osteonecrosis , preservation of 95% of hips , compared favorably with untreated historic controls of approximately 20% with 2 years hip preservation . as mentioned above , in the study published in 2005 by glueck et al there were 12 patients ( age , 36 9 years ; 5 women and 7 men ) who had osteonecrosis secondary to long - term and high - dose corticosteroid use ( 15 hips : 5 stage i and 10 stage ii ) . of which 12 patients continued using corticosteroids during the study , 2 patients had systemic lupus erythematosus ( sle ) . after enoxaparin treatment ( 60 mg / day for 3 months ) and with 108 weeks of follow - up , in 12 patients with secondary osteonecrosis , only 3 of 15 hips ( 20% ) had osteonecrosis that remained at ficat stages i and ii , whereas 12 ( 80% ) had osteonecrosis that had progressed to ficat stages iii and iv . when comparing the outcomes between the primary and secondary onfh , the percent of hips remaining at stages i and ii in patients with primary osteonecrosis ( 95% ) was much greater ( p < .001 ) than in patients with secondary osteonecrosis ( 20% ) . so they concluded that for osteonecrosis secondary to corticosteroid use , enoxaparin did not alter progression to ficat stages iii or iv . they conducted a prospective clinical trial for the prevention of steroid - associated onfh in sle patients using warfarin . in their study , 60 patients consisting of 5 males and 55 females ranging from 16 to 58 years of age were selected ; all of them were requested to be newly diagnosed as having sle and to require high - dose ( 40 mg / day ) prednisolone as the initial treatment including pulse therapy with 1000 mg / day of methylprednisolone for 3 days . plain radiography and mri were used first at 3 months after the beginning of steroid treatment and subsequently every year for > 5 years to make a diagnosis of onfh . patients received warfarin ( 15 mg / day , mean 2.9 mg / day ) starting together with steroid therapy for 3 to 5 months ( mean , 3.2 months ) . silent onfh developed in 19 of 58 hips ( 33% ) in the control group , compared to 13 of 62 hips ( 21% ) in the warfarin group , without significant difference ( p = .13 ) . however , symptomatic onfh developed in 8 of 58 hips ( 14% ) in the control group . in comparison , it appeared in only 3 of 62 hips ( 4.8% ) in the warfarin group , with only a trend toward significance ( p = .08 ) . the authors concluded that there was no statistically significant effect of anticoagulant therapy on the prevention of onfh induced by corticosteroid . besides , warfarin tended to prevent only symptomatic onfh other than silent onfh induced by corticosteroid . except 1 patient from the study group developed transient hematuria after 12 weeks of enoxaparin injection according to chotanaphuti et al . glueck et al studied 6 patients ( four men and two women ( 9 hips , 8 ficat stage ii , 1 stage i ) with familial thrombophilia ( 5 factor v leiden heterozygotes , 1 with resistance to activated protein c [ rapc ] ) . they used enoxaparin ( 60 mg / day ) for 3 months for every subjects , in 4 patients , coumadin was used in long term with the international normalized ratio targeted between 2 and 3 , and 1 patient was given pradaxa 150 mg twice per day after 90 days of enoxaparin , whereas in 1 patient , coumadin was later supplanted by xarelto , 20 mg / day . they found that after the anticoagulants therapy , 9 hips in the 6 patients ( 8 originally ficat ii and 1 ficat i ) remained unchanged , contrasted to untreated onfh , ficat stage ii , wherein 50% to 80% of hips progress to collapse ( ficat stages iii - iv ) within 2 years after diagnosis . within 3 , 3 , 3 , 9 , and 16 months after starting anticoagulation , 5 patients became pain - free and remained asymptomatic throughout follow - up , the rest 1 patient required prcocet for pain . these authors confirmed that long - term anticoagulation started before segmental collapse of the head of the femur ( ficat stages iii ) , in patients with thrombophilic factor v leiden or rapc , would be expected to stop the progression of idiopathic osteonecrosis and relieve symptoms , thus preventing the need for total hip replacement . thirty - six patients ( 25 men and 11 women ) diagnosed of idiopathic osteonecrosis of the hips with 49 hips been in the precollapsed stage were randomized into 2 groups averagely ; the study group consisting of 26 hips had been administered with 6000 u of enoxaparin daily for 12 weeks , whereas 23 hips in the control group had not received additional treatment . all patients had been radiographically evaluated with x - ray of the studied hips at 3 , 6 , 12 , 18 , and 24 months . at 24 months follow - up , 15 hips ( 57.7% ) from the study group remained in precollapse stage , whereas only 5 hips ( 21.7% ) in the control group remained in precollapse stage ( p = .042 ) . in the study group , 10 of 11 hips ( 90.9% ) that were in the ficat & arlet stage 0 or i at the time of enrollment have not progressed beyond stage ii , whereas only 5 of 15 hips ( 33.3% ) that were initially in stage ii had remained unchanged , and only 1 patient from the study group developed transient hematuria , which spontaneously subsided . they concluded that enoxaparin administration for the idiopathic osteonecrosis of the hip in precollapse stage can significantly prevent the progression of the disease in 24 months of follow - up . glueck et al also performed a study including patients with primary and secondary onfh . in their study , 16 patients ( age 45 8 years , 5 women and 11 men ) had primary osteonecrosis ( 25 hips : 13 stage i and 12 stage ii ) ; they were treated with enoxaparin ( 60 mg / day in a preloaded syringe ) administered for 12 weeks , with serial hip radiographs taken every 36 weeks to 108 weeks . during the study , 1 patient was lost to follow - up at 36 weeks , and 1 patient , on worsening of left hip osteonecrosis , stopped follow - up at 36 weeks despite no change in his right hip , and another patient progressed to ficat stage iii and iv osteonecrosis and received tha at 36 weeks . at 108 weeks or more of follow - up ( mean , 161 weeks ; range , 108216 weeks ) in 13 patients ( 20 hips ) with primary osteonecrosis , 19 hips ( 95% ) had no change from ficat stages i and ii osteonecrosis . at 108 weeks follow - up in patients with primary osteonecrosis , preservation of 95% of hips , compared favorably with untreated historic controls of approximately 20% with 2 years hip preservation . as mentioned above , in the study published in 2005 by glueck et al , secondary onfh patients were also included . there were 12 patients ( age , 36 9 years ; 5 women and 7 men ) who had osteonecrosis secondary to long - term and high - dose corticosteroid use ( 15 hips : 5 stage i and 10 stage ii ) . of which 12 patients continued using corticosteroids during the study , 2 patients had systemic lupus erythematosus ( sle ) . after enoxaparin treatment ( 60 mg / day for 3 months ) and with 108 weeks of follow - up , in 12 patients with secondary osteonecrosis , only 3 of 15 hips ( 20% ) had osteonecrosis that remained at ficat stages i and ii , whereas 12 ( 80% ) had osteonecrosis that had progressed to ficat stages iii and iv . when comparing the outcomes between the primary and secondary onfh , the percent of hips remaining at stages i and ii in patients with primary osteonecrosis ( 95% ) was much greater ( p < .001 ) than in patients with secondary osteonecrosis ( 20% ) . so they concluded that for osteonecrosis secondary to corticosteroid use , enoxaparin did not alter progression to ficat stages iii or iv . they conducted a prospective clinical trial for the prevention of steroid - associated onfh in sle patients using warfarin . in their study , 60 patients consisting of 5 males and 55 females ranging from 16 to 58 years of age were selected ; all of them were requested to be newly diagnosed as having sle and to require high - dose ( 40 mg / day ) prednisolone as the initial treatment including pulse therapy with 1000 mg / day of methylprednisolone for 3 days . plain radiography and mri were used first at 3 months after the beginning of steroid treatment and subsequently every year for > 5 years to make a diagnosis of onfh . patients received warfarin ( 15 mg / day , mean 2.9 mg / day ) starting together with steroid therapy for 3 to 5 months ( mean , 3.2 months ) . silent onfh developed in 19 of 58 hips ( 33% ) in the control group , compared to 13 of 62 hips ( 21% ) in the warfarin group , without significant difference ( p = .13 ) . however , symptomatic onfh developed in 8 of 58 hips ( 14% ) in the control group . in comparison , it appeared in only 3 of 62 hips ( 4.8% ) in the warfarin group , with only a trend toward significance ( p = .08 ) . the authors concluded that there was no statistically significant effect of anticoagulant therapy on the prevention of onfh induced by corticosteroid . besides , warfarin tended to prevent only symptomatic onfh other than silent onfh induced by corticosteroid . except 1 patient from the study group developed transient hematuria after 12 weeks of enoxaparin injection according to chotanaphuti et al . the overall goal of our review was to make a summary conclusion of whether anticoagulants could prevent or treat onfh before its collapse stage by synthesizing evidence from previous studies . in this review , we searched 3 databases , but only found 4 clinical trial articles published to determine the effect of anticoagulants on preventing and treating the onfh . two of them reported the result of enoxaparin for primary onfh , and one article was related to prevention of onfh using warfarin , whereas the last one included primary and secondary onfh with the treatment of enoxaparin . generally speaking , first , the numbers of the included studies and the subjects in each of the study were not so sufficient , the smallest study only consisted of 6 patients , and 2 of the 4 articles used observational noncontrolled methods . second , there was not a unified standard in dose and time of anticoagulants use either , and the follow - up time was different among the studies , so some clinical heterogeneity was very likely to exist . despite of the shortcomings mentioned above , the result of our review offered some useful information about the positive efficacy toward primary onfh and the unprotected efficacy toward secondary onfh with the use of anticoagulants . in addition , there were no severe adverse effects associated with anticoagulants treatment reported during follow - up in the included studies . moreover , to arrive at a convincing conclusion , further studies should be conducted to demonstrate the following aspects : the detailed indication of patient - specific factors should be further unified , including age , mean morbidity time , and life expectancy ; the strategy of intervention should be optimized , including timing of treatment initiation and anticoagulants therapy dose and duration ; the trial design should be randomized and double - blind when grouping , treating , and examining efficacy . this review not only showed a positive effect on primary onfh with the prevention and treatment of anticoagulants , but also suggested that for secondary onfh , anticoagulants can not play a protective role . so if we can use the anticoagulants to treat primary onfh before collapse ( ficat stages i or ii ) or before the pathology of necrosis , this disease will be possibly reversed . nevertheless , the lack of large - scale randomized , and double - blind studies should be noted ; confirmation from further meta - analysis with large - scale , well - designed randomized control trials is required . the authors thank the members of the department of epidemiology and bio - statistics , school of public health , peking university for help with the statistical analysis , and fan meng ( experienced nurse ) for helpful discussions .

abstractbackground : osteonecrosis of the femoral head ( onfh ) is a progressive disease , which mainly affects young adults and often necessitates total hip arthroplasty ( tha ) , so early interventions are critical to successfully protect hip joint from tha . in this review , our purpose was to determine the effects of anticoagulants for preventing and treating the primary and secondary onfh , respectively , before the collapse stage or before the pathology of necrosis.methods:we searched pubmed , embase , web of science databases for relevant studies . any observational or experimental studies that evaluated anticoagulants and onfh were our goal of searching the electric databases.results:four studies including a total of 218 hips were identified in this review , 2 of them were prospective studies which performed by 1 group , 1 was a retrospective study , and the last was a prospective comparative study.conclusions:our findings supported that the anticoagulants could be used for primary onfh . however , anticoagulants can not play a protective role on secondary onfh . moreover , there were no serious adverse effects reported in the studies after anticoagulants treatment . nevertheless , our present study with some limitations such as the limited sample size only provided limited quality of evidence ; confirmation from further systematic review or meta - analysis with large - scale , well - designed randomized control trials is required .

Introduction Materials and methods Search strategy Eligibility criteria Data extraction Statistical analysis Results Primary ONFH outcomes analysis Secondary ONFH outcomes analysis Adverse events analysis Discussion Conclusions Acknowledgments

osteonecrosis of the femoral head ( onfh ) , which is also called avascular necrosis of femoral head or aseptic necrosis of femoral head , is a progressive disease that can be divided into 2 categories . primary ( idiopathic ) osteonecrosis of hips is commonly associated with inherited hrombophilia or hypofibrinolysis or antiphospholipid antibodies syndrome , whereas onfh can also be secondary to multiple risk factors such as high - dose or long - term corticosteroids , alcoholism , decompression sickness , hip trauma including femoral neck fractures and hip dislocations , and so on . although the pathogenesis of this multifactorial disease has not been completely elucidated , a sequence for the development of onfh has been postulated by some scholars : osseous venous outflow obstruction is caused by venous thrombosis because of thrombophilia - hypofibrinolysis , circulating lipids , nitrogen bubble formation or direct interruption , leading to reduced arterial flow , ischemia , bone death , and collapse of the articular surface ( fig . and there were also some animal experiments or other experimental models of osteonecrosis confirming venous occlusion as a primary event . as this devastating disease mainly affects adults in their 3rd , 4th , or 5th decade of life , and often necessitates total hip arthroplasty ( tha ) at a young age , it is increasingly essential to find a feasible and less - invasive therapeutic regimen to save the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosis ( table 1 ) . various options for the management of onfh to prevent surgical procedures have been applied , including bisphosphonates , vasodilators , and biophysical modalities . recently , some scholars have investigated the effects of anticoagulants on relieving or reversing venous occlusion . anticoagulant drugs , which prevent blood coagulation by participating in physiological coagulation , could be classified into 4 kinds : indirect thrombin inhibitor ( heparin and enoxaparin , among others ) , direct thrombin inhibitor ( argatroban and hirudin , among others ) , vitamin k antagonists ( warfarin and dicoumaral , among others ) , and selective factor xa inhibitors such fondaparinux and ximelagatran . given this process characterized by bone death and reduced local blood flow , we performed a study with a purpose of determining the efficacy of anticoagulants for preventing and treating the primary and secondary onfh on its precollapse stage to improve the hip prognosis . this meta - analysis was carried out in accordance with the preferred reporting items for systematic reviews and meta - analyses reporting guidelines for the meta - analysis of intervention trials . ethical approval for this study was unnecessary because it was a review of existing literature and did not involve any handling of individual patient data . three electronic databases ( pubmed , embase , and web of science ) were searched and we used terms and boolean operators as follows : ( anticoagulants or anticoagulant drugs ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . we did not limit the year of publication , publication status , or language , and there was also no limitation on any particular study design : randomized or nonrandomized clinical trials , cohort , and case - control studies . in addition , we also checked the references of the articles manually to identify other potentially relevant publications . the study selection was then independently performed by 2 of the authors , and any different opinions were resolved through discussion . studies were considered eligible if they met the following criteria : the study enrolled patients with onfh ; the intervention considered in the study was the use of anticoagulants saving the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosisresults ; the efficiency before and after the treatment were at least evaluated by ficat or arlet stage . we extracted the following details from each article : the first author 's name , publication year , study design , the type of onfh , study population ( hips / patients ) , patient 's sex and age , follow - up time , interventions , outcomes , and adverse effects . three electronic databases ( pubmed , embase , and web of science ) were searched and we used terms and boolean operators as follows : ( anticoagulants or anticoagulant drugs ) and ( avascular necrosis or aseptic necrosis or osteonecrosis ) and ( femoral head ) . we did not limit the year of publication , publication status , or language , and there was also no limitation on any particular study design : randomized or nonrandomized clinical trials , cohort , and case - control studies . the study selection was then independently performed by 2 of the authors , and any different opinions were resolved through discussion . studies were considered eligible if they met the following criteria : the study enrolled patients with onfh ; the intervention considered in the study was the use of anticoagulants saving the hips before collapse ( ficat stages i or ii ) or before the pathology of necrosisresults ; the efficiency before and after the treatment were at least evaluated by ficat or arlet stage . we extracted the following details from each article : the first author 's name , publication year , study design , the type of onfh , study population ( hips / patients ) , patient 's sex and age , follow - up time , interventions , outcomes , and adverse effects . a total of 75 articles were initially searched from the pubmed , embase , and web of science databases . after duplicating , title screening , and abstract or full - text screening , 4 articles with a total of 218 hips were included in this review.figure 2 showed the process and details of our searching work . tables 2 and 3 provided the basic information and outcomes of the 4 studies , which included 2 prospective noncontrolled studies conducted by the same group during different follow - up periods and 2 comparative studies . all of the studies evaluated the efficacy of anticoagulants according to ficat or arlet stage identified by x - ray or magnetic resonance imaging ( mri ) or both . two articles studied the primary onfh and 1 was performed on secondary onfh , whereas the rest one included both of the types . data could not be pooled because of the methodological heterogeneity and limited number of the available controlled studies . the selection process of this review . characteristics of studies of the prevention and treatment of anticoagulants for onfh . methods and outcomes of studies of the prevention and treatment of anticoagulants for onfh . glueck et al studied 6 patients ( four men and two women ( 9 hips , 8 ficat stage ii , 1 stage i ) with familial thrombophilia ( 5 factor v leiden heterozygotes , 1 with resistance to activated protein c [ rapc ] ) . they found that after the anticoagulants therapy , 9 hips in the 6 patients ( 8 originally ficat ii and 1 ficat i ) remained unchanged , contrasted to untreated onfh , ficat stage ii , wherein 50% to 80% of hips progress to collapse ( ficat stages iii - iv ) within 2 years after diagnosis . within 3 , 3 , 3 , 9 , and 16 months after starting anticoagulation , 5 patients became pain - free and remained asymptomatic throughout follow - up , the rest 1 patient required prcocet for pain . these authors confirmed that long - term anticoagulation started before segmental collapse of the head of the femur ( ficat stages iii ) , in patients with thrombophilic factor v leiden or rapc , would be expected to stop the progression of idiopathic osteonecrosis and relieve symptoms , thus preventing the need for total hip replacement . thirty - six patients ( 25 men and 11 women ) diagnosed of idiopathic osteonecrosis of the hips with 49 hips been in the precollapsed stage were randomized into 2 groups averagely ; the study group consisting of 26 hips had been administered with 6000 u of enoxaparin daily for 12 weeks , whereas 23 hips in the control group had not received additional treatment . all patients had been radiographically evaluated with x - ray of the studied hips at 3 , 6 , 12 , 18 , and 24 months . at 24 months follow - up , 15 hips ( 57.7% ) from the study group remained in precollapse stage , whereas only 5 hips ( 21.7% ) in the control group remained in precollapse stage ( p = .042 ) . in the study group , 10 of 11 hips ( 90.9% ) that were in the ficat & arlet stage 0 or i at the time of enrollment have not progressed beyond stage ii , whereas only 5 of 15 hips ( 33.3% ) that were initially in stage ii had remained unchanged , and only 1 patient from the study group developed transient hematuria , which spontaneously subsided . they concluded that enoxaparin administration for the idiopathic osteonecrosis of the hip in precollapse stage can significantly prevent the progression of the disease in 24 months of follow - up . glueck et al also performed a study including patients with primary and secondary onfh . in their study , 16 patients ( age 45 8 years , 5 women and 11 men ) had primary osteonecrosis ( 25 hips : 13 stage i and 12 stage ii ) ; they were treated with enoxaparin ( 60 mg / day in a preloaded syringe ) administered for 12 weeks , with serial hip radiographs taken every 36 weeks to 108 weeks . during the study , 1 patient was lost to follow - up at 36 weeks , and 1 patient , on worsening of left hip osteonecrosis , stopped follow - up at 36 weeks despite no change in his right hip , and another patient progressed to ficat stage iii and iv osteonecrosis and received tha at 36 weeks . as mentioned above , in the study published in 2005 by glueck et al there were 12 patients ( age , 36 9 years ; 5 women and 7 men ) who had osteonecrosis secondary to long - term and high - dose corticosteroid use ( 15 hips : 5 stage i and 10 stage ii ) . of which 12 patients continued using corticosteroids during the study , 2 patients had systemic lupus erythematosus ( sle ) . when comparing the outcomes between the primary and secondary onfh , the percent of hips remaining at stages i and ii in patients with primary osteonecrosis ( 95% ) was much greater ( p < .001 ) than in patients with secondary osteonecrosis ( 20% ) . they conducted a prospective clinical trial for the prevention of steroid - associated onfh in sle patients using warfarin . in their study , 60 patients consisting of 5 males and 55 females ranging from 16 to 58 years of age were selected ; all of them were requested to be newly diagnosed as having sle and to require high - dose ( 40 mg / day ) prednisolone as the initial treatment including pulse therapy with 1000 mg / day of methylprednisolone for 3 days . silent onfh developed in 19 of 58 hips ( 33% ) in the control group , compared to 13 of 62 hips ( 21% ) in the warfarin group , without significant difference ( p = .13 ) . however , symptomatic onfh developed in 8 of 58 hips ( 14% ) in the control group . in comparison , it appeared in only 3 of 62 hips ( 4.8% ) in the warfarin group , with only a trend toward significance ( p = .08 ) . they used enoxaparin ( 60 mg / day ) for 3 months for every subjects , in 4 patients , coumadin was used in long term with the international normalized ratio targeted between 2 and 3 , and 1 patient was given pradaxa 150 mg twice per day after 90 days of enoxaparin , whereas in 1 patient , coumadin was later supplanted by xarelto , 20 mg / day . they found that after the anticoagulants therapy , 9 hips in the 6 patients ( 8 originally ficat ii and 1 ficat i ) remained unchanged , contrasted to untreated onfh , ficat stage ii , wherein 50% to 80% of hips progress to collapse ( ficat stages iii - iv ) within 2 years after diagnosis . these authors confirmed that long - term anticoagulation started before segmental collapse of the head of the femur ( ficat stages iii ) , in patients with thrombophilic factor v leiden or rapc , would be expected to stop the progression of idiopathic osteonecrosis and relieve symptoms , thus preventing the need for total hip replacement . thirty - six patients ( 25 men and 11 women ) diagnosed of idiopathic osteonecrosis of the hips with 49 hips been in the precollapsed stage were randomized into 2 groups averagely ; the study group consisting of 26 hips had been administered with 6000 u of enoxaparin daily for 12 weeks , whereas 23 hips in the control group had not received additional treatment . all patients had been radiographically evaluated with x - ray of the studied hips at 3 , 6 , 12 , 18 , and 24 months . at 24 months follow - up , 15 hips ( 57.7% ) from the study group remained in precollapse stage , whereas only 5 hips ( 21.7% ) in the control group remained in precollapse stage ( p = .042 ) . in the study group , 10 of 11 hips ( 90.9% ) that were in the ficat & arlet stage 0 or i at the time of enrollment have not progressed beyond stage ii , whereas only 5 of 15 hips ( 33.3% ) that were initially in stage ii had remained unchanged , and only 1 patient from the study group developed transient hematuria , which spontaneously subsided . they concluded that enoxaparin administration for the idiopathic osteonecrosis of the hip in precollapse stage can significantly prevent the progression of the disease in 24 months of follow - up . glueck et al also performed a study including patients with primary and secondary onfh . in their study , 16 patients ( age 45 8 years , 5 women and 11 men ) had primary osteonecrosis ( 25 hips : 13 stage i and 12 stage ii ) ; they were treated with enoxaparin ( 60 mg / day in a preloaded syringe ) administered for 12 weeks , with serial hip radiographs taken every 36 weeks to 108 weeks . during the study , 1 patient was lost to follow - up at 36 weeks , and 1 patient , on worsening of left hip osteonecrosis , stopped follow - up at 36 weeks despite no change in his right hip , and another patient progressed to ficat stage iii and iv osteonecrosis and received tha at 36 weeks . as mentioned above , in the study published in 2005 by glueck et al , secondary onfh patients were also included . there were 12 patients ( age , 36 9 years ; 5 women and 7 men ) who had osteonecrosis secondary to long - term and high - dose corticosteroid use ( 15 hips : 5 stage i and 10 stage ii ) . of which 12 patients continued using corticosteroids during the study , 2 patients had systemic lupus erythematosus ( sle ) . when comparing the outcomes between the primary and secondary onfh , the percent of hips remaining at stages i and ii in patients with primary osteonecrosis ( 95% ) was much greater ( p < .001 ) than in patients with secondary osteonecrosis ( 20% ) . in their study , 60 patients consisting of 5 males and 55 females ranging from 16 to 58 years of age were selected ; all of them were requested to be newly diagnosed as having sle and to require high - dose ( 40 mg / day ) prednisolone as the initial treatment including pulse therapy with 1000 mg / day of methylprednisolone for 3 days . silent onfh developed in 19 of 58 hips ( 33% ) in the control group , compared to 13 of 62 hips ( 21% ) in the warfarin group , without significant difference ( p = .13 ) . however , symptomatic onfh developed in 8 of 58 hips ( 14% ) in the control group . in comparison , it appeared in only 3 of 62 hips ( 4.8% ) in the warfarin group , with only a trend toward significance ( p = .08 ) . the overall goal of our review was to make a summary conclusion of whether anticoagulants could prevent or treat onfh before its collapse stage by synthesizing evidence from previous studies . in this review , we searched 3 databases , but only found 4 clinical trial articles published to determine the effect of anticoagulants on preventing and treating the onfh . two of them reported the result of enoxaparin for primary onfh , and one article was related to prevention of onfh using warfarin , whereas the last one included primary and secondary onfh with the treatment of enoxaparin . generally speaking , first , the numbers of the included studies and the subjects in each of the study were not so sufficient , the smallest study only consisted of 6 patients , and 2 of the 4 articles used observational noncontrolled methods . second , there was not a unified standard in dose and time of anticoagulants use either , and the follow - up time was different among the studies , so some clinical heterogeneity was very likely to exist . despite of the shortcomings mentioned above , the result of our review offered some useful information about the positive efficacy toward primary onfh and the unprotected efficacy toward secondary onfh with the use of anticoagulants . in addition , there were no severe adverse effects associated with anticoagulants treatment reported during follow - up in the included studies . moreover , to arrive at a convincing conclusion , further studies should be conducted to demonstrate the following aspects : the detailed indication of patient - specific factors should be further unified , including age , mean morbidity time , and life expectancy ; the strategy of intervention should be optimized , including timing of treatment initiation and anticoagulants therapy dose and duration ; the trial design should be randomized and double - blind when grouping , treating , and examining efficacy . this review not only showed a positive effect on primary onfh with the prevention and treatment of anticoagulants , but also suggested that for secondary onfh , anticoagulants can not play a protective role . so if we can use the anticoagulants to treat primary onfh before collapse ( ficat stages i or ii ) or before the pathology of necrosis , this disease will be possibly reversed . nevertheless , the lack of large - scale randomized , and double - blind studies should be noted ; confirmation from further meta - analysis with large - scale , well - designed randomized control trials is required . the authors thank the members of the department of epidemiology and bio - statistics , school of public health , peking university for help with the statistical analysis , and fan meng ( experienced nurse ) for helpful discussions .

the escherichia coli groe chaperonin system facilitates protein folding in vivo and in vitro in an atp - dependent manner ( for reviews see , for example , refs ( 13 ) ) . it comprises groel , an oligomer of 14 identical subunits that form two heptameric rings , stacked back - to - back , with a cavity at each end in which protein folding can take place in a protective environment , and its helper - protein groes , which is a homoheptameric single ring . the groe system is essential for the folding of only a small subset of e. coli proteins ( < 100 ) but what distinguishes groe clients from all other e. coli proteins remains unclear . obligate substrates or other non - native proteins can become encapsulated in the groel cavity when groes binds to the apical domains of a substrate- and atp - occupied groel ring . the substrates are then discharged into bulk solution , either folded or not , following groes dissociation that is triggered by atp hydrolysis in the groes - bound cis groel ring and atp binding to the opposite trans groel ring ( see refs ( 2 and 7 ) for detailed schemes of current models of the groe reaction cycle ) . the reaction cycle of groel is governed by the cooperative binding of atp that is positive within rings and negative between rings . the intraring positive allostery facilitates cycling of the groel rings between protein substrate acceptor and release states . inter - ring negative allostery ensures that the two rings can operate out - of - phase with respect to each other and that atp binding to one ring triggers groes release from the opposite ring . however , the role of the inter - ring allostery is less clear when the symmetric football - shaped groel groes2 complex ( and not the asymmetric groel groes complex ) is the active species of this nanomachine . despite more than two decades of intensive research , it remains unclear and controversial whether the cavity of the groel groes complex is only a passive cage in which aggregation is prevented but the folding pathway is unchanged or a chamber that has evolved to optimize the folding process itself . factors that could influence the folding reaction inside the groel cavity are steric confinement , the chemical nature of the cavity walls and the properties of the cavity - confined water , which may , in fact , be intimately linked to the steric and/or chemical effects of the confinement imposed by the groel interior surface . the extent of steric confinement and the chemical nature of the cavity walls are known from the crystal structure of the groel groes complex , but there is no available experimental data regarding the properties of the cavity water . specifically , insight into the diffusion dynamics of water within the groel cavity can offer critical clues about the groel surface water attraction and may allow us to hypothesize about the stability and folding potential of proteins entering the groel cavity . if water is interacting favorably with the interior surface of the groel cavity , as would be reflected in strongly retarded , rigidified , surface water dynamics , then a protein substrate that is encapsulated in the cavity will experience a strongly repulsive hydration barrier from the groel surface and , thus , tend to fold in order to bury its hydrophobic residues . by contrast , the hydrophobic nature of the cavity walls in groel s substrate acceptor state may be reflected in nonretarded , fast diffusing , surface water dynamics that disfavor substrate folding . equally interesting is the surface of the groes lid : is it strongly or weakly hydrated and do the hydration level and dynamics change upon formation of the groel the hydration properties of the groel cavity have been the focus of computational studies that indicated , for example , that groel s ability to assist folding scales with the affinity for water of the cavity s interior surface . however , direct experimental measurements of properties of confined water in the groel cavity have not yet been reported . in this study , we present the first such experimental measurements for water near the surface of free groes and the same surface when it faces the cavity of the groel groes complex . the experiments described here combine site - directed spin labeling ( sdsl ) with two state - of - the - art magnetic resonance techniques : electron - spin echo envelope modulation ( eseem ) and overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance ( odnp - nmr ) . a single site , tyr71 , in groes was replaced by site - directed mutagenesis with a cysteine to which a nitroxide spin label , n-(1-oxyl-2,2,5,5- tetramethyl pyrrolidinyl)-maleimide , was attached . this position was chosen since it is fully exposed to bulk water in unbound groes and , upon groel groes complex formation , faces the confined water inside the chaperonin cavity ( figure 1a , b ) . ( a ) side and ( b ) top views of single - ring groel in complex with spin - labeled groes . groel and groes in the crystal structure of the groel groes complex ( pdb code : 1aon ) are represented by space - filling ( in gray ) and ball - and - stick ( in magenta ) models , respectively . the sulfur , carbon , nitrogen , and oxygen atoms of the spin label are shown in orange , yellow , blue , and red , respectively . in panel a , the apical , intermediate and equatorial domains are designated by a , i , and e , respectively , and two subunits of groel were removed in order to reveal the cavity . in the single - ring groel groes complex , the spin - labels are exposed to confined water in the cavity and are not close to any residues of groel . importantly , the spin label at this position is sufficiently far - removed from residues in the cavity wall , with the closest residue being asn299 , whose c side - chain atom is about 17 away from the nitroxide oxygen . the single - ring ( sr1 ) version of groel with the mutation asp398 ala that slows atp hydrolysis considerably was studied here instead of wild - type groel in order to minimize dissociation of the labeled groes from groel . the cavity properties and intraring allostery of sr1 are similar to those of wild - type groel . eseem and odnp - nmr spectroscopy at x - band ( 10 ghz ) frequencies and a magnetic field of 0.35 t were employed to probe the properties of local water within the chaperonin cavity . in order to probe the amount of water in the vicinity of the spin label that protrudes into the cavity of the groes groel complex and can sense its upper region , the well - established eseem technique was employed for measuring hyperfine interactions between the electron spin of the label and nearby nuclear spins . when the hyperfine interaction is very weak , its isotropic part is zero and the anisotropic part can be described by the point dipole interaction between the electron spin and the nuclear spin , whose strength is inversely proportional to the cube of their distance , r. in such cases , this interaction is manifested as modulations in the electron spin echo decay that oscillate at a frequency equal to the larmor frequencies of the coupled nuclei , and the number of weakly coupled magnetic nuclei and their average distances from the electron spin are reflected in the modulation depth . by combining eseem of h nuclei in d2o solutions with spin labeling , it is possible to probe the number of d2o molecules in the vicinity of the spin - labeled residue cys71 ( up to about 8 ) without interferences from the protein protons . this method has been successfully used to derive the water penetration depth in membranes and water exposure of protein residues . eseem measurements are usually carried out in frozen solutions and can not probe the dynamic properties of protein surface water . to get information regarding dynamics under solution conditions at room temperature , we applied odnp - nmr relaxometry to probe the diffusion dynamics of water near the spin - labeled cys71 . odnp selectively amplifies the h nmr signal of the local hydration water around a specific spin label ( within 510 ) of a protein site by transferring polarization from the electron spin to the nearby moving water molecules using the same anisotropic hyperfine interaction mentioned above ( alternatively termed the electron - nuclear dipole odnp relies on the enhancement of the h nmr signal of water at 0.35 t and 15 mhz that is achieved by saturating the electron spin resonance ( esr ) transitions at 10 ghz . since only the h of water molecules that move fast ( relative to 10 ghz ) experience electron - h spin flip - flops that give rise to h nmr signal enhancement , odnp can be exploited to quantify local water diffusivity near the nitroxide spin label . the motion of hydration water is characterized by a translational diffusion correlation time ( c ) , which represents the time needed for water to diffuse near the spin label within a distance b ( typically 510 , as determined by the electron - h dipolar coupling field ) and is inversely proportional to the local diffusion coefficient ( d ) , i.e. , with c b / d . crucially , odnp , when combined with h nmr relaxation time measurements , can separate contributions of freely diffusively translating hydration water ( k , picosecond time scale ) from motional fluctuations that occur on a slower time scale ( klow , nanosecond time scale ) . weak protein surface water attraction will be reflected in small c and large d and large k values . strong protein surface water attraction will present the opposite trend of large c and small d and small k values . in addition , there can be contributions from strongly bound water on protein surfaces with lifetimes exceeding 1 ns , whose presence would be reflected in a large klow value that increases as the rotational tumbling of the protein is slowed , for example , upon immobilization or immersion in a viscous solvent . using this approach , the hydration dynamics landscape around lipid membranes and proteins has been mapped out recently with site - specificity . it should be noted that protein site - specific correlation times for hydration dynamics have also been measured using ultrafast laser methods that monitor the relaxation of water around an excited tryptophan electric dipole by probing time - resolved stokes shifts . in these studies , all the modes of electric dipolar rearrangements from fs to ps can be captured . time constants of several ps have generally been assigned in these studies to reorientation of water molecules and slower time constants of tens of ps to slow / bound water or collective translational motion . time scales of hundreds of ps have also been reported for specific protein sites . by contrast , values of c of hundreds of ps determined using odnp reflect , like in previous nmr relaxometry studies , only the translational diffusive motion of water in equilibrium . time scales derived from different physical measurements are , therefore , best compared in terms of a relative change ( e.g. , retardation factor ) to assess slow and fast water dynamics on or near a biological surface of interest . here , we report on measurements , using both the eseem and odnp techniques , that indicate that the formation of the groes groel complex does not induce significant changes in the local water density , level of hydration , dynamics of surface water , or the dynamics of the spin label itself compared to those of free groes . interestingly , we find that the water dynamics at the groes surface are minimally retarded relative to bulk water , unlike the significantly slowed water dynamics observed in cases of hydrophilic lipid membrane surfaces or representative protein surfaces . this implies that the groes surface is not attracting water significantly and that the groes surface - water vs bulk water water interaction is balanced , so that the interaction of other biomolecular constituents ( e.g. , protein substrates ) with the groes surface is relatively unhindered . the gene coding for groes fused to a his6-tag at its c - terminus and containing the tyr71 cys mutation was generated using the plasmid poa and the quick - change site - directed mutagenesis kit ( stratagene , la jolla , ca ) . the his6-tag was introduced in two steps using the forward ( and corresponding back ) primers : his - tag 1,5-caaa ggagagttatcaatgcaccatcaccatcaccatttgattcgtccattgcatgatcg-3 ; his - tag 2,5-gcaccatcaccatcaccataatattcgtccattgcatgatcg-3. the tyr71 cys mutation was introduced using the forward primer : 5-cgttattttcaacgatggctgcggtgtgaaatctgagaagatcg -3 and the corresponding back primer . dna sequencing of the entire groes gene was carried out to verify that the desired construct was obtained . groes was purified by growing e. coli tg1 cells bearing the plasmid described above overnight at 37 c in 2xty medium containing 50 g / ml ampicillin . the overnight culture was diluted 1:100 in 2xty medium containing 50 g / ml ampicillin , grown overnight at 37 c and harvested . the pellet was resuspended in 50 mm tris - hcl buffer ( ph 7.5 ) containing 10% ( w / v ) sucrose , centrifuged , and stored at 80 c until further use . it was then resuspended in 50 mm tris - hcl buffer ( ph 7.5 ) containing 0.5 m nacl , 10 mm -mercaptoethanol , 10 mm imidazole ( buffer a ) , and 1 mm phenylmethanesulphonylfluoride . the cells were disrupted by sonication and the lysate was clarified by centrifugation at 20,000 rpm for 30 min at 4 c . the supernatant was loaded on a 5 ml histrap hp column ( amersham pharmacia , uppsala , sweden ) , and groes was eluted using a 10500 mm imidazole gradient in buffer a. fractions were analyzed by sds - page and those containing groes were combined and concentrated using a vivaspin device ( sartorius , goettingen , germany ) with a 10 kda cutoff filter . the concentrated protein was transferred into 50 mm tris - hcl buffer ( ph 7.5 ) containing 10 mm kcl and 10 mm mgcl2 ( g10k buffer ) using a pd-10 desalting column ( ge healthcare , uppsala , sweden ) and then concentrated again . aliquots of protein were snap frozen in liquid nitrogen and stored at 80 c . purification of sr1 , a single - ring version of groel , with the asp398 ala mutation was carried out as described previously . a 50-fold molar excess of the 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy ( 3-maleimido - proxyl ) spin probe ( sigma ) was added to the groes tyr71 cys mutant in d2o g10k buffer and the suspension was then shaken for 16 h at 37 c . under these conditions , excess spin label was separated from the labeled groes by using microspin g-25 buffer exchange columns ( ge healthcare , uppsala , sweden ) . the labeled groes was divided into aliquots , snap - frozen in liquid nitrogen and stored at 80 c . eseem and odnp experiments were not carried out using the more standard s-(2,2,5,5-tetramethyl-2,5-dihydro-1h - pyrrol-3-yl)methylmethanesulfonothioate ( mtsl ) label since groes in complex with groel loses this label over time for reasons that are not clear . the esr and eseem experiments were carried out using an sr1groes complex that was prepared by incubating 1 mm atp with 12 m sr1 for 30 s and then adding labeled groes ( all in d2o g10k buffer ) and incubating for an additional 5 min . the molar ratio between sr1 and labeled groes was 1.5:1 , respectively , in order to ensure that all the labeled groes is groel bound . the odnp experiments were carried out using 240 m labeled or unlabeled groes and a 1.5 molar excess of sr1 in g10k buffer containing 4 mm atp and 21% ( w / v ) ficoll 70 where indicated . all cw x - band ( 9.5 ghz ) measurements were performed at room temperature ( 2225 c ) on a bruker elexsys e500 spectrometer , using round quartz capillaries ( 0.75 mm i.d . and 1 mm o.d . ) . eseem experiments were carried out at 80 k on a bruker elexsys e580 spectrometer ( 9.5 ghz ) using an er4118x - ms-5 probe - head with a split ring resonator ( 5 mm sample access ) on 5060 l sample volumes . the eseem experiments were carried out using the three - pulse sequence /2--/2-t-/2--echo , with a repetition time of 2.5 ms and a four - step phase cycling , in the presence of a magnetic field set to maximum echo intensity . the -value was optimized to maximize the modulation depth of h , i.e. , = 1/(2d ) while minimizing the modulation depth of h , i.e. = 1/(h ) , yielding = 208 ns , where h / d is the h or h larmor frequency , respectively . the time interval t was incremented in 20 ns steps starting at 60 ns for a total number of 250 points . the eseem modulation was isolated from the signal trace and its fourier transform ( ft - eseem ) as follows : ( 1 ) phase correction ; ( 2 ) normalization ; ( 3 ) division with a fifth - order polynomial obtained from fitting the echo decay during the time interval t ; ( 4 ) subtraction of unity ; and ( 5 ) apodization with a hamming window , zero filling to 4096 points , ft and cross - term averaging . we chose the intensity of the h peak , i(h ) , in the ft - eseem as a characteristic of the h eseem pattern that reflects the modulation depth and , in turn , indicates the deuterium density around the spin label . samples of 3.1 l in a 0.6 mm i.d . and 0.84 mm o.d . quartz capillary tube were analyzed by odnp as described before , using an nmr probe of a pass - through design built to fit inside a 3 mm i.d . and 6 mm o.d . quartz tube , which can be inserted into a high sensitivity ( i.e. , high q ) cavity ( er 4119hs - lc , bruker biospin ) . the samples were sealed inside the capillary with a protective layer of critoseal , followed by hot beeswax , and all odnp measurements were performed at 2024 c . for these measurements , a microwave source and a homebuilt amplifier supplied up to 6 w at the esr frequency ( 9.8 ghz ) , and the field was set on resonance with the central esr hyperfine transition , which was progressively saturated . the presence of spin labels has two effects : ( i ) with or without microwave irradiation , the spin labels lead to a faster nmr relaxation rate , r1 ( figure 2a , b ) ; and ( ii ) in the presence of saturating microwaves , the esr transition will cross - relax with the nmr transition of the h nuclei of water ( at a rate given by kcsl as described below ) , thereby leading to an enhanced h nmr signal ( figure 2c ) . these two effects were quantified by carrying out nmr inversion recovery experiments ( figure 2a , b ) and a series of basic nmr free induction decay ( fid ) experiments ( figure 2c ) over a range of microwave powers . in both cases , the resulting nmr signals were fourier transformed , baseline corrected , and integrated ( pulse sequences in figure 2a c yield the respective data in figure 2d f ) . the integrated ft nmr signal from the fid experiments ( figure 2c ) was normalized against the signal in the absence of microwave power to illustrate the increasingly larger enhancements ( i.e. e(p ) ) obtained with increasing powers of saturating microwaves ( figure 2f ) . the inversion recovery data ( figure 2e ) reflects the rate of recovery of the nuclear magnetization from the inverted state to equilibrium ( i.e. r1(p ) ) . finally , a control measurement is performed on a sample prepared without spin label . this consists of an inversion recovery experiment in the absence of microwave power ( figure 2a ) , which reflects the rate of recovery of magnetization to equilibrium in the absence of spin label , r1,0 ( note that here r1 and r1,0 refer to the inverses of the nmr spin lattice relaxation times , i.e. r1 = t11 and r1,0 = t1,01 ) . outline of the complete procedure for odnp data processing is shown for representative data . first , a variety of nmr measurements is carried out including an inversion recovery sequence acquired on a sample without spin label ( a ) , a series of inversion recovery sequences acquired with spin label and different microwave powers ( b ) , and a simple nmr spectrum acquired at different powers of esr - resonant microwaves ( c ) . the data corresponding to these pulse sequences are shown in panels d f . the inversion recovery curves ( d , e ) are fitted to determine the nmr relaxation rates r1,0 ( g ) and r1(p ) ( h ) . the latter multiplies 1 e(p ) ( f ) to yield ks(p ) ( i ) , which are fitted to an asymptotic curve ( shown as a solid line ) , allowing us to extrapolate it to full saturation of the esr transition and determine k ksmax . the multiple curves in panels f , h and i are for repeated experiments as indicated by the color code in panel g. the data shown in figure 2d f were further processed to obtain the spin label - dependent relaxation rates , or relaxivities , that offer insight into the dynamics of the hydration water , as explained in more detail elsewhere . the inversion recovery curves ( e.g. , figure 2d , e ) are fitted to obtain the nmr relaxation rates of samples without the spin label ( r1,0 , figure 2 g ) and with the spin label ( r1(p = 0 ) from figure 2h ) . the self - relaxation rate , kcsl , is obtained by subtracting r1,0 from r1(p = 0 ) ( i.e. , r1 in the absence of microwave power ) . the spin - label - driven proton self - relaxivity , k , is then obtained from the self - relaxation rate by normalizing against the spin label concentration ( csl ) . the cross - relaxivity , k , is determined from the data in figure 2i , which are obtained by multiplying 1 e(p ) , the amount of polarization transferred ( figure 2f ) , by the microwave power - dependent relaxation rate r1(p ) ( figure 2h ) and dividing by 659.3 ( the ratio of the esr to nmr resonance frequencies ) and the concentration csl . these data are then fitted to an asymptotic curve to obtain a value for the cross relaxivity , k ksmax ( smax 1 , as shown previously ) where the value of ks(p ) approaches complete saturation of the esr transition at high microwave power . the ratio of the relaxivities k and k yields the coupling factor , ( = k/k ) . given a specific field ( and therefore resonance frequency ) , the force - free hard - sphere ( ffhs ) model for translational dynamics provides a relationship that can be used to determine the translational correlation time , c , from the measured value of . in order to better understand the contribution of partially bound waters ( which are not well modeled by ffhs ) to the value of , the contribution from the fast waters ( i.e. , k ) can also be subtracted from the self - relaxivities ( k ) as follows:1where klow describes the slower time scale ( 15 mhz ) fluctuations of the dipolar interaction . the value of is related to the ratio between k and klow:2where 0 k/klow 1 . each measurement was repeated 24 times , and the standard deviations of the resulting values of , k , klow , and c are presented as errors ( i.e. , as value error ) . the x - band esr spectrum of the spin labeled groes ( sl - groes ) in figure 3 shows that the mobility of the spin label at position 71 on the groes surface ( figure 1 ) is restricted compared to a free spin probe and represents a single population , thus providing evidence that the spin label is attached to the protein . an estimate of 10 s for the rotational correlation time can be obtained from comparison to spectra simulated using easyspin and assuming isotropic motion . notably , the esr spectrum shows only very subtle broadening upon formation of the complex between sl - groes and sr1 , thus indicating that the mobility of the spin label hardly changes when it is encapsulated within the cavity . cw esr spectra of the spin label attached to free groes ( black ) and groes in complex with sr1 ( red ) at room temperature in d2o . ( a ) time domain traces of three - pulse eseem measured at 80 k and the corresponding ( b ) fourier transforms for free ( black ) and sr1-bound spin - labeled groes ( red ) . for more details , see materials and methods . the peak at the h frequency with intensity i(h ) shows two components , where the broad resonance is due to water molecules h - bonded to the nitroxide moiety and the narrow component , i(h)narrow , is due to more distant water molecules . the time domain and ft - eseem traces for the sl - groes and sl - groes sr1 complex samples are identical , thus indicating that there is no difference between the density of water near the spin label of free sl - groes vs sl - groes in complex with sr1 . this implies that the number of water molecules and their distances from the spin label are the same in the two samples as reflected in the same i(h ) = 42 . for comparison , we also measured the i(h ) value for a free spin label dissolved in d2o / glycerol - d8 ( 7:3 v / v ) and obtained i(h ) = 80 . here , the addition of the glycerol was essential to prevent ice formation and aggregation of the spin probe upon freezing . a ratio of 0.5 is found between the i(h ) values for the sl - groes by itself or in complex with sr1 and the free spin label . assuming that glycerol - d8 does not affect significantly the h density in the sample ( as glycerol was not present in the protein samples ) and in the vicinity of the spin label , we can compare this value to the values of 0.54 and 0.18 that were obtained for the most exposed and buried mtsl - labeled sites , respectively , in the light harvesting protein complex iib of photosystem ii . this is consistent with the spin label attached to groes being exposed to bulk or the cavity water . currently , there is no reliable theoretical model for extracting the actual water distribution in the vicinity of the spin probe , in the case of d2o solutions , from fitting the experimental data . consequently , the data are often fitted to a model based on assuming a spherical distribution of nh nuclei around the spin label at an effective distance r. we chose not to use such a model as it is not realistic and preferred , instead , to interpret the experimental i(h ) values on a comparative basis . representative r1 and r10 data , as well as all the original h nmr signal enhancement measurements as a function of microwave power , e(p ) , are shown in figure 2 . these data were collected for three samples : free groes in g10k buffer , groes in complex with sr1 , and groes in a ficoll 70 solution . from these data , k ksmax values were extracted , as well as the klow values using eq 1 , the coupling factor , , and the translational diffusion correlation time , c ( see table 1 ) . the ratios between the k,klow , , and c values for the spin label tethered to groes and the free spin label in bulk solution are presented in table 1 and figure 5 . it can be seen that the values of these ratios are the same , within error , for sl - groes and the sl - groes therefore , we will first discuss the meaning of the resulting average values and the fact that the value of k remains completely unaltered the key result presented here . the meaning of very small changes in klow / klow , bulk that impact the value of and c ( eq 2 ) will be discussed below . interestingly , the value of klow / klow , bulk that represents the contribution from slow time scale fluctuations is approximately 1 , thereby indicating that it is likely that there is no bound water at the sl - groes surface . this , by itself , is an interesting result as it is typical to find some contribution from bound water near protein surfaces , unlike at the surfaces of lipid membranes that are known to have minimal or no contribution from bound water . all the k/k,bulk values are 0.4 0.07 and , thus , reflect modest retardation and comparatively fast diffusive motion of the surface water hydrating the sl - groes surface . these data clearly illustrate that the decrease in the values relative to those of bulk water and the retardation of surface water dynamics as reflected in c/c , bulk originate exclusively from changes in the contribution of fast moving , loosely bound , surface water , as reflected in k. moreover , the calculated value of 23 for the retardation factor , c/c , bulk , is exceptionally small compared to typical retardation factors of 510 or larger , as found for solvent - exposed protein surfaces of tau , apomyoglobin and other biomolecular or polymer surfaces ( figure 6 ) . all of these trends point to a highly lubricated , weakly hydrated , protein surface of sl - groes . this weak hydration does not change , within error , upon complexation with sr1 . to further test this conclusion , the measurements of water dynamics were repeated for sl - groes in the presence of 21% ( w / v ) ficoll 70 , a known viscogen that does not interact with the protein surface but slows the overall protein tumbling time by increasing the bulk water viscosity by about 10-fold at 21% ( w / v ) concentration . interestingly , the values for klow / klow , bulk , , /bulk , and c/c , bulk are all , within error , unaltered , suggesting that there is no bound water population whose effect is masked due to fast protein tumbling in the absence of ficoll 70 . the contribution from fast moving water , as reflected in k/k,bulk , also remains unaltered and , in keeping with previous observations on lipid surfaces , remains unaffected by the increase in the bulk solvent viscosity induced by ficoll 70 , thus confirming that this polymeric viscogen does not interact with the groes surface . bar plot of the values of the various odnp measurements for free groes in aqueous buffer , groes in complex with sr1 and groes in the presence of ficoll 70 . shown are values of the cross - relaxivity , k ( blue ) , the slow - motion component of the self - relaxivity , klow = 5/3k 7/3k ( green ) , the coupling factor , ( red ) , and the translational correlation time , c ( cyan ) , which is determined by applying the ffhs model . for simplicity , all quantities are normalized by the appropriate bulk values : k,bulk = 95.4 s m , klow , bulk = 366 s m , bulk = 0.27 , k = 353 s m , and c , bulk = 54 ps . for derivation of the relaxivity values , see the text and figure 2 . plot of the coupling factor measurement , , as a function of the modeled translational correlation time , c . the data points for the odnp measurements for free groes , groes in complex with sr1 , and groes in the presence of ficoll 70 are in brown , red , and green , respectively . the ffhs model gives a fixed relationship , (c ) , for measurements at 0.35 t ( corresponding to 15 mhz nuclear larmor frequency ) that is illustrated by the solid gray line . the gray symbols indicate previous odnp measurements for a variety of proteins , small peptides , lipids , and dna that are grouped ( in brown text , to the right ) according to the location of the spin label . as explained previously , measurements in the zone designated buried were for labels attached within the core of a lipid bilayer , globular protein , or compact polymer system ; in the zone designated surface for labels attached to the surfaces of proteins or other polymer ; in the intermediate zone for labels attached near but not at the surface of , for example , a lipid bilayer ; and in the bulk zone for small molecule nitroxides freely dispersed in water or certain highly charged polymers such as dna . interestingly , the value of klow / klow , bulk for the groes / sr1 complex is found to be somewhat higher ( and may exceed the error of measurement ) than the corresponding values for groes with or without ficoll 70 ( figure 5 ) . the increase in klow leads to a slightly larger apparent retardation factor , thereby indicating slower hydration dynamics ( see c/c , bulk in figure 5 ) . to understand the subtle meaning of these changes , we recall that odnp is sensitive to fluctuations in the spin spin dipolar interaction between water and the spin label that is attached to the surface of groes . the value of k samples fluctuations with time constants of tens of picoseconds and faster ( i.e. , 10 ghz fluctuations ) . fluctuations on this time scale are typically associated with water molecules freely diffusing past a spin label . therefore , the change in klow observed here does not reflect a change in the dynamics of freely translationally diffusing hydration water since such a change would also alter the value of k. rather , a selective increase in klow , as observed here , indicates an increase in slower fluctuations , with time constants as low as 10 ns ( i.e. , 15 mhz fluctuations ) . fluctuations on this time scale can arise either when , for example , water molecules near the spin label bind partially ( for ns or tens of ns ) to the surface of groes as it tumbles in solution or when water molecules chemically exchange with labile protons on the protein surface near the spin label . thus , it is possible that groes / sr1 either might trap a limited number of partially bound water molecules or may engage the water in chemical exchange . because the value of klow is the same ( within error ) for groes with or without ficoll 70 , this limited population of bound or exchanging waters would only be present in the chaperone complex and not on the surface of free groes . however , most importantly , because the change in klow is small ( 2-fold at most ) , we can assume that these changes indicate the presence of relatively few bound or exchanging water molecules . even these small changes do not arise from changes in the freely translating water inside the nanocavity , as indicated by the consistent k value . we conclude that the sl - groes surface is very weakly hydrated with highly mobile surface water , with no contribution of surface bound water , thus representing an unusual protein surface . there are indications that , upon formation of the sl - groes sr1 complex , a very select and small number of water molecules either bind partially to the cavity surface or engage in chemical exchange with it . however , it is clear that the majority of the water molecules continue to exhibit the same unusually high mobility and weak hydration even when confined inside the sl - groes sr1 cavity . this implies that the repulsive hydration barrier for a substrate to approach the groes surface is very small and that the substrate experiences a bulk water - like environment , even upon confinement within the cavity of the sl - groes a previous computational study suggested that the folding potential of proteins within the chaperonin cavity is enhanced owing to the hydrophilicity of the cavity inner surface , as measured by the density of surface water . when employing odnp methods , a high hydrophilicity of a protein surface would be reflected in retarded surface water diffusivity because of the attraction of water to the protein surface . however , we observe rather unusually fast dynamics of water on the cavity - facing surface of groes , both when it is free and when it is in complex with groel . odnp - nmr does yield very slightly different results for the groes / sr1 complex due to the presence of a small number of bound water molecules or labile protons on the inner surface of the cavity but does not yield results suggesting an overall slowing of the hydration water . the fast dynamics seen here have been seen for the surfaces of unstructured polymers but have not been observed before in cases of proteins and lipid membranes ( see figure 6 ) . these unexpectedly fast diffusion dynamics of the surface hydration water implies a low repulsive barrier for the substrate to approach ( and leave ) the groes surface as well as a low folding potential for the substrate near the groes surface . this suggestion that the groes lid confers a low protein folding potential is in agreement with the finding that replacing tyr71 in groes with charged residues enhances the groel - assisted folding of gfp . our observation that the cavity - facing surface of the groes lid has a low folding potential is also in agreement with the report that nonfolded substrate proteins can approach the lid and escape from the cage . in this study , the properties of the chaperonin cavity - confined water were studied using eseem and odnp by attaching a spin label to a cysteine in the tyr71 cys groes mutant . this residue is fully exposed to bulk water in free groes and can probe the confined water in the upper region of the cavity in the groel groes complex . previous work has shown that replacement of tyr71 in groes with positively or negatively charged residues enhances groe - assisted gfp folding , thereby indicating that the position we labeled senses a region of the cavity that is of functional importance . our main findings are that both the density and the dynamics of the water in the vicinity of the spin label are the same in free and sr1-bound groes , and that the properties of the cavity - confined water are similar to those of bulk water . these findings are consistent with the claim that the folding process inside the groel cage is similar to that in bulk solution , i.e. , that the groel cavity is a passive cage in which folding is not accelerated and may even be slowed down . it should be borne in mind , however , that the dynamics of the surface water closer to the bottom of the groel cavity may be vastly different ( e.g. , slower ) than those of water at the top . future studies need to be designed for probing the properties of water at the bottom of the cage and in the presence of nonfolded substrates .

atp - dependent binding of the chaperonin groel to its cofactor groes forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution . it has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement , interactions with the cavity walls , and differences between the properties of cavity - confined and bulk water . however , experimental data regarding the cavity - confined water are lacking . here , we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the tyr71 cys groes mutant obtained using two magnetic resonance techniques : electron - spin echo envelope modulation and overhauser dynamic nuclear polarization . residue 71 in groes is fully exposed to bulk water in free groes and to confined water within the cavity of the groel groes complex . our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation , thus indicating that bulk water - exposed and cavity - confined groes surface water share similar properties . interestingly , the diffusion dynamics of water near the groes surface are found to be unusually fast relative to other protein surfaces studied . the implications of these findings for chaperonin - assisted folding mechanisms are discussed .

Introduction Materials and Methods Results and Discussion Conclusions

the escherichia coli groe chaperonin system facilitates protein folding in vivo and in vitro in an atp - dependent manner ( for reviews see , for example , refs ( 13 ) ) . obligate substrates or other non - native proteins can become encapsulated in the groel cavity when groes binds to the apical domains of a substrate- and atp - occupied groel ring . the substrates are then discharged into bulk solution , either folded or not , following groes dissociation that is triggered by atp hydrolysis in the groes - bound cis groel ring and atp binding to the opposite trans groel ring ( see refs ( 2 and 7 ) for detailed schemes of current models of the groe reaction cycle ) . however , the role of the inter - ring allostery is less clear when the symmetric football - shaped groel groes2 complex ( and not the asymmetric groel groes complex ) is the active species of this nanomachine . despite more than two decades of intensive research , it remains unclear and controversial whether the cavity of the groel groes complex is only a passive cage in which aggregation is prevented but the folding pathway is unchanged or a chamber that has evolved to optimize the folding process itself . factors that could influence the folding reaction inside the groel cavity are steric confinement , the chemical nature of the cavity walls and the properties of the cavity - confined water , which may , in fact , be intimately linked to the steric and/or chemical effects of the confinement imposed by the groel interior surface . the extent of steric confinement and the chemical nature of the cavity walls are known from the crystal structure of the groel groes complex , but there is no available experimental data regarding the properties of the cavity water . specifically , insight into the diffusion dynamics of water within the groel cavity can offer critical clues about the groel surface water attraction and may allow us to hypothesize about the stability and folding potential of proteins entering the groel cavity . if water is interacting favorably with the interior surface of the groel cavity , as would be reflected in strongly retarded , rigidified , surface water dynamics , then a protein substrate that is encapsulated in the cavity will experience a strongly repulsive hydration barrier from the groel surface and , thus , tend to fold in order to bury its hydrophobic residues . by contrast , the hydrophobic nature of the cavity walls in groel s substrate acceptor state may be reflected in nonretarded , fast diffusing , surface water dynamics that disfavor substrate folding . equally interesting is the surface of the groes lid : is it strongly or weakly hydrated and do the hydration level and dynamics change upon formation of the groel the hydration properties of the groel cavity have been the focus of computational studies that indicated , for example , that groel s ability to assist folding scales with the affinity for water of the cavity s interior surface . however , direct experimental measurements of properties of confined water in the groel cavity have not yet been reported . in this study , we present the first such experimental measurements for water near the surface of free groes and the same surface when it faces the cavity of the groel groes complex . the experiments described here combine site - directed spin labeling ( sdsl ) with two state - of - the - art magnetic resonance techniques : electron - spin echo envelope modulation ( eseem ) and overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance ( odnp - nmr ) . this position was chosen since it is fully exposed to bulk water in unbound groes and , upon groel groes complex formation , faces the confined water inside the chaperonin cavity ( figure 1a , b ) . groel and groes in the crystal structure of the groel groes complex ( pdb code : 1aon ) are represented by space - filling ( in gray ) and ball - and - stick ( in magenta ) models , respectively . in the single - ring groel groes complex , the spin - labels are exposed to confined water in the cavity and are not close to any residues of groel . importantly , the spin label at this position is sufficiently far - removed from residues in the cavity wall , with the closest residue being asn299 , whose c side - chain atom is about 17 away from the nitroxide oxygen . eseem and odnp - nmr spectroscopy at x - band ( 10 ghz ) frequencies and a magnetic field of 0.35 t were employed to probe the properties of local water within the chaperonin cavity . in order to probe the amount of water in the vicinity of the spin label that protrudes into the cavity of the groes groel complex and can sense its upper region , the well - established eseem technique was employed for measuring hyperfine interactions between the electron spin of the label and nearby nuclear spins . when the hyperfine interaction is very weak , its isotropic part is zero and the anisotropic part can be described by the point dipole interaction between the electron spin and the nuclear spin , whose strength is inversely proportional to the cube of their distance , r. in such cases , this interaction is manifested as modulations in the electron spin echo decay that oscillate at a frequency equal to the larmor frequencies of the coupled nuclei , and the number of weakly coupled magnetic nuclei and their average distances from the electron spin are reflected in the modulation depth . by combining eseem of h nuclei in d2o solutions with spin labeling , it is possible to probe the number of d2o molecules in the vicinity of the spin - labeled residue cys71 ( up to about 8 ) without interferences from the protein protons . to get information regarding dynamics under solution conditions at room temperature , we applied odnp - nmr relaxometry to probe the diffusion dynamics of water near the spin - labeled cys71 . odnp selectively amplifies the h nmr signal of the local hydration water around a specific spin label ( within 510 ) of a protein site by transferring polarization from the electron spin to the nearby moving water molecules using the same anisotropic hyperfine interaction mentioned above ( alternatively termed the electron - nuclear dipole odnp relies on the enhancement of the h nmr signal of water at 0.35 t and 15 mhz that is achieved by saturating the electron spin resonance ( esr ) transitions at 10 ghz . since only the h of water molecules that move fast ( relative to 10 ghz ) experience electron - h spin flip - flops that give rise to h nmr signal enhancement , odnp can be exploited to quantify local water diffusivity near the nitroxide spin label . the motion of hydration water is characterized by a translational diffusion correlation time ( c ) , which represents the time needed for water to diffuse near the spin label within a distance b ( typically 510 , as determined by the electron - h dipolar coupling field ) and is inversely proportional to the local diffusion coefficient ( d ) , i.e. here , we report on measurements , using both the eseem and odnp techniques , that indicate that the formation of the groes groel complex does not induce significant changes in the local water density , level of hydration , dynamics of surface water , or the dynamics of the spin label itself compared to those of free groes . interestingly , we find that the water dynamics at the groes surface are minimally retarded relative to bulk water , unlike the significantly slowed water dynamics observed in cases of hydrophilic lipid membrane surfaces or representative protein surfaces . , protein substrates ) with the groes surface is relatively unhindered . the presence of spin labels has two effects : ( i ) with or without microwave irradiation , the spin labels lead to a faster nmr relaxation rate , r1 ( figure 2a , b ) ; and ( ii ) in the presence of saturating microwaves , the esr transition will cross - relax with the nmr transition of the h nuclei of water ( at a rate given by kcsl as described below ) , thereby leading to an enhanced h nmr signal ( figure 2c ) . the multiple curves in panels f , h and i are for repeated experiments as indicated by the color code in panel g. the data shown in figure 2d f were further processed to obtain the spin label - dependent relaxation rates , or relaxivities , that offer insight into the dynamics of the hydration water , as explained in more detail elsewhere . the cross - relaxivity , k , is determined from the data in figure 2i , which are obtained by multiplying 1 e(p ) , the amount of polarization transferred ( figure 2f ) , by the microwave power - dependent relaxation rate r1(p ) ( figure 2h ) and dividing by 659.3 ( the ratio of the esr to nmr resonance frequencies ) and the concentration csl . the x - band esr spectrum of the spin labeled groes ( sl - groes ) in figure 3 shows that the mobility of the spin label at position 71 on the groes surface ( figure 1 ) is restricted compared to a free spin probe and represents a single population , thus providing evidence that the spin label is attached to the protein . notably , the esr spectrum shows only very subtle broadening upon formation of the complex between sl - groes and sr1 , thus indicating that the mobility of the spin label hardly changes when it is encapsulated within the cavity . cw esr spectra of the spin label attached to free groes ( black ) and groes in complex with sr1 ( red ) at room temperature in d2o . the time domain and ft - eseem traces for the sl - groes and sl - groes sr1 complex samples are identical , thus indicating that there is no difference between the density of water near the spin label of free sl - groes vs sl - groes in complex with sr1 . assuming that glycerol - d8 does not affect significantly the h density in the sample ( as glycerol was not present in the protein samples ) and in the vicinity of the spin label , we can compare this value to the values of 0.54 and 0.18 that were obtained for the most exposed and buried mtsl - labeled sites , respectively , in the light harvesting protein complex iib of photosystem ii . this is consistent with the spin label attached to groes being exposed to bulk or the cavity water . currently , there is no reliable theoretical model for extracting the actual water distribution in the vicinity of the spin probe , in the case of d2o solutions , from fitting the experimental data . consequently , the data are often fitted to a model based on assuming a spherical distribution of nh nuclei around the spin label at an effective distance r. we chose not to use such a model as it is not realistic and preferred , instead , to interpret the experimental i(h ) values on a comparative basis . the ratios between the k,klow , , and c values for the spin label tethered to groes and the free spin label in bulk solution are presented in table 1 and figure 5 . it can be seen that the values of these ratios are the same , within error , for sl - groes and the sl - groes therefore , we will first discuss the meaning of the resulting average values and the fact that the value of k remains completely unaltered the key result presented here . interestingly , the value of klow / klow , bulk that represents the contribution from slow time scale fluctuations is approximately 1 , thereby indicating that it is likely that there is no bound water at the sl - groes surface . all the k/k,bulk values are 0.4 0.07 and , thus , reflect modest retardation and comparatively fast diffusive motion of the surface water hydrating the sl - groes surface . these data clearly illustrate that the decrease in the values relative to those of bulk water and the retardation of surface water dynamics as reflected in c/c , bulk originate exclusively from changes in the contribution of fast moving , loosely bound , surface water , as reflected in k. moreover , the calculated value of 23 for the retardation factor , c/c , bulk , is exceptionally small compared to typical retardation factors of 510 or larger , as found for solvent - exposed protein surfaces of tau , apomyoglobin and other biomolecular or polymer surfaces ( figure 6 ) . to further test this conclusion , the measurements of water dynamics were repeated for sl - groes in the presence of 21% ( w / v ) ficoll 70 , a known viscogen that does not interact with the protein surface but slows the overall protein tumbling time by increasing the bulk water viscosity by about 10-fold at 21% ( w / v ) concentration . interestingly , the values for klow / klow , bulk , , /bulk , and c/c , bulk are all , within error , unaltered , suggesting that there is no bound water population whose effect is masked due to fast protein tumbling in the absence of ficoll 70 . the contribution from fast moving water , as reflected in k/k,bulk , also remains unaltered and , in keeping with previous observations on lipid surfaces , remains unaffected by the increase in the bulk solvent viscosity induced by ficoll 70 , thus confirming that this polymeric viscogen does not interact with the groes surface . as explained previously , measurements in the zone designated buried were for labels attached within the core of a lipid bilayer , globular protein , or compact polymer system ; in the zone designated surface for labels attached to the surfaces of proteins or other polymer ; in the intermediate zone for labels attached near but not at the surface of , for example , a lipid bilayer ; and in the bulk zone for small molecule nitroxides freely dispersed in water or certain highly charged polymers such as dna . interestingly , the value of klow / klow , bulk for the groes / sr1 complex is found to be somewhat higher ( and may exceed the error of measurement ) than the corresponding values for groes with or without ficoll 70 ( figure 5 ) . to understand the subtle meaning of these changes , we recall that odnp is sensitive to fluctuations in the spin spin dipolar interaction between water and the spin label that is attached to the surface of groes . therefore , the change in klow observed here does not reflect a change in the dynamics of freely translationally diffusing hydration water since such a change would also alter the value of k. rather , a selective increase in klow , as observed here , indicates an increase in slower fluctuations , with time constants as low as 10 ns ( i.e. this implies that the repulsive hydration barrier for a substrate to approach the groes surface is very small and that the substrate experiences a bulk water - like environment , even upon confinement within the cavity of the sl - groes a previous computational study suggested that the folding potential of proteins within the chaperonin cavity is enhanced owing to the hydrophilicity of the cavity inner surface , as measured by the density of surface water . when employing odnp methods , a high hydrophilicity of a protein surface would be reflected in retarded surface water diffusivity because of the attraction of water to the protein surface . however , we observe rather unusually fast dynamics of water on the cavity - facing surface of groes , both when it is free and when it is in complex with groel . odnp - nmr does yield very slightly different results for the groes / sr1 complex due to the presence of a small number of bound water molecules or labile protons on the inner surface of the cavity but does not yield results suggesting an overall slowing of the hydration water . these unexpectedly fast diffusion dynamics of the surface hydration water implies a low repulsive barrier for the substrate to approach ( and leave ) the groes surface as well as a low folding potential for the substrate near the groes surface . this suggestion that the groes lid confers a low protein folding potential is in agreement with the finding that replacing tyr71 in groes with charged residues enhances the groel - assisted folding of gfp . our observation that the cavity - facing surface of the groes lid has a low folding potential is also in agreement with the report that nonfolded substrate proteins can approach the lid and escape from the cage . in this study , the properties of the chaperonin cavity - confined water were studied using eseem and odnp by attaching a spin label to a cysteine in the tyr71 cys groes mutant . this residue is fully exposed to bulk water in free groes and can probe the confined water in the upper region of the cavity in the groel groes complex . previous work has shown that replacement of tyr71 in groes with positively or negatively charged residues enhances groe - assisted gfp folding , thereby indicating that the position we labeled senses a region of the cavity that is of functional importance . our main findings are that both the density and the dynamics of the water in the vicinity of the spin label are the same in free and sr1-bound groes , and that the properties of the cavity - confined water are similar to those of bulk water . these findings are consistent with the claim that the folding process inside the groel cage is similar to that in bulk solution , i.e. it should be borne in mind , however , that the dynamics of the surface water closer to the bottom of the groel cavity may be vastly different ( e.g. future studies need to be designed for probing the properties of water at the bottom of the cage and in the presence of nonfolded substrates .

collaborative synergies in science , engineering and pharmaceutical research have led to the development of medical technologies , drugs and rehabilitation protocols to diagnose , treat and manage critical illnesses such as cancer , heart diseases and neurological disorders improving healthcare and life expectancy . though successful , these advances are delivered mainly in managed clinical environments such as physician offices and hospitals for diagnosis , treatment and rehabilitation . the traditional research and technology development synergies have been somewhat limited by a lack of cross - disciplinary interaction among the academic and federal research , industry , regulatory , and clinical communities . furthermore , these traditional approaches not only have prolonged the time from research and innovation to clinical translation and acceptance , they have also significantly contributed to overall higher healthcare costs . the global challenge of providing quality healthcare with preventive , personalized and precision medicine at affordable cost calls for a collaborative paradigm with active partnership among all stakeholder groups including researchers and technology developers , healthcare providers , patients , regulatory sectors , and payors in treating patients effectively and helping them stay healthy . it is critical to further develop this paradigm with advanced technological and communication protocols that connect patients with clinicians and provide seamless healthcare from point - of - care to clinics , hospitals and intensive care units . most biomedical research is constrained within academic , federal or industrial sectors where clinical needs and challenges are identified by researchers without prominent participation from healthcare providers , and data from patient responses on specific clinical protocols and procedures . even though major advances have recently been made in the development of devices for environment and lifestyle sensing , cancer diagnosis and monitoring , less progress has been made in the detection of infectious and non - communicable diseases , and other applications . a collaborative global platform involving researchers with active participation from clinicians and industry in a data - rich environment with patient - centered information databases could develop the much - needed synergy and foundation for development and implementation of future poc technologies in diverse clinical or semi - clinical settings for quality global healthcare . poc devices have the potential to transform medicine by making laboratory tests simpler , faster , more affordable , and portable , even in regions with little laboratory infrastructure . such devices can deliver large amounts of continuous or real - time data to researchers , clinics , and public health agencies . with these advances come enormous challenges associated with standardization and data integration . an initiative of bringing stakeholder groups to discuss global healthcare challenges and future trends in biomedical poc technologies for monitoring , diagnosis and therapeutic interventions was launched in partnership of engineering in medicine and biology society ( embs ) of the institute of electrical and electronics engineers ( ieee ) and national institutes of health ( nih ) with the first special topic international conference on point of - care healthcare technologies in 2013 in bangalore , india . this initiative was followed by second special topic conference on healthcare innovations and point - of - care technologies held in 2014 the nih - ieee strategic conference on healthcare innovations and point - of - care technologies for precision medicine ( hi - poct ) held at the niaid conference center on november 9 - 10 , 2015 brought representatives from all stakeholder groups to discuss critical issues in developing and implementing point - of - care technologies . to address the grand challenge of providing preventive , personalized and precision medicine based quality global healthcare at affordable cost , specifically with poc technologies , we started an initiative of bringing stakeholder groups in partnership of engineering in medicine and biology society ( embs ) of the institute of electrical and electronics engineers ( ieee ) and national institutes of health ( nih ) . the first ieee international conference on point - of - care healthcare technologies was held in bangalore , india on january 16 - 18 , 2013 . the conference provided an international forum with clinicians , healthcare providers , industry experts , innovators , researchers , and students to discuss clinical needs and technology solutions toward commercialization and translation to clinical applications across different environments and infrastructures . the conference , with sponsorships and participation from nih , ibm , phillips , kyoto hospital , apollo hospital , and other healthcare agencies also focused on challenges in developing and using point - of - care technologies for quality global healthcare . this meeting was then followed by the ieee conference on healthcare innovations and poc technologies held in seattle on october 8 - 10 , 2014 that further emphasized collaborative opportunities and resources for applications of poc technologies in developing and developed economies to address regional priorities for clinical needs with technology innovations in medical devices , translational engineering , information and communication technologies , infrastructure support , and patient and clinician acceptance of poc healthcare technologies . it was emphasized that poc developers focus on regional needs towards the overall goal of global healthcare since the environment and social behavior vary according to a region s economic growth , gross domestic product as well as geographical location . these regional need priorities include pre - natal monitoring , hypertension , infectious and chronic disease management and monitoring , therapeutic intervention in treatment of critical diseases such as cancer , rehabilitation , and medical information system to allow remote and evidence based medicine . discussion in previous conferences led to the development of a patient - centered data rich environment with technology innovation , information processing and communication protocols towards acceptances from patients , clinicians and payors . figure 1 shows that data privacy , security and integrity must be integrated with poc technologies to be effective and accepted in the clinical environment . the precision medicine initiative ( pmi ) , announced during the january 2015 state of the union address , is designed to enable a new era of medicine through research , technology , and policies that empower patients , researchers , and providers to work together toward development of individualized treatments . in keeping with these goals , the nih - ieee 2015 strategic conference on healthcare innovations and point - of - care technologies for precision medicine held on november 9 - 10 , 2015 at the national institute of allergy and infectious disease ( niaid ) conference center , bethesda , brought together patients , clinicians , engineers , industry leaders , and representatives of government to discuss clinical needs for expeditious , continuous , and/or real - time physiological and lifestyle data and emerging technologies to enable the delivery of accurate and affordable personalized medicine . the nih - ieee 2015 strategic conference on healthcare innovations and poc technologies for precision medicine was planned and organized by representatives from the ieee engineering in medicine & biology society ( embs ) , national institute of biomedical imaging and bioengineering ( nibib ) , the bill & melinda gates foundation ( bmgf ) , the national cancer institute ( nci ) , the niaid , the national heart , lung , and blood institute ( nhlbi ) , the point of care technologies research network ( poctrn ) , cvs caremark , path , and arizona state university . the conference was attended by more than 218 representatives from academic , federal , industry , clinical , fda and entrepreneurial communities . the distribution of stakeholders is shown in table 1.table 1percentage of conference attendees representing major stakeholder groups.stakeholder groupattendance percentageacademic researchers and students29%federal agencies and research labs18%industry representatives27%clinical researchers and practitioners26% the conference program included invited keynote and panel presentations on clinical needs , enabling poc technologies for monitoring , regulatory issues for poc devices , business models for sustainable poc businesses and funding opportunities on the first day . four breakout sessions on the second day addressed strategic needs and challenges with the following topics : 1.poc technologies in resource - limited settings2.clinical use and acceptance of poc technologies3.patient education and acceptance of poc technologies4.comprehensive diagnostic evaluation and validation of poc devices poc technologies in resource - limited settings clinical use and acceptance of poc technologies patient education and acceptance of poc technologies comprehensive diagnostic evaluation and validation of poc devices the conference featured 81 oral and poster presentations on current poc technologies for a spectrum of clinical applications including diagnosis and monitoring of cancer , infectious diseases , neurological disorders , hypertension and cardiac arrhythmia . the conference concluded with a panel discussion that included reports from breakout sessions , future plans and specific recommendations . a detailed report of the conference discussion and presentations is available on online on the website url . the overall goal of this strategic conference was to encourage and support the next generation of scientists to pursue creative new advances for detecting , measuring , and analyzing a wide range of biological information including molecular , genomic , cellular , clinical , behavioral , physiological , and environmental parameters and to provide opportunities for stakeholders to explore collaborations and synergies to accelerate healthcare system development , validation , deployment , and adoption of point - of - care technologies ( poct ) for precision medicine to improve global healthcare at affordable cost . it is important to improve accessibility and quality of healthcare while reducing costs and , optimally , to develop technologies that are networked among patient informational and clinical management systems to enable precision medicine in various environments including low - resource settings . challenges that the poc field is beginning to address include early detection and prevention of diseases , anywhere access to healthcare , equity in healthcare outcomes , therapeutic interventions , and increased care for chronic diseases in an aging population . the concept of precision medicine is not new , but pocts will be very important for its future advancement . in 2004 a commentary laid out the potential use of genomics for a prospective cohort study of genes and environment in the united states however , the cost of sequencing at that time was prohibitive . since then , the cost of sequencing has decreased , and therefore its use has increased , both substantially . in 2009 , the health information technology for economic and clinical health ( hitech ) act directed the office of the national coordinator for health information technology ( onc ) to promote the adoption of electronic health records ( ehrs ) . in parallel , the poc field has seen a large increase in the uptake of wearable sensors and mobile technologies . for example , a short - term goal of the precision medicine initiative ( pmi ) may be to apply personalized strategies to cancer treatments , but the longer - term goal is to apply the same principles to all areas of future medicine . the pmi will support research to create new approaches to detect , measure , and analyze a wide variety of molecular , genomic , cellular , physiological , behavioral , and environmental variables , test these approaches in small pilot studies , and utilize the most promising approaches in larger numbers of people over longer periods of time . the plenary sessions focused on the importance of collecting more data and making it available for biomedical research to allow longitudinal studies for healthcare and preventive medicine . it is important to include social and health risk behavior parameters such physical activity , smoking , sun exposure , environmental exposures , and dietary intake in computer models for risk assessment to determine the target screening - group for poc devices to collect healthcare related data . innovative poc devices to collect data may include the affinity dual hormone sensor , transdermal alcohol sensor , non - contact electrocardiogram sensor , edible adherence sensor , real - time food intake sensor , and continuous pulse oximetry but in each case the data must be intelligently integrated to provide feedback and to incentivize people to lead healthier lives . through special panel sessions , the conference focused on innovative poc technologies addressing clinical needs in treatment , monitoring and therapeutic intervention . researchers and clinicians from cancer centers presented clinical case studies of using poc technologies in detection and treatment critical diseases such as cancers . a physician and a cancer surviving patient discussed their experience as a living proof of recent progress in the treatment of chronic myelogenous leukemia ( cml ) . because of treatment advances over the past two decades , cml has become a survivable , chronic condition . geneexpert qpcr analyzer , one of the successful systems from cepheid ( http://www.cepheid.com/us/ ) , is a low cost device that can be used to diagnose cml and monitor patients receiving tyrosine kinase inhibitors . the genexpert devices are portable , user - friendly , cartridge - based devices for automated sample preparation and multiplex quantitative reverse transcription - pcr ( qrt - pcr ) . they can accommodate a large number of clinical sample types ( e.g. , blood , plasma , serum , urine , stool , formalin - fixed paraffin - embedded tissue , sputum , and vaginal and skin swabs ) and deliver test results within a few hours . the devices can perform 18 fda - approved tests and 23 tests that are available outside the united states . genexpert poct is in widespread use in developing countries : more than 9,000 systems have been deployed worldwide ; at least one device is present in all but 10 countries . the genexpert omni is an especially small and portable unit that can connect with cell phone towers for automated remote transmission to a data cloud and can accelerate clinical decision - making in the field . in another presentation from the researchers and technology developers at path medical center ( http://www.path.org/ ) , performance and effectiveness of several poc testing devices were discussed . laboratories in rural china reported 84 percent sensitivity and 87.5 percent specificity using care hpv , and field tests in india , uganda , and nicaragua reported similar results . several enabling technologies were presented that currently are in clinical / field evaluation of low - cost poc diagnosis for critical global challenges including optical devices for cervical cancer diagnosis , and acoustofluidics devices using acoustic waves to manipulate microfluidic flow for sputum analysis for the diagnosis and treatment of asthma , tuberculosis , lung cancer , cystic fibrosis , and chronic obstructive pulmonary disease . nih funded center for point - of - care tests for sexually transmitted diseases has been focusing on developing home - based urine and self - collected swab tests that are as sensitive as laboratory tests . currently , poc tests are being developed for chlamydia , gonorrhea , trichomonas , syphilis , herpes simplex virus ( hsv ) , and hiv . potential advantages include immediate treatment before patients leave the clinic ( with no loss to follow - up ) , decreased chance for the disease to spread , and potential for counseling . tests for trichomonas include the osomrapid tv antigen test ( immuno - chromatographic detection ) and the amplivueassay ( colorimetric detection of amplified dna on a lateral - flow strip ) . the syphilis healthcheck uses rapid immune - chromatography to detect human anti - treponemal antibodies . chembio diagnostic systems offers a dual path platform test that simultaneously tests for syphilis and hiv . rapid poc tests for hiv include the oraquick advance , uni - gold recombigen , alere determine , insti , clearviewcomplete , and clearviewstat pak . the nih and the viral hemorrhagic fever consortium ( vhfc ) have been interested in the development of new rapid poc diagnostic tests for lassa fever and ebola . such poc tests and devices can provide a cost - effective means to achieving a large public health benefit in resource - limited settings and can upload data to healthcare providers through smartphones , and information and communication technology ( ict ) . wearable sensors along with smartphones and ict can provide effective screening tests on high - risk patients at homes or nursing facilities . wearable sensors as a part of the poc monitoring devices produce large data sets with specific challenges on the accuracy , reliability , integrity and security of the data for meaningful analysis for clinical / healthcare use . in addition , for better effectiveness it is critical that there are interoperability standards to connect devices with ehr / emr following common data formats . as professional societies and industry stakeholders get involved with the development of standards for poc devices , they also need to work with regulatory agencies towards bringing validated devices in the market for reliable clinical / healthcare use . open ice , ( based on a published standard , american society for testing and materials ( astm ) f2761 ) provides a standards - based system architecture to allow for safety and performance and the required interoperability . booz allen hamilton is involved in the development of advanced data analytics and interface systems to convert data from wearable sensors and telemedicine data sources to perform clinical studies . however , integrity and authentication of data provided by the patients is a continued concern of mobile devices based data management . the data analysis at the poc information processing platforms such as smartphones is essential for managing large data sets to allow only meaningful data uploads to clinical / healthcare providers when needed . it is still an undefined challenge to determine the reliability and associated liabilities of poc diagnostics and transmission of the required amount of data to clinical / healthcare facilities or ehr / emr systems . though the top healthcare challenges and causes of death vary from low - income to middle and high income communities across developing and developed nations , there are common global healthcare challenges involving infection and communicable disease , and non - communicable diseases including ischemia and heart diseases , cancer , diabetes , strokes and neurological disorders . the trends in poc technology development are critically focused on molecular , cellular , microfluidic , acoustics based tests , and non - communicable disease screening technologies as well as biosensors based monitoring . the data collection and informatics for real - time data analysis are critical for timely poc diagnostics , alerts and preventive , personalized and prcised healthcare . thus , poc technologies may be used for screening and monitoring of high - risk patients with reliable diagnostics and data analytics for meaningful clinical use . the meaningful use of the accurate , reliable and secured data can improve the clinical management , treatment and therapeutic intervention , and help providing quality healthcare ( figure 2 ) . the poc technologies combined with ict can outreach patients for adherence and behavior change towards preventive medicine and staying healthy .. changing workflows to test and treat patients in a single visit was suggested to increase efficiency . 2.application and impact of poc technologies in personalized , preventive and precision ( ppp ) medicine . application and impact of poc technologies in personalized , preventive and precision ( ppp ) medicine . translational of poc technologies from research and innovation stage to clinical validation and commercialization should be adopted by various stakeholders including investors , industry , clinicians , patients and regulation agencies . healthcare systems should be scalable and standardized in terms of setup , process , and systems , creating challenges so that they can be connected to medical records and healthcare databases . system - level considerations to maximize adoption and sustainability in the clinical environment are data integration , financial modeling , workflow design , and clinical outcomes . data quality assurance , data integration , and data integrity solutions are hampered by the fact that the field is evolving at a rapid pace . glucose monitors , for example , are being provided by many vendors using a plethora of proprietary formats . fundamentally , for researchers to get more value out of available data , policies are needed to direct standardization . the future strategic directions may be focused on developing opportunities and platforms to facilitate co - invention of future poc technologies through active collaborations among all stakeholders . the intent is to help deliver quality global healthcare with emphasis on personalized and preventive medicine . specific tactics to build strategic alliances and partnerships may be focused on the following . 1.development a global healthcare collaborative network ( ghcn ) to develop databases of specific clinical needs to prioritize needs and define required specifications for potential technology and solutions and healthcare protocols.2.development an open forum for researchers and all stakeholders to address clinical needs with potential technological solutions and to discuss challenges of the required clinical response.3.development of protocols on data standards and interoperability requirements , including policies for data quality , integrity , consistency , connectivity , and data sharing for translation of enabling technologies and potential solutions in different environments including under - resourced , rural , and remote clinical facilities.4.development of protocols on integration of interoperable devices , device to device communication , and data exchange and integration with ehr / emr systems.5.development of training protocols and means at multiple levels to educate end users to adapt technological solutions for clinical applications . development a global healthcare collaborative network ( ghcn ) to develop databases of specific clinical needs to prioritize needs and define required specifications for potential technology and solutions and healthcare protocols . development an open forum for researchers and all stakeholders to address clinical needs with potential technological solutions and to discuss challenges of the required clinical response . development of protocols on data standards and interoperability requirements , including policies for data quality , integrity , consistency , connectivity , and data sharing for translation of enabling technologies and potential solutions in different environments including under - resourced , rural , and remote clinical facilities . development of protocols on integration of interoperable devices , device to device communication , and data exchange and integration with ehr / emr systems . development of training protocols and means at multiple levels to educate end users to adapt technological solutions for clinical applications . the jtehm special issue on healthcare innovations and poc technologies presents selected papers from the presentations held at the nih - ieee 2015 strategic conference on healthcare innovations and poc technologies for precision medicine and those submitted in response of the open call for papers . these papers are available through open access jtehm website http://health.embs.org/. national institute of biomedical imaging and bioengineering initiated a poctrn program to emphasize multidisciplinary partnerships and close facilitation translation of poc technologies from early stage of development into clinical testing and patient use . the paper , national institute of biomedical imaging and bioengineering point - of - care technology research network : advancing precision medicine by ford carleton , steven schachter , john a. parrish , john m. collins , ben crocker , ronald dixon , susan edgman - levitan , kent lewandrowski , james stahl , catherine klapperich , mario cabodi , charlotte a. gaydos , anne m. rompalo , yukari manabe , tza - huei wang , r rothman , chris d. geddes , lea widdice , joany jackman , rishi a. mathura , and tiffani bailey lash present a detailed introduction of the poctrn program and the three currently funded centers as examples of academic - based organizations that support collaborations across disciplines , institutions , and geographic regions to successfully drive innovative solutions from concept to patient care . these three centers have promoted the development and translation of various poc technologies to clinical validation and pre - commercialization . another nih initiative on global healthcare is described in the paper , the national institutes of health affordable cancer technologies program : improving access to resource - appropriate technologies for cancer detection , diagnosis , monitoring and treatment in low- and middle - income countries by paul c. pearlman , rao divi , michael gwede , pushpa tandon , brian s. sorg , miguel r. ossandon , lokesh agrawal , vinay pai , houston baker , and tiffani bailey lash . this paper specifically focuses on issues related to point - of - care ( poc ) technologies in cancer detection , diagnosis , monitoring , and treatment in the developed world for potential impact in the low - and - middle - income countries ( lmic ) . among several poc innovations , the paper explores infectious etiologies in many preventable and treatable cancers in lmics , such as cervical cancer , one of the most common causes of cancer death for women in many lmics . as mentioned above , it is critical for poc devices to be interoperable and easy to connect to data communication and analytics systems . they must be validated and celebrated with specific standards to provide accurate and reliable information for meaningful use in healthcare . sandy weininger , michael b. jaffe , michael robkin , tracy rausch , david arney , and julian m. goldman argue on this important challenge in their paper , the importance of state and context in safe interoperable medical systems describing the roles of devices with respect to interactions such as human user workflows and device to device communication in clinical environment . point - of - care technologies may also provide an effective solution on monitoring and management of patients with high risks of heart , lung , blood , and sleep ( hlbs ) disorders . hlbs disorders can be better diagnosed and treated with the monitoring of transient variables including physiological monitors and activity level . mary emma gorham bigelow , brian g. jamieson , chi on chui , yufei mao , kyeong - sik shin , tony jun huang , po - hsun huang , liqiang ren , bishow adhikari , jue chen , and erin iturriaga , describe three poc technologies in their paper , point - of - care technologies for the advancement of precision medicine in heart , lung , blood , and sleep disorders . the paper rightly points out the critical importance of hlbs in healthcare as cardiovascular disease ( cvd ) and strokes accounted for $ 195.6 billion in healthcare costs in united states in 2011 . cardiovascular disease ( cvd ) remains the leading cause of death in the united states , accounting for 1 in 3 deaths . semiconductor electronic label - free assay ( selfa ) technology is being developed and commercialized as a sample - to - answer poc diagnostic device by selfa , inc . diagnostic biochips has demonstrated the continuous monitoring of the chemotherapeutic agent doxorubicin in whole blood . the electrochemical sensors are embedded in a standard iv catheter , or the smartiv blood monitoring platform . such technologies can provide personalized delivery of therapies based on individual pharmacokinetics and pharmacodynamics . the acoustofluidic technology platform described in this paper is the beginning of use of poc devices in developing personalized treatment strategies to asthmatic patients in both research and community settings . these three technologies are now in translational pre - commercialization phase with a strong potential clinical impact . akilan palaniswami , shazia khan , sultan siebel erdem , and tayyaba hasan present a poc system to perform fluorescent microfluidic assay designed to rapidly determine antibiotic susceptibility for a range of bacterial pathogens that commonly occur in patients seen in clinical practice . in their paper , guiding empiric treatment for serious bacterial infections via point of care b - lactamase characterization , authors evaluates the potential application of their assay technology in the selection of the appropriate antibiotic for personalized and precision medicine reducing the societal threat of antibiotic resistance . the paper , elucidating the hemodynamic origin of ballistocardiographic forces : towards improved monitoring of cardiovascular health at home by abdul qadir javaid , hazar ashouri , srini tridandapani , and omer t. inan focuses on the mathematical modeling between the bcg signal and impedance cardiography ( icg ) , and arterial blood pressure ( abp ) waveforms , to monitor cardiac output and blood pressure asynchronously . this study can lead to the development of poc tools for monitoring heart failure ( hf ) patients at home following the discharge from the hospital , with the goal of potentially predicting exacerbations and thus reducing unnecessary re - hospitalizations . considering the continuously increasing trends in longevity and more older people living alone , the monitoring of their wellness at home is an important global issue . as authors , johanna austin , hiroko h. dodge , thomas riley , peter g. jacobs , stephen thielke , and jeffrey kaye explain in their paper , a smart - home system to unobtrusively and continuously assess loneliness in older adults , older adults often suffer from loneliness associated with increased morbidity and mortality , decreased sleep quality , and increased risk of cognitive decline . they present a system to measure loneliness by assessing in - home behavior using wireless motion and contact sensors , phone monitors , and computer software as well as algorithms developed to assess key behaviors of interest . this approach , if successful , can help in improving quality of life of older adults living alone at home . while the poc devices have to provide meaningful information for clinical or patient management , they have to be inexpensive , easy - to - use and interoperable with some form of calibration capabilities . the poc devices are often compared with more expensive and sophisticated devices used in the hospitals under trained healthcare providers . it is not clear what could be an acceptable accuracy , sensitivity and specificity for meaningful use in healthcare . matthew thompson , bernhard weigl , annette fitzpatrick , and nicole ide argue on the issues beyond accuracy in their paper , more than just accuracy : a novel method to incorporate multiple test attributes in evaluating diagnostic tests including point of care tests . they suggest a novel way to weigh different attributes to devise standards so that multiple stakeholders can use to visualize test attributes , their interactions , and impacts on individual and population outcomes as needed for their respective specific application . the poc technology development and translation to patient - centric environment for healthcare applications may lead to critical global solutions for better personalized , preventive and precision medicine . however , there is a wide spectrum of challenges including clinical acceptance , payor acceptance and patient adherence . these issues are closely correlated with the way poc technology may be used in patient or clinical management for diagnosis , treatment , therapeutic intervention , and/or rehabilitation . they also raise a challenging need of involving multiple stakeholders such as clinicians , industry , patients , and others in the early phases of research and development towards a successful technology translation in healthcare . it is not clear how clinicians , investors and other stakeholders can collaboratively participate in early phases of development of specifications of clinical needs and potential technology solutions across the globe as the regional needs and technology acceptances are often not the same in developing and developed economies among diverse income levels and regulatory infrastructures . a summary of panel discussions from the ieee - nih 2015 strategic conference on healthcare innovations and poc technologies for precision medicine has been presented here with comments on future trends and challenges . this special issue of selected papers demonstrates needs , significance , and translational research of some of the potential poc technologies . it is clear that we need to continue co - inventing the future of poc technology with multiple stakeholder groups towards the development of a global paradigm to address critical needs , and provide quality healthcare at affordable cost .

recent advances in biosensors , medical instrumentation , and information processing and communication technologies ( ict ) have enabled significant improvements in healthcare . however , these technologies have been mainly applied in clinical environments , such as hospitals and healthcare facilities , under managed care by well - trained and specialized individuals . the global challenge of providing quality healthcare at affordable cost leads to the proposed paradigm of p reventive , personalized , and precision medicine that requires a seamless use of technology and infrastructure support for patients and healthcare providers at point - of - care ( poc ) locations including homes , semi or pre - clinical facilities , and hospitals . the complexity of the global healthcare challenge necessitates strong collaborative interdisciplinary synergies involving all stakeholder groups including academia , federal research institutions , industry , regulatory agencies , and clinical communities . it is critical to evolve with collaborative efforts on the translation of research to technology development toward clinical validation and potential healthcare applications . this special issue is focused on technology innovation and translational research for poc applications with potential impact in improving global healthcare in the respective areas . some of these papers were presented at the nih - ieee strategic conference on healthcare innovations and poc technologies for precision medicine ( hi - poct ) held at the nih on november 910 , 2015 . the papers included in the special issue provide a spectrum of critical issues and collaborative resources on translational research of advanced poc devices and ict into global healthcare environment .

Introduction Background: POC for Global Quality Helathcare Challenge Procedures and Methods: Conference Program Overall Goal: Clinical Impact Enabling POC Technologies Future Trends and Challenges Special Issue Conclusion

though successful , these advances are delivered mainly in managed clinical environments such as physician offices and hospitals for diagnosis , treatment and rehabilitation . the traditional research and technology development synergies have been somewhat limited by a lack of cross - disciplinary interaction among the academic and federal research , industry , regulatory , and clinical communities . the global challenge of providing quality healthcare with preventive , personalized and precision medicine at affordable cost calls for a collaborative paradigm with active partnership among all stakeholder groups including researchers and technology developers , healthcare providers , patients , regulatory sectors , and payors in treating patients effectively and helping them stay healthy . it is critical to further develop this paradigm with advanced technological and communication protocols that connect patients with clinicians and provide seamless healthcare from point - of - care to clinics , hospitals and intensive care units . most biomedical research is constrained within academic , federal or industrial sectors where clinical needs and challenges are identified by researchers without prominent participation from healthcare providers , and data from patient responses on specific clinical protocols and procedures . even though major advances have recently been made in the development of devices for environment and lifestyle sensing , cancer diagnosis and monitoring , less progress has been made in the detection of infectious and non - communicable diseases , and other applications . a collaborative global platform involving researchers with active participation from clinicians and industry in a data - rich environment with patient - centered information databases could develop the much - needed synergy and foundation for development and implementation of future poc technologies in diverse clinical or semi - clinical settings for quality global healthcare . poc devices have the potential to transform medicine by making laboratory tests simpler , faster , more affordable , and portable , even in regions with little laboratory infrastructure . an initiative of bringing stakeholder groups to discuss global healthcare challenges and future trends in biomedical poc technologies for monitoring , diagnosis and therapeutic interventions was launched in partnership of engineering in medicine and biology society ( embs ) of the institute of electrical and electronics engineers ( ieee ) and national institutes of health ( nih ) with the first special topic international conference on point of - care healthcare technologies in 2013 in bangalore , india . this initiative was followed by second special topic conference on healthcare innovations and point - of - care technologies held in 2014 the nih - ieee strategic conference on healthcare innovations and point - of - care technologies for precision medicine ( hi - poct ) held at the niaid conference center on november 9 - 10 , 2015 brought representatives from all stakeholder groups to discuss critical issues in developing and implementing point - of - care technologies . to address the grand challenge of providing preventive , personalized and precision medicine based quality global healthcare at affordable cost , specifically with poc technologies , we started an initiative of bringing stakeholder groups in partnership of engineering in medicine and biology society ( embs ) of the institute of electrical and electronics engineers ( ieee ) and national institutes of health ( nih ) . the first ieee international conference on point - of - care healthcare technologies was held in bangalore , india on january 16 - 18 , 2013 . the conference provided an international forum with clinicians , healthcare providers , industry experts , innovators , researchers , and students to discuss clinical needs and technology solutions toward commercialization and translation to clinical applications across different environments and infrastructures . the conference , with sponsorships and participation from nih , ibm , phillips , kyoto hospital , apollo hospital , and other healthcare agencies also focused on challenges in developing and using point - of - care technologies for quality global healthcare . this meeting was then followed by the ieee conference on healthcare innovations and poc technologies held in seattle on october 8 - 10 , 2014 that further emphasized collaborative opportunities and resources for applications of poc technologies in developing and developed economies to address regional priorities for clinical needs with technology innovations in medical devices , translational engineering , information and communication technologies , infrastructure support , and patient and clinician acceptance of poc healthcare technologies . these regional need priorities include pre - natal monitoring , hypertension , infectious and chronic disease management and monitoring , therapeutic intervention in treatment of critical diseases such as cancer , rehabilitation , and medical information system to allow remote and evidence based medicine . discussion in previous conferences led to the development of a patient - centered data rich environment with technology innovation , information processing and communication protocols towards acceptances from patients , clinicians and payors . the precision medicine initiative ( pmi ) , announced during the january 2015 state of the union address , is designed to enable a new era of medicine through research , technology , and policies that empower patients , researchers , and providers to work together toward development of individualized treatments . in keeping with these goals , the nih - ieee 2015 strategic conference on healthcare innovations and point - of - care technologies for precision medicine held on november 9 - 10 , 2015 at the national institute of allergy and infectious disease ( niaid ) conference center , bethesda , brought together patients , clinicians , engineers , industry leaders , and representatives of government to discuss clinical needs for expeditious , continuous , and/or real - time physiological and lifestyle data and emerging technologies to enable the delivery of accurate and affordable personalized medicine . the nih - ieee 2015 strategic conference on healthcare innovations and poc technologies for precision medicine was planned and organized by representatives from the ieee engineering in medicine & biology society ( embs ) , national institute of biomedical imaging and bioengineering ( nibib ) , the bill & melinda gates foundation ( bmgf ) , the national cancer institute ( nci ) , the niaid , the national heart , lung , and blood institute ( nhlbi ) , the point of care technologies research network ( poctrn ) , cvs caremark , path , and arizona state university . the conference was attended by more than 218 representatives from academic , federal , industry , clinical , fda and entrepreneurial communities . the distribution of stakeholders is shown in table 1.table 1percentage of conference attendees representing major stakeholder groups.stakeholder groupattendance percentageacademic researchers and students29%federal agencies and research labs18%industry representatives27%clinical researchers and practitioners26% the conference program included invited keynote and panel presentations on clinical needs , enabling poc technologies for monitoring , regulatory issues for poc devices , business models for sustainable poc businesses and funding opportunities on the first day . four breakout sessions on the second day addressed strategic needs and challenges with the following topics : 1.poc technologies in resource - limited settings2.clinical use and acceptance of poc technologies3.patient education and acceptance of poc technologies4.comprehensive diagnostic evaluation and validation of poc devices poc technologies in resource - limited settings clinical use and acceptance of poc technologies patient education and acceptance of poc technologies comprehensive diagnostic evaluation and validation of poc devices the conference featured 81 oral and poster presentations on current poc technologies for a spectrum of clinical applications including diagnosis and monitoring of cancer , infectious diseases , neurological disorders , hypertension and cardiac arrhythmia . a detailed report of the conference discussion and presentations is available on online on the website url . the overall goal of this strategic conference was to encourage and support the next generation of scientists to pursue creative new advances for detecting , measuring , and analyzing a wide range of biological information including molecular , genomic , cellular , clinical , behavioral , physiological , and environmental parameters and to provide opportunities for stakeholders to explore collaborations and synergies to accelerate healthcare system development , validation , deployment , and adoption of point - of - care technologies ( poct ) for precision medicine to improve global healthcare at affordable cost . it is important to improve accessibility and quality of healthcare while reducing costs and , optimally , to develop technologies that are networked among patient informational and clinical management systems to enable precision medicine in various environments including low - resource settings . in 2004 a commentary laid out the potential use of genomics for a prospective cohort study of genes and environment in the united states however , the cost of sequencing at that time was prohibitive . for example , a short - term goal of the precision medicine initiative ( pmi ) may be to apply personalized strategies to cancer treatments , but the longer - term goal is to apply the same principles to all areas of future medicine . the pmi will support research to create new approaches to detect , measure , and analyze a wide variety of molecular , genomic , cellular , physiological , behavioral , and environmental variables , test these approaches in small pilot studies , and utilize the most promising approaches in larger numbers of people over longer periods of time . the plenary sessions focused on the importance of collecting more data and making it available for biomedical research to allow longitudinal studies for healthcare and preventive medicine . it is important to include social and health risk behavior parameters such physical activity , smoking , sun exposure , environmental exposures , and dietary intake in computer models for risk assessment to determine the target screening - group for poc devices to collect healthcare related data . innovative poc devices to collect data may include the affinity dual hormone sensor , transdermal alcohol sensor , non - contact electrocardiogram sensor , edible adherence sensor , real - time food intake sensor , and continuous pulse oximetry but in each case the data must be intelligently integrated to provide feedback and to incentivize people to lead healthier lives . researchers and clinicians from cancer centers presented clinical case studies of using poc technologies in detection and treatment critical diseases such as cancers . several enabling technologies were presented that currently are in clinical / field evaluation of low - cost poc diagnosis for critical global challenges including optical devices for cervical cancer diagnosis , and acoustofluidics devices using acoustic waves to manipulate microfluidic flow for sputum analysis for the diagnosis and treatment of asthma , tuberculosis , lung cancer , cystic fibrosis , and chronic obstructive pulmonary disease . nih funded center for point - of - care tests for sexually transmitted diseases has been focusing on developing home - based urine and self - collected swab tests that are as sensitive as laboratory tests . potential advantages include immediate treatment before patients leave the clinic ( with no loss to follow - up ) , decreased chance for the disease to spread , and potential for counseling . the nih and the viral hemorrhagic fever consortium ( vhfc ) have been interested in the development of new rapid poc diagnostic tests for lassa fever and ebola . such poc tests and devices can provide a cost - effective means to achieving a large public health benefit in resource - limited settings and can upload data to healthcare providers through smartphones , and information and communication technology ( ict ) . wearable sensors as a part of the poc monitoring devices produce large data sets with specific challenges on the accuracy , reliability , integrity and security of the data for meaningful analysis for clinical / healthcare use . in addition , for better effectiveness it is critical that there are interoperability standards to connect devices with ehr / emr following common data formats . as professional societies and industry stakeholders get involved with the development of standards for poc devices , they also need to work with regulatory agencies towards bringing validated devices in the market for reliable clinical / healthcare use . the data analysis at the poc information processing platforms such as smartphones is essential for managing large data sets to allow only meaningful data uploads to clinical / healthcare providers when needed . it is still an undefined challenge to determine the reliability and associated liabilities of poc diagnostics and transmission of the required amount of data to clinical / healthcare facilities or ehr / emr systems . though the top healthcare challenges and causes of death vary from low - income to middle and high income communities across developing and developed nations , there are common global healthcare challenges involving infection and communicable disease , and non - communicable diseases including ischemia and heart diseases , cancer , diabetes , strokes and neurological disorders . the trends in poc technology development are critically focused on molecular , cellular , microfluidic , acoustics based tests , and non - communicable disease screening technologies as well as biosensors based monitoring . the meaningful use of the accurate , reliable and secured data can improve the clinical management , treatment and therapeutic intervention , and help providing quality healthcare ( figure 2 ) . 2.application and impact of poc technologies in personalized , preventive and precision ( ppp ) medicine . application and impact of poc technologies in personalized , preventive and precision ( ppp ) medicine . translational of poc technologies from research and innovation stage to clinical validation and commercialization should be adopted by various stakeholders including investors , industry , clinicians , patients and regulation agencies . system - level considerations to maximize adoption and sustainability in the clinical environment are data integration , financial modeling , workflow design , and clinical outcomes . 1.development a global healthcare collaborative network ( ghcn ) to develop databases of specific clinical needs to prioritize needs and define required specifications for potential technology and solutions and healthcare protocols.2.development an open forum for researchers and all stakeholders to address clinical needs with potential technological solutions and to discuss challenges of the required clinical response.3.development of protocols on data standards and interoperability requirements , including policies for data quality , integrity , consistency , connectivity , and data sharing for translation of enabling technologies and potential solutions in different environments including under - resourced , rural , and remote clinical facilities.4.development of protocols on integration of interoperable devices , device to device communication , and data exchange and integration with ehr / emr systems.5.development of training protocols and means at multiple levels to educate end users to adapt technological solutions for clinical applications . development a global healthcare collaborative network ( ghcn ) to develop databases of specific clinical needs to prioritize needs and define required specifications for potential technology and solutions and healthcare protocols . development an open forum for researchers and all stakeholders to address clinical needs with potential technological solutions and to discuss challenges of the required clinical response . development of protocols on data standards and interoperability requirements , including policies for data quality , integrity , consistency , connectivity , and data sharing for translation of enabling technologies and potential solutions in different environments including under - resourced , rural , and remote clinical facilities . the jtehm special issue on healthcare innovations and poc technologies presents selected papers from the presentations held at the nih - ieee 2015 strategic conference on healthcare innovations and poc technologies for precision medicine and those submitted in response of the open call for papers . national institute of biomedical imaging and bioengineering initiated a poctrn program to emphasize multidisciplinary partnerships and close facilitation translation of poc technologies from early stage of development into clinical testing and patient use . the paper , national institute of biomedical imaging and bioengineering point - of - care technology research network : advancing precision medicine by ford carleton , steven schachter , john a. parrish , john m. collins , ben crocker , ronald dixon , susan edgman - levitan , kent lewandrowski , james stahl , catherine klapperich , mario cabodi , charlotte a. gaydos , anne m. rompalo , yukari manabe , tza - huei wang , r rothman , chris d. geddes , lea widdice , joany jackman , rishi a. mathura , and tiffani bailey lash present a detailed introduction of the poctrn program and the three currently funded centers as examples of academic - based organizations that support collaborations across disciplines , institutions , and geographic regions to successfully drive innovative solutions from concept to patient care . these three centers have promoted the development and translation of various poc technologies to clinical validation and pre - commercialization . another nih initiative on global healthcare is described in the paper , the national institutes of health affordable cancer technologies program : improving access to resource - appropriate technologies for cancer detection , diagnosis , monitoring and treatment in low- and middle - income countries by paul c. pearlman , rao divi , michael gwede , pushpa tandon , brian s. sorg , miguel r. ossandon , lokesh agrawal , vinay pai , houston baker , and tiffani bailey lash . this paper specifically focuses on issues related to point - of - care ( poc ) technologies in cancer detection , diagnosis , monitoring , and treatment in the developed world for potential impact in the low - and - middle - income countries ( lmic ) . among several poc innovations , the paper explores infectious etiologies in many preventable and treatable cancers in lmics , such as cervical cancer , one of the most common causes of cancer death for women in many lmics . as mentioned above , it is critical for poc devices to be interoperable and easy to connect to data communication and analytics systems . sandy weininger , michael b. jaffe , michael robkin , tracy rausch , david arney , and julian m. goldman argue on this important challenge in their paper , the importance of state and context in safe interoperable medical systems describing the roles of devices with respect to interactions such as human user workflows and device to device communication in clinical environment . point - of - care technologies may also provide an effective solution on monitoring and management of patients with high risks of heart , lung , blood , and sleep ( hlbs ) disorders . mary emma gorham bigelow , brian g. jamieson , chi on chui , yufei mao , kyeong - sik shin , tony jun huang , po - hsun huang , liqiang ren , bishow adhikari , jue chen , and erin iturriaga , describe three poc technologies in their paper , point - of - care technologies for the advancement of precision medicine in heart , lung , blood , and sleep disorders . in their paper , guiding empiric treatment for serious bacterial infections via point of care b - lactamase characterization , authors evaluates the potential application of their assay technology in the selection of the appropriate antibiotic for personalized and precision medicine reducing the societal threat of antibiotic resistance . the paper , elucidating the hemodynamic origin of ballistocardiographic forces : towards improved monitoring of cardiovascular health at home by abdul qadir javaid , hazar ashouri , srini tridandapani , and omer t. inan focuses on the mathematical modeling between the bcg signal and impedance cardiography ( icg ) , and arterial blood pressure ( abp ) waveforms , to monitor cardiac output and blood pressure asynchronously . the poc devices are often compared with more expensive and sophisticated devices used in the hospitals under trained healthcare providers . it is not clear what could be an acceptable accuracy , sensitivity and specificity for meaningful use in healthcare . matthew thompson , bernhard weigl , annette fitzpatrick , and nicole ide argue on the issues beyond accuracy in their paper , more than just accuracy : a novel method to incorporate multiple test attributes in evaluating diagnostic tests including point of care tests . the poc technology development and translation to patient - centric environment for healthcare applications may lead to critical global solutions for better personalized , preventive and precision medicine . however , there is a wide spectrum of challenges including clinical acceptance , payor acceptance and patient adherence . they also raise a challenging need of involving multiple stakeholders such as clinicians , industry , patients , and others in the early phases of research and development towards a successful technology translation in healthcare . it is not clear how clinicians , investors and other stakeholders can collaboratively participate in early phases of development of specifications of clinical needs and potential technology solutions across the globe as the regional needs and technology acceptances are often not the same in developing and developed economies among diverse income levels and regulatory infrastructures . a summary of panel discussions from the ieee - nih 2015 strategic conference on healthcare innovations and poc technologies for precision medicine has been presented here with comments on future trends and challenges . this special issue of selected papers demonstrates needs , significance , and translational research of some of the potential poc technologies . it is clear that we need to continue co - inventing the future of poc technology with multiple stakeholder groups towards the development of a global paradigm to address critical needs , and provide quality healthcare at affordable cost .

most intracranial aneurysms occur at arterial branching points ( bifurcations , side - branches , or perforators ) . simple covered stents are associated with the highest rate of complete aneurysm occlusion . however , their use in the human intracranial circulation is limited by access issues and the possibility of occluding small perforating end - arteries . it is important to assess the patency of normal arterial branches arising from the covered segments of the artery after implantation of simple covered stents . we established rabbit carotid aneurysms by intraluminal incubation of elastase within an endovascularly trapped segment of the proximal common carotid artery ( cca ) , and we implanted hybrid stents equipped with micropores in these aneurysms . besides studying the occlusive effect of the hybrid stents in the aneurysms , we examined the patency of the side - branches arising from the right subclavian artery ( sa ) , both angiographically and histologically . all experiments were performed on japanese white rabbits ( 3.54.2 kg ) and conducted in accordance with the principles of laboratory animal care ( formulated by the national society for medical research , chicago , il ) and the guide for the care and use of laboratory animals ( nih publication no . 12010 ) was approved by the ethics committee of the national cerebral and cardiovascular center research institute . the aneurysms were first created and then subjected to stent implantation two to four weeks later under general anesthesia , which was induced by the intramuscular injection of 0.2 ml / kg ketamine chloride ( 10% ) and 0.3 ml / kg xylazine , and additional dosage was determined on the basis of the animal s movements . a stainless mold ( diameter , 1 mm ) was dipped in a tetrahydrofuran solution and allowed to dry . subsequently , a balloon - expandable coronary - artery bare stent ( momo stent : diameter , 3 mm ; length , 20 mm ; japan stent technology , okayama , japan ) was mounted on the cover film of the mold , and again , it was dipped in the solution and dried . the thickness of the cover film was restricted to approximately 30 m . micropores were then made in the cover film using a krf excimer laser apparatus ( l4500 ; hamamatsu photonics , shizuoka , japan ) . micropores are circular , with a pore diameter and interpore distance of 100 m and 250 m , respectively , such that they achieve an opening ratio of 23.6% ( calculated ) after full expansion . the outer surface of the film of the microporous stent grafts was coated with argatroban ( 500 g / cm ) , which was applied using a methanol solution ( 1% w / v ) ; the solvent was subsequently volatilized for physical adsorption . the fabricated hybrid stent was then remounted on the delivery balloon system ( thrill slalom pta dilatation balloon catheter ; diameter , 3 mm ; length , 2 cm ; cordis , j & j , europa , n. v. , netherlands ) . aneurysms were constructed in 10 female rabbits by employing a previously described method , with a few simple modifications . the right cca was surgically isolated , ligated distally , and controlled proximally with 2.0 silk sutures before a 5-fr detachable sheath introducer ( medikit , tokyo , japan ) was induced and passed retrograde to the midportion of the cca ( approximately 3 cm cephalad to the origin of the cca ) . a hemostatic valve of the sheath ( medikit , tokyo , japan ) was detached , and a rotational and hemostatic triple connecter was connected to the sheath placed in the cca . through the sheath , an occlusive microballoon ( naviballoon ; kaneka , tokyo , japan ) ( titan percutaneous transluminal coronal angioplasty ( ptca ) dilatation balloon catheter : diameter , 4.0 mm ; length , 9 mm ; cordis , j & j , miami , florida , usa ) and subsequently a microcatheter ( excelsior sl10 ; striker , tokyo , japan ) were advanced near the cca origin ( figure 2(b ) ) . porcine elastase ( sigma , tokyo , japan ) was mixed with nonionic contrast medium to obtain a 25% dilution . elastase ( 6.8 units / mg/0.25 cc ) was injected and incubated in the isolated cca for 20 min , while the balloon was inflated at the origin of the right cca . the cca was ligated with a 2.0 silk thread after sheath removal , and the skin was closed by discontinuous knots with a similar thread . endovascular treatment was performed via the right femoral artery , at two to four weeks after the creation of the aneurysm . after pharmacological dilation of the artery with 8 mg of papaverine chloride , a 19-g puncture needle was used to introduce the 0.032 mandrel ; then , a 4-fr 10-cm sheath ( clinical supply , tokyo , japan ) was advanced into the right femoral artery under fluoroscopic guidance . the 4-fr catheter was navigated into the brachiocephalic artery ( bca ) with a 0.035 guidewire . digital subtraction angiography ( dsa ) showed the bca , sa , and aneurysm . a balloon catheter ( thrill slalom pta dilatation balloon catheter ; diameter , 3 mm ; length , 2 cm ; cordis , j & j , europa , n.v . , netherlands ) crimped with our hybrid stent was passed through the sheath and navigated into the bca . using fluoroscopy and road mapping , we advanced the hybrid stent over the balloon catheter across the aneurysmal neck and inflated the balloon . the femoral artery was ligated with two sutures of a 2.0 silk thread previously placed around the artery , and the skin was closed discontinuously with a similar thread . after the observation period , angiography was performed using a 4-fr sheath placed at the left cca in all animals . a 19-g puncture needle was used to introduce the 0.032 mandrel ; then , a 4-fr , 10-cm long sheath ( clinical supply , tokyo , japan ) was advanced to the left proximal cca under fluoroscopic guidance . occlusion of the aneurysms and patency of preoperatively detected side - branches of the artery was evaluated angiographically . thereafter , the animals were subjected to euthanasia by intravenous injection of potassium chloride . the bca , the right sa , vertebral artery ( va ) , some other arterial branches , and the treated aneurysm were resected en bloc and placed directly in a 4% paraformaldehyde phosphate - buffered saline solution for rapid fixation . some of the samples were evaluated histologically to determine the patency of the branches . histological examination was performed in some cases , by using a cross - sectional sample ( hematoxylin and eosin ( h&e ) stain ) obtained from the stented portion of the aneurysm to confirm the patency of the side - branches . a stainless mold ( diameter , 1 mm ) was dipped in a tetrahydrofuran solution and allowed to dry . subsequently , a balloon - expandable coronary - artery bare stent ( momo stent : diameter , 3 mm ; length , 20 mm ; japan stent technology , okayama , japan ) was mounted on the cover film of the mold , and again , it was dipped in the solution and dried . the thickness of the cover film was restricted to approximately 30 m . micropores were then made in the cover film using a krf excimer laser apparatus ( l4500 ; hamamatsu photonics , shizuoka , japan ) . micropores are circular , with a pore diameter and interpore distance of 100 m and 250 m , respectively , such that they achieve an opening ratio of 23.6% ( calculated ) after full expansion . the outer surface of the film of the microporous stent grafts was coated with argatroban ( 500 g / cm ) , which was applied using a methanol solution ( 1% w / v ) ; the solvent was subsequently volatilized for physical adsorption . the fabricated hybrid stent was then remounted on the delivery balloon system ( thrill slalom pta dilatation balloon catheter ; diameter , 3 mm ; length , 2 cm ; cordis , j & j , europa , n. v. , netherlands ) . aneurysms were constructed in 10 female rabbits by employing a previously described method , with a few simple modifications . the right cca was surgically isolated , ligated distally , and controlled proximally with 2.0 silk sutures before a 5-fr detachable sheath introducer ( medikit , tokyo , japan ) was induced and passed retrograde to the midportion of the cca ( approximately 3 cm cephalad to the origin of the cca ) . a hemostatic valve of the sheath ( medikit , tokyo , japan ) was detached , and a rotational and hemostatic triple connecter was connected to the sheath placed in the cca . through the sheath , an occlusive microballoon ( naviballoon ; kaneka , tokyo , japan ) ( titan percutaneous transluminal coronal angioplasty ( ptca ) dilatation balloon catheter : diameter , 4.0 mm ; length , 9 mm ; cordis , j & j , miami , florida , usa ) and subsequently a microcatheter ( excelsior sl10 ; striker , tokyo , japan ) were advanced near the cca origin ( figure 2(b ) ) . porcine elastase ( sigma , tokyo , japan ) was mixed with nonionic contrast medium to obtain a 25% dilution . elastase ( 6.8 units / mg/0.25 cc ) was injected and incubated in the isolated cca for 20 min , while the balloon was inflated at the origin of the right cca . the cca was ligated with a 2.0 silk thread after sheath removal , and the skin was closed by discontinuous knots with a similar thread . endovascular treatment was performed via the right femoral artery , at two to four weeks after the creation of the aneurysm . after pharmacological dilation of the artery with 8 mg of papaverine chloride , a 19-g puncture needle was used to introduce the 0.032 mandrel ; then , a 4-fr 10-cm sheath ( clinical supply , tokyo , japan ) was advanced into the right femoral artery under fluoroscopic guidance . the 4-fr catheter was navigated into the brachiocephalic artery ( bca ) with a 0.035 guidewire . digital subtraction angiography ( dsa ) showed the bca , sa , and aneurysm . a balloon catheter ( thrill slalom pta dilatation balloon catheter ; diameter , 3 mm ; length , 2 cm ; cordis , j & j , europa , n.v . , netherlands ) crimped with our hybrid stent was passed through the sheath and navigated into the bca . using fluoroscopy and road mapping , we advanced the hybrid stent over the balloon catheter across the aneurysmal neck and inflated the balloon . the femoral artery was ligated with two sutures of a 2.0 silk thread previously placed around the artery , and the skin was closed discontinuously with a similar thread . after the observation period , angiography was performed using a 4-fr sheath placed at the left cca in all animals . a 19-g puncture needle was used to introduce the 0.032 mandrel ; then , a 4-fr , 10-cm long sheath ( clinical supply , tokyo , japan ) was advanced to the left proximal cca under fluoroscopic guidance . occlusion of the aneurysms and patency of preoperatively detected side - branches of the artery was evaluated angiographically . thereafter , the animals were subjected to euthanasia by intravenous injection of potassium chloride . the bca , the right sa , vertebral artery ( va ) , some other arterial branches , and the treated aneurysm were resected en bloc and placed directly in a 4% paraformaldehyde phosphate - buffered saline solution for rapid fixation . some of the samples were evaluated histologically to determine the patency of the branches . histological examination was performed in some cases , by using a cross - sectional sample ( hematoxylin and eosin ( h&e ) stain ) obtained from the stented portion of the aneurysm to confirm the patency of the side - branches . a balloon catheter crimped with our hybrid stent was easily and smoothly passed through the sheath , navigated into the bca and right sa via the aorta , and inflated at the neck of the aneurysm . the hybrid stent covered and occluded the aneurysm instantly or after a few minutes . all the side - branches of the right sa observed preoperatively appeared patent ( without any significant stenosis ) on the angiograms obtained at one , six , and 12 months . the stent struts were completely embedded within the cover film , thereby indicating that the luminal surface of the stent graft was smooth and flat ( figure 1(a ) and ( b ) ) . it was possible to shrink the stent grafts by using a hand - held crimping device , without any damage to the cover film . micropores ( with a pore diameter and interpore distance of 100 m and 250 m , respectively , to accomplish an opening ratio of 23.6% after full expansion ) are placed in a polyurethane membrane of approximately 30-m thickness on a bare coronary stent ( a coronary momo stent , japan stent technology ) . ( a ) macroscopic image and ( b ) scanning electron microscopic image . a hybrid stent is shown . micropores ( with a pore diameter and interpore distance of 100 m and 250 m , respectively , to accomplish an opening ratio of 23.6% after full expansion ) are placed in a polyurethane membrane of approximately 30-m thickness on a bare coronary stent ( a coronary momo stent , japan stent technology ) . a 5-fr detachable sheath introducer , later connected with a rotational and hemostatic triple connector , was easily inserted into the affected cca . the size and shape of the aneurysms were variable , although they were originally funnel - shaped . at 12 months , all aneurysms but one were completely occluded , with four aneurysms being occluded at one month ; three , at three months ( figure 3(a ) and ( b ) ) ; and two , at 12 months ( figure 4(a ) and ( b ) ) . at the 12-month follow - up , one aneurysm remained patent because of distal movement of the hybrid stent itself from the aneurysm neck . figure 2.a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . branches such as the vertebral artery , internal thoracic artery , and costocervical artery are indicated ( a ) . entrapment system in the right cca . a microballoon and microcatheter through a 5-fr detachable sheath ( c ) . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . figure 3.at three months after occlusion , the aneurysm was still occluded ( before ( a ) and after ( b ) ) . side - branches , such as the vertebral artery and costocervical artery , were patent . an arrow shows a stent graft placed a cross the aneurysmal neck ( b ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . figure 4.at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . an arrow shows a stent graft placed across the aneurysmal neck ( b ) . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . branches such as the vertebral artery , internal thoracic artery , and costocervical artery are indicated ( a ) . entrapment system in the right cca . a microballoon and microcatheter through a 5-fr detachable sheath ( c ) . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . at three months after occlusion , the aneurysm was still occluded ( before ( a ) and after ( b ) ) . side - branches , such as the vertebral artery and costocervical artery , were patent . an arrow shows a stent graft placed a cross the aneurysmal neck ( b ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . an arrow shows a stent graft placed across the aneurysmal neck ( b ) . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . the side - branches were the right va , internal thoracic artery , costocervical artery , and other small branches . all the side - branches detected on the angiogram before the operation were visible after the occlusion and before the rabbits were killed ( table 1 ; figures 3(a , b ) and 4(a , b ) ) . h&e staining revealed that the orifice of the branch from the right sa was patent ( figure 4(c ) ) . table 1.results.monthsnumbersaneurysmbranchesocclusionpatency144all333all1232allall aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized.vertebral artery , internal thoracic artery , costocervical artery , etc.open in one , due to stent migration . all aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized . the stent struts were completely embedded within the cover film , thereby indicating that the luminal surface of the stent graft was smooth and flat ( figure 1(a ) and ( b ) ) . it was possible to shrink the stent grafts by using a hand - held crimping device , without any damage to the cover film . micropores ( with a pore diameter and interpore distance of 100 m and 250 m , respectively , to accomplish an opening ratio of 23.6% after full expansion ) are placed in a polyurethane membrane of approximately 30-m thickness on a bare coronary stent ( a coronary momo stent , japan stent technology ) . ( a ) macroscopic image and ( b ) scanning electron microscopic image . a hybrid stent is shown . micropores ( with a pore diameter and interpore distance of 100 m and 250 m , respectively , to accomplish an opening ratio of 23.6% after full expansion ) are placed in a polyurethane membrane of approximately 30-m thickness on a bare coronary stent ( a coronary momo stent , japan stent technology ) . a 5-fr detachable sheath introducer , later connected with a rotational and hemostatic triple connector , was easily inserted into the affected cca . the size and shape of the aneurysms were variable , although they were originally funnel - shaped . at 12 months , all aneurysms but one were completely occluded , with four aneurysms being occluded at one month ; three , at three months ( figure 3(a ) and ( b ) ) ; and two , at 12 months ( figure 4(a ) and ( b ) ) . at the 12-month follow - up , one aneurysm remained patent because of distal movement of the hybrid stent itself from the aneurysm neck . figure 2.a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . branches such as the vertebral artery , internal thoracic artery , and costocervical artery are indicated ( a ) . entrapment system in the right cca . a microballoon and microcatheter through a 5-fr detachable sheath ( c ) . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . figure 3.at three months after occlusion , the aneurysm was still occluded ( before ( a ) and after ( b ) ) . side - branches , such as the vertebral artery and costocervical artery , were patent . an arrow shows a stent graft placed a cross the aneurysmal neck ( b ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . figure 4.at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . an arrow shows a stent graft placed across the aneurysmal neck ( b ) . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . branches such as the vertebral artery , internal thoracic artery , and costocervical artery are indicated ( a ) . approach to the right cca via a rotational and hemostatic triple connector . a microcatheter and microballoon are inserted from each hemostatic valve ( b ) . entrapment system in the right cca . a microballoon and microcatheter through a 5-fr detachable sheath ( c ) . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . at three months after occlusion , the aneurysm was still occluded ( before ( a ) and after ( b ) ) . side - branches , such as the vertebral artery and costocervical artery , were patent . an arrow shows a stent graft placed a cross the aneurysmal neck ( b ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . an arrow shows a stent graft placed across the aneurysmal neck ( b ) . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . the side - branches were the right va , internal thoracic artery , costocervical artery , and other small branches . all the side - branches detected on the angiogram before the operation were visible after the occlusion and before the rabbits were killed ( table 1 ; figures 3(a , b ) and 4(a , b ) ) . staining revealed that the orifice of the branch from the right sa was patent ( figure 4(c ) ) . table 1.results.monthsnumbersaneurysmbranchesocclusionpatency144all333all1232allall aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized.vertebral artery , internal thoracic artery , costocervical artery , etc.open in one , due to stent migration . all aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized . although guglielmi detachable coil systems ( gdc coils ) have been widely accepted and used in the treatment of intracranial aneurysms , primary stenting of aneurysms by using porous stents or stent grafts and implantation of coils after stenting are emerging techniques in endovascular treatment . most intracranial aneurysms occur at branch points ( bifurcation , small side - branches , and perforating arteries ) . the maintenance of the patency of normal arterial branches originating from the covered segment of the artery after stenting across the lesion is a major concern . the salient issues influencing the use of stent grafting for aneurysms in humans include the delivery of the stent graft , degree of cover porosity , configuration of the aneurysm , curvature of the parent vessel , and presence or absence of perforating end - arteries . a simple bare stent has no efficacious occlusive effect on aneurysms , although it raises a few concerns about the occlusion of side - branches . in contrast , a simple covered stent has sufficient occlusive effect on the aneurysms , but it can occlude the side - branches . to compensate for the disadvantages of simple covered stents , we have been developing a hybrid stent . this hybrid stent consists of the commercially available balloon - expandable coronary - artery stent with a thin spu film ( by dip - coating method ) on which micropores have been created by the excimer laser ablation technique , followed by coating with argatroban . we have developed a hybrid stent with micropores to prevent early parent artery occlusion by more early endothelialization and administration of argatroban , and mid- to long - term parent artery stenosis by control of intimal hyperplasia after aneurysm occlusion , compared with those in a simple covered stent . in the current study , we established elastase - induced rabbit carotid aneurysms , all of which were occluded by our hybrid stents until 12 months , and all the small side - branches detected preoperatively remained patent without significant stenosis or occlusion even after implantation of the hybrid stents . the hybrid stent was smoothly navigated into the affected bca and sa from the femoral artery , as easily as the bare stents . our microporous membrane induces early endothelialization capable of secreting tissue plasminogen within one week of stent graft implantation , thereby avoiding early thrombosis and controlling mid- to long - term hyperplasia . polyurethane has excellent elastomeric properties and is clinically used as a material for manufacturing blood pumps and arterial grafts ; it also exhibits no toxicity or little biodegradation . argatroban , an arginine - derived synthetic low - molecular - weight compound that binds to thrombin , competitively inhibits fibrinogen cleavage and the platelet activation stimulated by thrombin . it has been used for preparing antithrombogenic surfaces in percutaneous transluminal angioplasty ( pta ) devices , including balloon catheters and stents . in this study , cerebral thromboembolic events were not evaluated using magnetic resonance imaging ( mri ) . less intimal hyperplasia of the parent artery was focally noted in the group treated with hybrid stents without antiplatelets . if this device is clinically used , antiplatelet therapy with one or two drugs will be necessary for approximately one to three months , as per the conventional therapy after vascular stenting . the aneurysms were created experimentally by intraluminal incubation of elastase within an endovascularly trapped segment of the proximal portion of the cca , with slight modifications to the method . the cca branches from the bca of the rabbit , and it was ligated beyond the point of elastase incubation . endoluminal digestion of the internal elastic lamina , with spreading of elastase up to the adventitia , results in a thin - walled aneurysm , as observed in humans . aneurysm formation occurred over a two - week period , after which the animals were treated with the respective therapies . the rabbit aneurysm model with elastase has some characteristic features : ( 1 ) long - term patency in untreated animals , ( 2 ) simulating aneurysm morphologies that place a high shear stress on the aneurysmal neck , ( 3 ) similar size in aneurysm diameter and parent artery diameter , ( 4 ) maintenance of the integrity of the endothelium of the aneurysmal cavity , and ( 5 ) short construction time and easy reproduction . the rabbit aneurysm is histologically similar to the human saccular aneurysm and was useful for the current study . mechanisms of branch occlusion by stents include the longitudinal snow - plowing effect , stent jailing ( early stage ) , and in - stent neointimal growth ( late stage ) during or after stent deployment . radial force in the stent strut might be less than that in the intracranial pta stent , which reduced the longitudinal redistribution of the plaque . our hybrid stent had a porosity appropriate for the regulation of the in - stent neointima growth even at 12 months ( personal communication ) . the arteriovenous pressure gradient is the driving force of the blood flow through the branches and perforators under normal conditions . additionally , long - term remodeling of the artery and its lumen , in response to the presence of the intraluminal prosthesis , is less likely to result in complete occlusion of the jailed branch . experimental evidence in dogs showed that vessels ( small muscular branches from the va ) comparable to human perforators tend to remain patent if less than 50% of the ostial diameter is covered with the stent strut . if the diameter of struts is 100 m , complete occlusion of the perforators may not occur , but the exposed stent wire covering the perforators will remain a potential source of thromboembolic complications in canine carotid aneurysm models . the diameter of stent struts in our hybrid stent was less than 50 m . fibrotic tissue growth around the filaments extended into the origin of the branches ( external arteries ) without narrowing them significantly . intracranial self - expanding stents generate a radial force too weak to require coverage with cover materials , although some of them , such as neuroform or enterprise , are recommended . therefore , we selected balloon - expandable coronary - artery stents for the preparation of hybrid stents . the branch originating from the aneurysm sac , which is treated with a flow diverter ( pipeline embolization device ) , is kept patent in the presence of a flow demand through it . the significant shrinkage of the aneurysm gives the branch an infundibular - like appearance at its origin , or the branch is represented by a tortuous takeoff from the parent artery in place of the initial aneurysm . a slow occlusion process for the treatment of ruptured aneurysms is yet to be developed . the stent developed by us is a hybrid of a bare stent and a simple covered stent ; this hybrid nature helps regulate the porosity and enables early intraluminal endothelialization and late inhibition of the intimal hyperplasia . experimentally fabricated canine carotid aneurysms were occluded with the placement of our hybrid , self - expanding , stent grafts at one , six , and 12 months ( personal communication ) . a porosity of 23.6% was found to be suitable in the presence of less neointimal hyperplasia . on the basis of the porosity and demand of the blood flow by vessels , side - branches or perforating arteries can be protected if the stenotic area generated by the hybrid stents is less than 50% . in this study , the patency of all the side - branches was maintained , along with the occlusion of the aneurysms , although the side - branches were not as narrow as the intracranial perforators . if the use of hybrid stents for aneurysmal occlusion is planned , the number of associated perforators should be less to ensure safety . in the examined aneurysmal model using stent grafts , all small side - branches of the patent artery were patent without significant stenosis . thus , our hybrid stent represents a step forward in the clinical management of aneurysms . the long - term results obtained in an animal model indicate that hybrid stents may be effective for aneurysm repair while maintaining side - branch patency . ccacommon carotid arteryicainternal carotid arterybcabrachiocephalic arterysasubclavian arteryvavertebral arterypcoaposterior communicating arteryophaophthalmic arteryachaanterior choroidal arterypicaposterior inferior cerebellar arteryptcapercutaneous transluminal coronal angioplastydsadigital subtraction angioplastyh & estain hematoxylin and eosin stain common carotid artery internal carotid artery brachiocephalic artery posterior communicating artery anterior choroidal artery posterior inferior cerebellar artery percutaneous transluminal coronal angioplasty digital subtraction angioplasty stain hematoxylin and eosin stain

objectivewe sought to determine the patency of normal arterial branches from the covered segments of an artery after stenting.backgroundmost intracranial aneurysms occur at arterial branching points ( bifurcations , side - branches , or perforators ) . the post - stenting patency of normal arterial branches from the covered segments of the artery is important . we have previously developed a hybrid stent with micropores to prevent early parent artery occlusion by more early endothelialization , and mid- to long - term parent artery stenosis by control of intimal hyperplasia after aneurysm occlusion.methodswe created aneurysms in 10 rabbits by distal ligation and intraluminal incubation of elastase within an endovascularly trapped proximal segment of the common carotid artery . all animals were treated with hybrid stents having micropores . four animals were observed for one month and three each for three and 12 months . the patency of the side - branches of the subclavian artery was evaluated angiographically and in some cases , histologically.resultsaneurysms were completely occluded at all time points other than 12 months . the subclavian artery and brachiocephalic artery were patent , without significant stenosis . all the side - branches of the subclavian artery detected on the preoperative angiogram remained patent at the final assessment.conclusionthe use of hybrid stents for aneurysm repair and side - branch patency seems to be effective , as per the long - term results obtained in an animal model .

Introduction Materials and methods Fabrication of hybrid stents ( Preparation of aneurysms in rabbits ( Endovascular technique Final angiography and harvesting Histology Results Stent graft and its remounting Fabrication of carotid aneurysms ( Occlusion of the aneurysm ( Preservation of side-branches Discussion Conclusion Abbreviations

most intracranial aneurysms occur at arterial branching points ( bifurcations , side - branches , or perforators ) . it is important to assess the patency of normal arterial branches arising from the covered segments of the artery after implantation of simple covered stents . we established rabbit carotid aneurysms by intraluminal incubation of elastase within an endovascularly trapped segment of the proximal common carotid artery ( cca ) , and we implanted hybrid stents equipped with micropores in these aneurysms . besides studying the occlusive effect of the hybrid stents in the aneurysms , we examined the patency of the side - branches arising from the right subclavian artery ( sa ) , both angiographically and histologically . subsequently , a balloon - expandable coronary - artery bare stent ( momo stent : diameter , 3 mm ; length , 20 mm ; japan stent technology , okayama , japan ) was mounted on the cover film of the mold , and again , it was dipped in the solution and dried . the femoral artery was ligated with two sutures of a 2.0 silk thread previously placed around the artery , and the skin was closed discontinuously with a similar thread . occlusion of the aneurysms and patency of preoperatively detected side - branches of the artery was evaluated angiographically . some of the samples were evaluated histologically to determine the patency of the branches . histological examination was performed in some cases , by using a cross - sectional sample ( hematoxylin and eosin ( h&e ) stain ) obtained from the stented portion of the aneurysm to confirm the patency of the side - branches . subsequently , a balloon - expandable coronary - artery bare stent ( momo stent : diameter , 3 mm ; length , 20 mm ; japan stent technology , okayama , japan ) was mounted on the cover film of the mold , and again , it was dipped in the solution and dried . the femoral artery was ligated with two sutures of a 2.0 silk thread previously placed around the artery , and the skin was closed discontinuously with a similar thread . occlusion of the aneurysms and patency of preoperatively detected side - branches of the artery was evaluated angiographically . the bca , the right sa , vertebral artery ( va ) , some other arterial branches , and the treated aneurysm were resected en bloc and placed directly in a 4% paraformaldehyde phosphate - buffered saline solution for rapid fixation . some of the samples were evaluated histologically to determine the patency of the branches . histological examination was performed in some cases , by using a cross - sectional sample ( hematoxylin and eosin ( h&e ) stain ) obtained from the stented portion of the aneurysm to confirm the patency of the side - branches . a balloon catheter crimped with our hybrid stent was easily and smoothly passed through the sheath , navigated into the bca and right sa via the aorta , and inflated at the neck of the aneurysm . all the side - branches of the right sa observed preoperatively appeared patent ( without any significant stenosis ) on the angiograms obtained at one , six , and 12 months . at 12 months , all aneurysms but one were completely occluded , with four aneurysms being occluded at one month ; three , at three months ( figure 3(a ) and ( b ) ) ; and two , at 12 months ( figure 4(a ) and ( b ) ) . at the 12-month follow - up , one aneurysm remained patent because of distal movement of the hybrid stent itself from the aneurysm neck . figure 2.a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . side - branches , such as the vertebral artery and costocervical artery , were patent . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . figure 4.at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . side - branches , such as the vertebral artery and costocervical artery , were patent . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . the side - branches were the right va , internal thoracic artery , costocervical artery , and other small branches . all the side - branches detected on the angiogram before the operation were visible after the occlusion and before the rabbits were killed ( table 1 ; figures 3(a , b ) and 4(a , b ) ) . table 1.results.monthsnumbersaneurysmbranchesocclusionpatency144all333all1232allall aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized.vertebral artery , internal thoracic artery , costocervical artery , etc.open in one , due to stent migration . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized . at 12 months , all aneurysms but one were completely occluded , with four aneurysms being occluded at one month ; three , at three months ( figure 3(a ) and ( b ) ) ; and two , at 12 months ( figure 4(a ) and ( b ) ) . at the 12-month follow - up , one aneurysm remained patent because of distal movement of the hybrid stent itself from the aneurysm neck . figure 2.a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . side - branches , such as the vertebral artery and costocervical artery , were patent . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . figure 4.at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . a schema of the aneurysm occlusion in the rabbit using a hybrid stent ( arrows ) implanted through the femoral artery . cca : common carotid artery ; va : vertebral artery ; ita : internal thoracic artery ; sa : subclavian artery . side - branches , such as the vertebral artery and costocervical artery , were patent . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery . at 12 months after occlusion , the aneurysm was completely occluded ( pre ( a ) and post ( b ) ) , while the vertebral artery , internal thoracic artery , and costocervical artery remained patent . a cross - sectional specimen ( h&e stain ) showed that the origin of the branch ( arrows ) from the subclavian artery was patent at 12 months ( c ) . cca : common carotid artery ; va : vertebral artery ; bca : brachiocephalic artery ; sa : subclavian artery ; h&e stain : hematoxylin and eosin stain . the side - branches were the right va , internal thoracic artery , costocervical artery , and other small branches . all the side - branches detected on the angiogram before the operation were visible after the occlusion and before the rabbits were killed ( table 1 ; figures 3(a , b ) and 4(a , b ) ) . table 1.results.monthsnumbersaneurysmbranchesocclusionpatency144all333all1232allall aneurysms but one were occluded at 12 months : four aneurysms at one month ; three at three months ; and two at 12 months . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized.vertebral artery , internal thoracic artery , costocervical artery , etc.open in one , due to stent migration . at 12 months , one aneurysm was patent because of distal movement of the stent graft itself from the aneurysm neck at the initial stent placement . side - branches were the right vertebral artery , internal thoracic artery , costocervical artery , and other small branches . all side - branches drawn preoperatively on the angiogram were visible after occlusion and before the rabbits were euthanized . most intracranial aneurysms occur at branch points ( bifurcation , small side - branches , and perforating arteries ) . the maintenance of the patency of normal arterial branches originating from the covered segment of the artery after stenting across the lesion is a major concern . the salient issues influencing the use of stent grafting for aneurysms in humans include the delivery of the stent graft , degree of cover porosity , configuration of the aneurysm , curvature of the parent vessel , and presence or absence of perforating end - arteries . in contrast , a simple covered stent has sufficient occlusive effect on the aneurysms , but it can occlude the side - branches . to compensate for the disadvantages of simple covered stents , we have been developing a hybrid stent . this hybrid stent consists of the commercially available balloon - expandable coronary - artery stent with a thin spu film ( by dip - coating method ) on which micropores have been created by the excimer laser ablation technique , followed by coating with argatroban . we have developed a hybrid stent with micropores to prevent early parent artery occlusion by more early endothelialization and administration of argatroban , and mid- to long - term parent artery stenosis by control of intimal hyperplasia after aneurysm occlusion , compared with those in a simple covered stent . in the current study , we established elastase - induced rabbit carotid aneurysms , all of which were occluded by our hybrid stents until 12 months , and all the small side - branches detected preoperatively remained patent without significant stenosis or occlusion even after implantation of the hybrid stents . the hybrid stent was smoothly navigated into the affected bca and sa from the femoral artery , as easily as the bare stents . our microporous membrane induces early endothelialization capable of secreting tissue plasminogen within one week of stent graft implantation , thereby avoiding early thrombosis and controlling mid- to long - term hyperplasia . less intimal hyperplasia of the parent artery was focally noted in the group treated with hybrid stents without antiplatelets . the aneurysms were created experimentally by intraluminal incubation of elastase within an endovascularly trapped segment of the proximal portion of the cca , with slight modifications to the method . the cca branches from the bca of the rabbit , and it was ligated beyond the point of elastase incubation . endoluminal digestion of the internal elastic lamina , with spreading of elastase up to the adventitia , results in a thin - walled aneurysm , as observed in humans . aneurysm formation occurred over a two - week period , after which the animals were treated with the respective therapies . the rabbit aneurysm model with elastase has some characteristic features : ( 1 ) long - term patency in untreated animals , ( 2 ) simulating aneurysm morphologies that place a high shear stress on the aneurysmal neck , ( 3 ) similar size in aneurysm diameter and parent artery diameter , ( 4 ) maintenance of the integrity of the endothelium of the aneurysmal cavity , and ( 5 ) short construction time and easy reproduction . mechanisms of branch occlusion by stents include the longitudinal snow - plowing effect , stent jailing ( early stage ) , and in - stent neointimal growth ( late stage ) during or after stent deployment . our hybrid stent had a porosity appropriate for the regulation of the in - stent neointima growth even at 12 months ( personal communication ) . additionally , long - term remodeling of the artery and its lumen , in response to the presence of the intraluminal prosthesis , is less likely to result in complete occlusion of the jailed branch . therefore , we selected balloon - expandable coronary - artery stents for the preparation of hybrid stents . the significant shrinkage of the aneurysm gives the branch an infundibular - like appearance at its origin , or the branch is represented by a tortuous takeoff from the parent artery in place of the initial aneurysm . experimentally fabricated canine carotid aneurysms were occluded with the placement of our hybrid , self - expanding , stent grafts at one , six , and 12 months ( personal communication ) . on the basis of the porosity and demand of the blood flow by vessels , side - branches or perforating arteries can be protected if the stenotic area generated by the hybrid stents is less than 50% . in this study , the patency of all the side - branches was maintained , along with the occlusion of the aneurysms , although the side - branches were not as narrow as the intracranial perforators . if the use of hybrid stents for aneurysmal occlusion is planned , the number of associated perforators should be less to ensure safety . in the examined aneurysmal model using stent grafts , all small side - branches of the patent artery were patent without significant stenosis . the long - term results obtained in an animal model indicate that hybrid stents may be effective for aneurysm repair while maintaining side - branch patency .

risk prediction models have become a main focus in epidemiological research in the past years . although a large number of prediction models exists , of which some have already been integrated in treatment strategies or health promotion programs , there is an ongoing effort to improve prediction models by the use of new risk markers . for the evaluation of such model extensions , the net reclassification improvement ( nri ) was proposed by pencina et al . in 2008 as an addition to the evaluation of discrimination , e.g. by comparing receiver operating characteristic curves . the nri is based on the calculation of the amount of correctly and incorrectly reclassified cases and non - cases comparing classification of individuals into a priori defined risk categories in terms of their predicted risk between two nested models . since its publication it has been used in a growing number of studies , however , there is a large heterogeneity in its use , presentation , and interpretation [ 2 , 3 ] . especially with regard to testing statistical significance of nri estimates , there remains uncertainty . pencina discussed that even small nri values ( < 0.01 ) might produce statistically significant p values and pepe et al . points out that valid methods for inference for the nri do not exist . in a recent review of nri measures , kerr et al . however , confidence intervals provide precision estimates and are preferable , not only for the overall nri , but also for its components . the nri components do not reflect an overall improvement but rather improvement among cases and non - cases separately . therefore , our aim was to introduce a method to calculate a confidence ellipse around the two components of the nri which reflects the precision of the estimates and can help interpret the magnitude and variability of the observed effects . extension of prediction models with additional risk factors usually leads to changes in predicted risk for individual study participants . when predefined risk categories are used , this is reflected by upward and downward movements across these risk categories from the reference to the extended model . this reclassification is used for the calculation of the nri which considers proportions of upward and downward movements separately for cases and non - cases ( 1 ) .1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } nri_{cases } & = p\left ( { up|case } \right ) - p\left ( { down|case } \right ) , \\ nri_{{non { - } cases } } & = p\left ( { down|non { - } case } \right ) - p\left ( { up|non { - } case } \right ) , \\ nri & = nri_{cases } + nri_{{non { - } cases } } \\ & = p\left ( { up|case } \right ) - p\left ( { down|case } \right ) + p\left ( { down|non { - } case } \right ) - p\left ( { up|non { - } case } \right ) \\ & = \left [ { p_{up , cases } - p_{down , cases } } \right ] + \left [ { p_{{down , non { - } cases } } - p_{{up , non { - } cases } } } \right ] \\ \end{aligned}$$\end{document}nricases = pup|case - pdown|case , nrinon - cases = pdown|non - case - pup|non - case , nri = nricases+nrinon - cases = pup|case - pdown|case+pdown|non - case - pup|non - case = pup , cases - pdown , cases+pdown , non - cases - pup , non - cases the corresponding standard error for the nri and its components was defined by pencina et al . and depends on the standard error of cases , which often is a much smaller group:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$se\left ( { \widehat{nri } } \right ) = \sqrt { se\left ( { \widehat{nri } _ { cases } } \right)^{2 } + se\left ( { \widehat{nri } _ { non - cases } } \right)^{2 } } , $ $ \end{document}senri^=senri^cases2+senri^non - cases2,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$se\left ( { \widehat{nri}_{cases } } \right ) = \sqrt { \frac{{\hat{p } _ { up , cases } + \hat{p } _ { down , cases } - \left ( { \hat{p } _ { up , cases } - \hat{p } _ { down , cases } } \right)^{2 } } } { { n_{cases } } } } , $ $ \end{document}senri^cases = p^up , cases+p^down , cases - p^up , cases - p^down , cases2ncases,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$se\left ( { \widehat{nri } _ { non - cases } } \right ) = \sqrt { \frac{{\hat{p } _ { up , non - cases } + \hat{p } _ { down , non - cases } - \left ( { \hat{p } _ { { up , non { - } cases } } - \hat{p } _ { down , non - cases } } \right)^{2 } } } { { n_{non - cases } } } } .$$\end{document}senri^non - cases = p^up , non - cases+p^down , non - cases - p^up , non - cases - p^down , non - cases2nnon - cases . as such , the nri is the sum of the single components ( nricases , nrinon - cases ) reflecting improvement among cases or improvement among non - cases or both . thereby , the overall measure does not include evaluation of improvement among cases or non - cases separately . absolute risks are derived from regression models ; either logistic regression or cox - regression with the disease as the outcome variable . pencina already suggested to report cis for the nri and used the bootstrap method for their construction . calculation of cis would be informative not only for the overall nri but also for the single components . besides the bootstrapping method , cis can be calculated with a formula related to the construction of cis for independent proportions according to agresti ; this approach will be applied further on . the standard errors for the overall nri and its single components were defined before , so that the cis can be defined as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left [ { \widehat{nri } - z_{1 - \frac{\alpha } { 2 } } se\left ( { \widehat{nri } } \right),\widehat{nri } + z_{1 - \frac{\alpha } { 2 } } se(\widehat{nri } ) } \right]$$\end{document}nri^-z1-2senri^,nri^+z1-2se(nri^)with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z_{1 - \frac{\alpha } { 2}}$$\end{document}z1-2 as the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left ( { 1 - \frac{\alpha } { 2 } } \right)$$\end{document}1-2-quantile of the standard normal distribution . the cis for nricases and nrinon - cases can be calculated with the same method . while cis of the two nri components , nricases and nrinon - cases , can be interpreted individually , this again would not allow an easy interpretation in terms of the overall improvement . to overcome this problem , we propose to use a confidence ellipse which allows evaluating the single components nricases and nrinon - cases in combination . we introduce the following notation : let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta = ( \theta_{1 , } \theta_{2 } ) $ $ \end{document}=(1,2 ) be the parameter consisting of the nri components , i.e.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta_{1 } = nri_{cases } = p\left ( { up |case } \right ) - p\left ( { down |case } \right)\;{\text{and}}$$\end{document}1=nricases = pup|case - pdown|caseand\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta_{2 } = nri_{{non { - } cases } } = p\left ( { down |non { - } case } \right ) - p\left ( { up |non { - } case } \right)$$\end{document}2=nrinon - cases = pdown|non - case - pup|non - casewe define the following probabilities\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{1 } = { \text{p}}\left ( { up \cap case } \right),\ , p_{2 } = { \text{p}}\left ( { down \cap case } \right),\ , p_{3 } = { \text{p}}\left ( { up \cap non { - } case } \right),\ , p_{4 } = { \text{p}}\left ( { down \cap non { - } case } \right)\;{\text{and}}\ ; p_{5 } = { \text{p}}\left ( { case } \right)$$\end{document}p1=pupcase , p2=pdowncase , p3=pupnon - case , p4=pdownnon - caseandp5=pcaseand can write \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta$$\end{document} as a function of these probabilities:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta = \left ( { \theta_{1 } , \theta_{2 } } \right ) = g\left ( \varvec{p } \right ) = \left ( { g_{1 } \left ( \varvec{p } \right),g_{2 } \left ( \varvec{p } \right ) } \right ) = \left ( { \frac{{p_{1 } - p_{2 } } } { { p_{5 } } } , \frac{{p_{4 } - p_{3 } } } { { 1 - p_{5 } } } } \right).$$\end{document}=1,2=gp = g1p , g2p = p1-p2p5,p4-p31-p5 . consequently , the maximum likelihood estimates of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta_{1}$$\end{document}1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta_{2}$$\end{document}2 are given by the relative frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p}_{j } = v_{j } /n$$\end{document}p^j = vj / n ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n = n_{cases } + n_{{non { - } cases}}$$\end{document}n = ncases+nnon - cases ) as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\theta } _ { 1 } = \frac{{\hat{p}_{1 } - \hat{p}_{2 } } } { { \hat{p}_{5 } } } \;{\text{and}}\ ; \hat{\theta } _ { 2 } = \frac{{\hat{p}_{4 } - \hat{p}_{3 } } } { { 1 - \hat{p}_{5 } } } $ $ \end{document}^1=p^1-p^2p^5and^2=p^4-p^31-p^5with up down total case \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{1 } $ $ \end{document}v1 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{2 } $ $ \end{document}v2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{5 } $ $ \end{document}v5 non - case \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{3 } $ $ \end{document}v3 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{4 } $ $ \end{document}v4 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n - v_{5 } $ $ \end{document}n - v5 applying the multivariate central limit theorem to the vector of relative frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\varvec{p } } = ( \hat{p}_{1 } , \hat{p}_{2 } , \hat{p}_{3 } , \hat{p}_{4 } , \hat{p}_{5 } ) ^{t}$$\end{document}p^=(p^1,p^2,p^3,p^4,p^5)t we get , that for a large sample size \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n the distribution of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt n ( \hat{\varvec{p } } - \varvec{p})$$\end{document}n(p^-p ) can be approximated by a five - dimensional normal distribution , i.e.,2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt n \left ( { \hat{\varvec{p } } - \varvec{p } } \right ) { \mathop \to \limits^ { d } } { \text{n}}_{5 } \left ( { 0,a(\varvec{p } ) } \right).$$\end{document}np^-pdn50,a(p).here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(\varvec{p})$$\end{document}a(p ) is the covariance matrix of the limit distribution . it depends on the underlying probabilities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{j}$$\end{document}pj and can be computed as:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a\left ( \varvec{p } \right ) = \left ( { \begin{array}{*{20}l } { p_{1 } ( 1 - p_{1 } ) } \hfill & { - p_{1 } p_{2 } } \hfill & { - p_{1 } p_{3 } } \hfill & { - p_{1 } p_{4 } } \hfill & { p_{1 } ( 1 - p_{5 } ) } \hfill \\ { - p_{1 } p_{2 } } \hfill & { p_{2 } ( 1 - p_{2 } ) } \hfill & { - p_{2 } p_{3 } } \hfill & { - p_{2 } p_{4 } } \hfill & { p_{2 } ( 1 - p_{5 } ) } \hfill \\ { - p_{1 } p_{3 } } \hfill & { - p_{2 } p_{3 } } \hfill & { p_{3 } ( 1 - p_{3 } ) } \hfill & { - p_{3 } p_{4 } } \hfill & { - p_{3 } p_{5 } } \hfill \\ { - p_{1 } p_{4 } } \hfill & { - p_{2 } p_{4 } } \hfill & { - p_{3 } p_{4 } } \hfill & { p_{4 } ( 1 - p_{4 } ) } \hfill & { - p_{4 } p_{5 } } \hfill \\ { p_{1 } ( 1 - p_{5 } ) } \hfill & { p_{2 } ( 1 - p_{5 } ) } \hfill & { - p_{3 } p_{5 } } \hfill & { - p_{4 } p_{5 } } \hfill & { p_{5 } ( 1 - p_{5 } ) } \hfill \\ \end{array } } \right).$$\end{document}ap = p1(1-p1)-p1p2-p1p3-p1p4p1(1-p5)-p1p2p2(1-p2)-p2p3-p2p4p2(1-p5)-p1p3-p2p3p3(1-p3)-p3p4-p3p5-p1p4-p2p4-p3p4p4(1-p4)-p4p5p1(1-p5)p2(1-p5)-p3p5-p4p5p5(1-p5 ) . with the help of the so - called delta method we can derive from ( 2 ) the asymptotic variance of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\theta } = ( \hat{\theta } _ { 1 } , \hat{\theta } _ { 2 } ) $ $ \end{document}^=(^1,^2 ) . here we use , that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\theta } = g(\hat{\varvec{p}})$$\end{document}^=g(p^ ) . to derive the asymptotic variance of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\theta } $ $ \end{document}^ one has to multiply the matrix of partial derivatives of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g$$\end{document}g with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(\varvec{p})$$\end{document}a(p ) . this leads to\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt n \left [ { \left ( { \begin{array}{*{20}c } { \hat{\theta } _ { 1 } } \\ { \hat{\theta } _ { 2 } } \\ \end{array } } \right ) - \left ( { \begin{array}{*{20}c } { \theta_{1 } } \\ { \theta_{2 } } \\ \end{array } } \right ) } \right ] { \mathop \to \limits^ { d } } { \text{n}}_{2 } \left ( { 0,w(\varvec{p } ) } \right)$$\end{document}n^1^2-12dn20,w(p)with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w(\varvec{p } ) = \left ( { \begin{array}{*{20}c } { w_{1 } } & 0 \\ 0 & { w_{2 } } \\ \end{array } } \right)$$\end{document}w(p)=w100w2 and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w_{1 } = \frac{{p_{1 } + p_{2 } } } { { p_{5}^{2 } } } - \frac{{\left ( { p_{1 } - p_{2 } } \right)^{2 } } } { { p_{5}^{3 } } } , w_{2 } = \frac{{p_{3 } + p_{4 } } } { { ( 1 - p_{5 } ) ^{2 } } } - \frac{{\left ( { p_{3 } - p_{4 } } \right)^{2 } } } { { ( 1 - p_{5 } ) ^{3 } } } .$$\end{document}w1=p1+p2p52-p1-p22p53,w2=p3+p4(1-p5)2-p3-p42(1-p5)3 . the asymptotic normality of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{\theta } $ $ \end{document}^ implies that3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\left ( { \hat{\theta } - \theta } \right)^{t } w^ { - 1 } \left ( { \hat{\varvec{p } } } \right)\left ( { \hat{\theta } - \theta } \right){\mathop \to \limits^ { d } } \chi_{2}^{2}$$\end{document}n^-tw-1p^^-d22with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi_{2}^{2}$$\end{document}22 , the chi squared distribution with two degrees of freedom and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w^ { - 1 } \left ( { \hat{\varvec{p } } } \right)$$\end{document}w-1p^ is the inverse of the matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w(\varvec{p})$$\end{document}w(p ) . because of the diagonal structure of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w(\varvec{p})$$\end{document}w(p ) and with asymptotic result from ( 3 ) we can define a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left ( { 1 - \alpha } \right)$$\end{document}1- confidence ellipse for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\theta$$\end{document} as\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { { \theta \in [ - 1,1]^{2 } \mid \frac{{\left ( { \hat{\theta } _ { 1 } - \theta_{1 } } \right)^{2 } } } { { \hat{w}_{1 } } } + \frac{{\left ( { \hat{\theta } _ { 2 } - \theta_{2 } } \right)^{2 } } } { { \hat{w}_{2 } } } \le \frac{{\chi_{2;1 - \alpha } ^{2 } } } { n } } \right\}.$$\end{document}[-1,1]2^1-12w^1+^2-22w^221-2n . the determination of the confidence ellipse allows to determine the simultaneous precision of the nri estimates for cases and non - cases . using previous notation and the following relationships \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p}_{up , cases } = \hat{p}_{1 } /\hat{p}_{5}$$\end{document}p^up , cases = p^1/p^5 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p}_{down , cases } = \hat{p}_{2 } /\hat{p}_{5}$$\end{document}p^down , cases = p^2/p^5 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p}_{{up , non { - } cases } } = \hat{p}_{3 } /(1 - \hat{p}_{5})$$\end{document}p^up , non - cases = p^3/(1-p^5 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p}_{up , cases } = \hat{p}_{4 } /(1 - \hat{p}_{5})$$\end{document}p^up , cases = p^4/(1-p^5 ) , the confidence ellipse can also be defined with the following equation.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\ { { \theta \in [ - 1,1]^{2 } \mid \left ( { \frac{{\widehat{nri}_{cases } - \theta_{1 } } } { { se\left ( { \widehat{nri } _ { cases } } \right ) } } } \right)^{2 } + \left ( { \frac{{\widehat{nri}_{{non { - } cases } } - \theta_{2 } } } { { se\left ( { \widehat{nri } _ { { non { - } cases } } } \right ) } } } \right)^{2 } \le \chi_{2;1 - \alpha } ^{2 } } \right\}.$$\end{document}[-1,1]2nri^cases-1senri^cases2+nri^non - cases-2senri^non - cases221-2 . the european prospective investigation into cancer and nutrition ( epic)-potsdam study is a prospective cohort study initially including 27,548 participants aged 3565 years . details of recruitment and follow - up procedures were described previously [ 7 , 8 ] . briefly , within a median follow - up time of 7 years , 849 participants out of 25,167 participants free of diabetes at baseline developed incident diabetes . on this basis , the german diabetes risk score ( gdrs ) was developed using cox - regression . with the gdrs the 5-year risk for developing future type 2 diabetes can be calculated using information on lifestyle and anthropometric factors , diet and physical activity . we used data from 21,846 participants ( 727 cases ) who had also information on family history of diabetes available . the extended model additionally included family history ; this model was compared with the reference model . table 1 shows the reclassification of cases and non - cases due to model extension based on the use of 5 predefined risk categories.table 1reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) n ( % ) gdrs + family historytotal1 : low2 : still low3 : increased4 : high5 : very high cases 1 : low 21 ( 2.89 ) 7 ( 0.96)28 ( 3.85)2 . still low13 ( 1.79 ) 102 ( 14.03 ) 30 ( 4.13)145 ( 19.94)3 . increased32 ( 4.40 ) 176 ( 24.21 ) 61 ( 8.39)269 ( 37.00)4 . high29 ( 3.99 ) 146 ( 20.08 ) 36 ( 4.95)211 ( 29.02)5 . very high15 ( 2.06 ) 59 ( 8.12 ) 74 ( 10.18)total34 ( 4.68)141 ( 19.39)235 ( 32.32)222 ( 30.54)95 ( 13.07)727 ( 100 ) non - cases 1 . low 9001 ( 42.62 ) 625 ( 2.96)9626 ( 45.58)2 . still low1415 ( 6.70 ) 4220 ( 19.98 ) 672 ( 3.18)6307 ( 29.86)3 . increased858 ( 4.06 ) 2613 ( 12.37 ) 387 ( 1.83)3858 ( 18.27)4 . high269 ( 1.27 ) 782 ( 3.70 ) 98 ( 0.46)1149 ( 5.44)5 . very high40 ( 0.19 ) 139 ( 0.66 ) 179 ( 0.85)total10,416 ( 49.32)5703 ( 27.0)3554 ( 16.83)1209 ( 5.72)237 ( 1.12)21,119 ( 100 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 based on the asymptotic method we determined 95 % cis for nricases and nrinon - cases ( fig . 1 ) . taking into account the large number of non - cases it is obvious that estimation of nrinon - cases was much more precise than of nricases . the calculation of cis for single components does not allow evaluating both components in combination.fig . 1confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . nri for cases and nri for non - cases were calculated by comparing the german diabetes risk score extended with family history of diabetes with the initial german diabetes risk score ; five predefined risk categories were used for calculation ; the confidence ellipse was defined with the accepted 1 and 2 values for = 0.05 . the area within the ellipse defines the area of accepting the null hypotheses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0 } : nri_{cases } = \theta_{1}$$\end{document}h0:nricases=1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = \theta_{2}$$\end{document}nrinon - cases=2 and the area outside the ellipse is the area of not accepting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0}$$\end{document}h0 . the black dot is the estimated for nri cases and nri non - cases . the horizontal and vertical reference lines display the lower and upper confidence limits of nri non - cases ( 0.0318 , 0.0440 ) and of nri cases the black diamond displays the discussed example for null hypotheses : nri cases = 0.02 and nri non - cases = 0.035 confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . nri for cases and nri for non - cases were calculated by comparing the german diabetes risk score extended with family history of diabetes with the initial german diabetes risk score ; five predefined risk categories were used for calculation ; the confidence ellipse was defined with the accepted 1 and 2 values for = 0.05 . the area within the ellipse defines the area of accepting the null hypotheses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0 } : nri_{cases } = \theta_{1}$$\end{document}h0:nricases=1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = \theta_{2}$$\end{document}nrinon - cases=2 and the area outside the ellipse is the area of not accepting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0}$$\end{document}h0 . the black dot is the estimated for nri cases and nri non - cases . the horizontal and vertical reference lines display the lower and upper confidence limits of nri non - cases ( 0.0318 , 0.0440 ) and of nri cases ( 0.0219 , 0.1019 ) respectively . the black diamond displays the discussed example for null hypotheses : nri cases = 0.02 and nri non - cases = 0.035 therefore , we computed a confidence ellipse for nricases and nrinon - cases to reflect precision of their estimates in combination and which also allows to evaluate the area of acceptable values . figure 1 shows cis for the single components ( vertical and horizontal lines ) as well as the confidence ellipse , both approaches were based on the five risk categories described before . when constructing cis for the components separately , nricases ( 0.0619 ) has a ci of 0.02190.1019 . therefore , the value 0.02 lies outside of this interval while the nrinon - cases ( 0.0379 ) had a ci ranging from 0.0318 to 0.0440 thus including a value of 0.035 . using both cis separately would therefore lead to the conclusion that nricases is significantly higher than 0.02 while nrinon - cases is not significantly higher than 0.035 . however , examining the vector ( 0.02 , 0.035 ) within the confidence ellipse we can see that it is located inside the area of the ellipse . thus , the confidence ellipse indicates that when evaluated together neither is the nricases different from 0.02 nor is the nrinon - cases different from 0.035 . this example clearly indicates that evaluating single nri components separately might result in different decisions than evaluating the single components in combination by the use of confidence ellipses . these results were based on the asymptotic method for both the calculation of cis and of the confidence ellipse . the european prospective investigation into cancer and nutrition ( epic)-potsdam study is a prospective cohort study initially including 27,548 participants aged 3565 years . details of recruitment and follow - up procedures were described previously [ 7 , 8 ] . briefly , within a median follow - up time of 7 years , 849 participants out of 25,167 participants free of diabetes at baseline developed incident diabetes . on this basis , the german diabetes risk score ( gdrs ) was developed using cox - regression . with the gdrs the 5-year risk for developing future type 2 diabetes can be calculated using information on lifestyle and anthropometric factors , diet and physical activity . we used data from 21,846 participants ( 727 cases ) who had also information on family history of diabetes available . the extended model additionally included family history ; this model was compared with the reference model . table 1 shows the reclassification of cases and non - cases due to model extension based on the use of 5 predefined risk categories.table 1reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) n ( % ) gdrs + family historytotal1 : low2 : still low3 : increased4 : high5 : very high cases 1 : low 21 ( 2.89 ) 7 ( 0.96)28 ( 3.85)2 . still low13 ( 1.79 ) 102 ( 14.03 ) 30 ( 4.13)145 ( 19.94)3 . increased32 ( 4.40 ) 176 ( 24.21 ) 61 ( 8.39)269 ( 37.00)4 . high29 ( 3.99 ) 146 ( 20.08 ) 36 ( 4.95)211 ( 29.02)5 . very high15 ( 2.06 ) 59 ( 8.12 ) 74 ( 10.18)total34 ( 4.68)141 ( 19.39)235 ( 32.32)222 ( 30.54)95 ( 13.07)727 ( 100 ) non - cases 1 . low 9001 ( 42.62 ) 625 ( 2.96)9626 ( 45.58)2 . still low1415 ( 6.70 ) 4220 ( 19.98 ) 672 ( 3.18)6307 ( 29.86)3 . increased858 ( 4.06 ) 2613 ( 12.37 ) 387 ( 1.83)3858 ( 18.27)4 . high269 ( 1.27 ) 782 ( 3.70 ) 98 ( 0.46)1149 ( 5.44)5 . very high40 ( 0.19 ) 139 ( 0.66 ) 179 ( 0.85)total10,416 ( 49.32)5703 ( 27.0)3554 ( 16.83)1209 ( 5.72)237 ( 1.12)21,119 ( 100 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 based on the asymptotic method we determined 95 % cis for nricases and nrinon - cases ( fig . 1 ) . taking into account the large number of non - cases it is obvious that estimation of nrinon - cases was much more precise than of nricases . the calculation of cis for single components does not allow evaluating both components in combination.fig . 1confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . nri for cases and nri for non - cases were calculated by comparing the german diabetes risk score extended with family history of diabetes with the initial german diabetes risk score ; five predefined risk categories were used for calculation ; the confidence ellipse was defined with the accepted 1 and 2 values for = 0.05 . the area within the ellipse defines the area of accepting the null hypotheses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0 } : nri_{cases } = \theta_{1}$$\end{document}h0:nricases=1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = \theta_{2}$$\end{document}nrinon - cases=2 and the area outside the ellipse is the area of not accepting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0}$$\end{document}h0 . the black dot is the estimated for nri cases and nri non - cases . the horizontal and vertical reference lines display the lower and upper confidence limits of nri non - cases ( 0.0318 , 0.0440 ) and of nri cases the black diamond displays the discussed example for null hypotheses : nri cases = 0.02 and nri non - cases = 0.035 confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . nri for cases and nri for non - cases were calculated by comparing the german diabetes risk score extended with family history of diabetes with the initial german diabetes risk score ; five predefined risk categories were used for calculation ; the confidence ellipse was defined with the accepted 1 and 2 values for = 0.05 . the area within the ellipse defines the area of accepting the null hypotheses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0 } : nri_{cases } = \theta_{1}$$\end{document}h0:nricases=1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = \theta_{2}$$\end{document}nrinon - cases=2 and the area outside the ellipse is the area of not accepting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{0}$$\end{document}h0 . the black dot is the estimated for nri cases and nri non - cases . the horizontal and vertical reference lines display the lower and upper confidence limits of nri non - cases ( 0.0318 , 0.0440 ) and of nri cases the black diamond displays the discussed example for null hypotheses : nri cases = 0.02 and nri non - cases = 0.035 therefore , we computed a confidence ellipse for nricases and nrinon - cases to reflect precision of their estimates in combination and which also allows to evaluate the area of acceptable values . figure 1 shows cis for the single components ( vertical and horizontal lines ) as well as the confidence ellipse , both approaches were based on the five risk categories described before . when constructing cis for the components separately , nricases ( 0.0619 ) has a ci of 0.02190.1019 . therefore , the value 0.02 lies outside of this interval while the nrinon - cases ( 0.0379 ) had a ci ranging from 0.0318 to 0.0440 thus including a value of 0.035 . using both cis separately would therefore lead to the conclusion that nricases is significantly higher than 0.02 while nrinon - cases is not significantly higher than 0.035 . however , examining the vector ( 0.02 , 0.035 ) within the confidence ellipse we can see that it is located inside the area of the ellipse . thus , the confidence ellipse indicates that when evaluated together neither is the nricases different from 0.02 nor is the nrinon - cases different from 0.035 . this example clearly indicates that evaluating single nri components separately might result in different decisions than evaluating the single components in combination by the use of confidence ellipses . these results were based on the asymptotic method for both the calculation of cis and of the confidence ellipse . the use of the nri is informative for the evaluation of improvements of prediction models when taking into account the obvious limitations associated with the use of categories and cut - offs . given that no established cut - offs for the nri exist which allow interpreting its value as being meaningful from a clinical or public health point of view , reliance solely on significance testing has been frequently adopted in reclassification analyses . as recommended in a recent review of the nri methods , it is preferable to investigate model improvement separately for cases or non - cases . a general framework for testing the two components of the overall nri , nricases and nrinon - cases , has previously been laid out by pencina et al . . however , a major drawback of examining single components in isolation is that the results can not be interpreted in terms of the overall model improvement . we note that recent recommendations suggest not applying statistical testing at all [ 2 , 3 ] . a particularly appealing approach is based on using the confidence ellipse which reflects the 2-dimensional nature of the situation . our empirical example indicates that confidence ellipses can be useful in reflecting both , the precision of the nri estimation as well as putting the results in the context of overall improvement . our proposed method of confidence ellipses is also flexible here as it can be applied to evaluating extensions of prediction models using equal or different weights as well as thresholds of acceptable model improvement for cases and non - cases as already discussed by greenland . in conclusion , confidence ellipses might be particularly useful in the context of evaluating overall or case- versus non - case - specific model improvement as they allow evaluating varying acceptable values of the nri components in combination and also reflect the precision of their estimates .

the net reclassification improvement ( nri ) has become a popular metric for evaluating improvement in disease prediction models through the past years . the concept is relatively straightforward but usage and interpretation has been different across studies . while no thresholds exist for evaluating the degree of improvement , many studies have relied solely on the significance of the nri estimate . however , recent studies recommend that statistical testing with the nri should be avoided . we propose using confidence ellipses around the estimated values of event and non - event nris which might provide the best measure of variability around the point estimates . our developments are illustrated using practical examples from epic - potsdam study .

Background Definition of NRI Confidence ellipse for two components of NRI Empirical data Study population Calculation of Confidence Intervals and Confidence ellipses Discussion

for the evaluation of such model extensions , the net reclassification improvement ( nri ) was proposed by pencina et al . very high40 ( 0.19 ) 139 ( 0.66 ) 179 ( 0.85)total10,416 ( 49.32)5703 ( 27.0)3554 ( 16.83)1209 ( 5.72)237 ( 1.12)21,119 ( 100 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 based on the asymptotic method we determined 95 % cis for nricases and nrinon - cases ( fig . 1confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . the horizontal and vertical reference lines display the lower and upper confidence limits of nri non - cases ( 0.0318 , 0.0440 ) and of nri cases the black diamond displays the discussed example for null hypotheses : nri cases = 0.02 and nri non - cases = 0.035 confidence ellipse for the two - dimensional estimate = ( 1 , 2 ) of the calculated nri cases and nri non - cases , epic - potsdam study . table 1 shows the reclassification of cases and non - cases due to model extension based on the use of 5 predefined risk categories.table 1reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) n ( % ) gdrs + family historytotal1 : low2 : still low3 : increased4 : high5 : very high cases 1 : low 21 ( 2.89 ) 7 ( 0.96)28 ( 3.85)2 . very high40 ( 0.19 ) 139 ( 0.66 ) 179 ( 0.85)total10,416 ( 49.32)5703 ( 27.0)3554 ( 16.83)1209 ( 5.72)237 ( 1.12)21,119 ( 100 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 reclassification table by cases and non - cases resulting from adding family history of diabetes to the german drs ( gdrs ) , epic - potsdam cohort ( n = 21,846 ) risk categories were created according to score points of the german diabetes risk score : low risk : < 410 points ( 5-year risk < 0.88 % ) ; still low : 410<510 ( 0.88<2.37 % ) ; increased risk : 510<610 ( 2.37<6.30 % ) ; high risk : 610<710 ( 6.30<16.21 % ) ; very high risk : 710 ( 16.21 % ) nri measures were calculated as follows \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{cases } = ( \left ( { 0.96 + 4.13 + 8.39 + 4.95 } \right ) - \left ( { 1.79 + 4.40 + 3.99 + 2.06 } \right))/100 = ( 18.43 - 12.24)/100 = 0.0619$$\end{document}nricases=(0.96 + 4.13 + 8.39 + 4.95 - 1.79 + 4.40 + 3.99 + 2.06)/100=(18.43 - 12.24)/100=0.0619 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri_{{non { - } cases } } = ( \left ( { 6.70 + 4.06 + 1.27 + 0.19 } \right ) - \left ( { 2.96 + 3.18 + 1.83 + 0.46 } \right))/100 = ( 12.22 - 8.43)/100 = 0.0379$$\end{document}nrinon - cases=(6.70 + 4.06 + 1.27 + 0.19 - 2.96 + 3.18 + 1.83 + 0.46)/100=(12.22 - 8.43)/100=0.0379 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$nri = 0.0619 + 0.0379 = 0.0998$$\end{document}nri=0.0619 + 0.0379=0.0998 based on the asymptotic method we determined 95 % cis for nricases and nrinon - cases ( fig . in conclusion , confidence ellipses might be particularly useful in the context of evaluating overall or case- versus non - case - specific model improvement as they allow evaluating varying acceptable values of the nri components in combination and also reflect the precision of their estimates .

resistin and other molecules commonly called adipokines , such as tnf- , il-6 , and visfatin , are produced and secreted by both adipocytes and macrophages , and have been suggested to be important players in the pathogenesis of insulin resistance and atherosclerosis [ 1 , 2 ] . increased production of these adipokines occurs with expanding obesity , particularly visceral obesity , by both the adipocytes and the nonfat cells , mostly macrophages that infiltrate the adipose tissue [ 3 , 4 ] . indeed , the common origin of macrophages and adipocytes has been well documented as well as their overlapping biology and function ( accumulation of lipids , expression and secretion of similar cytokines , such as tnf- , il-6 , resistin , visfatin , and expression of genes involved in lipid metabolism ) [ 4 , 5 ] . furthermore , recruitment and accumulation of macrophages in the adipose tissue are characteristics of obesity . interestingly , in humans , the expression of resistin and visfatin is predominantly detected in the macrophages populating the adipose tissue . several studies have also demonstrated increased expression and release of the proinflammatory cytokines , tnf- , il-1 , and il-6 , from the adipocytes or the macrophages infiltrating the adipose tissue [ 3 , 7 ] . notably , resistin belongs to a family of molecules called found in inflammatory zone ( fizz ) , since a member of this family was found to be expressed in bronchial epithelial cells during allergic pulmonary inflammation in mice . in human mononuclear cells , where resistin is highly expressed , its expression is markedly upregulated by lipopolysaccharide ( lps ) and the proinflammatory cytokines ( il-6 , tnf- , and il-1 ) , acting via the nf-b - dependent pathway . furthermore , intravenous administration of endotoxin in humans markedly increased circulating resistin levels [ 9 , 10 ] . in agreement with these experimental data , patients with severe inflammatory disease have significantly higher serum resistin concentrations , while patients with rheumatoid arthritis ( ra ) show increased resistin levels in their inflamed joints that correlate with markers of inflammation . furthermore , ppar agonists and hmg - coa reductase inhibitors ( statins ) , both recognized for their anti - inflammatory properties , decrease macrophage resistin mrna and protein secretion , apparently via the nf-b pathway [ 12 , 13 ] . consistent with the proinflammatory nature of resistin , human resistin powerfully stimulates tnf- , il-1 , il-6 , and il-12 expression in human pbmc and murine macrophages , an effect that can be abrogated by an nf-b inhibitor , indicating the importance of this signalling pathway in the resistin - mediated inflammation . resistin also acts on endothelial cells and promotes their activation , by inducing the expression and release of monocyte chemoattractant protein ( mcp)-1 , endothelin-1 ( et-1 ) , and adhesion molecules , such as vascular cell adhesion molecule -1 ( vcam-1 ) and intracellular adhesion molecule-1 ( icam-1 ) , while at the same time adiponectin abrogates this effect . moreover , resistin is secreted by the macrophages localizing to the atheromas , thereby promoting atherosclerosis in humans . indeed , patients with coronary artery disease ( cad ) have higher serum resistin levels that correlate with markers of inflammation , and are predictive of coronary atherosclerosis in humans [ 18 , 19 ] . high serum resistin levels have also been proposed as predictors of early atherosclerosis in obese children . while resistin 's role as an inflammatory marker in human 's and rodent 's physiology has been well documented , its role in obesity and insulin resistance in humans is still under debate . it is important that human resistin is expressed in much higher levels in macrophages than in adipocytes , quite the opposite from the case of mice , in which resistin clearly impairs insulin sensitivity [ 22 , 23 ] . several human studies have failed to confirm any relationship of circulating resistin levels with bmi , insulin sensitivity , or other metabolic parameters [ 2426 ] . however , there have been reports of increased serum resistin levels in individuals with obesity and/or type 2 diabetes [ 2729 ] . in the present study , we examined whether resistin tnf- , il-6 , and il-1 mrna levels from human peripheral mononuclear cells are altered in type 2 diabetes and whether they correlate with circulating resistin levels and with indices of obesity or insulin resistance . the study was approved by the alexandra hospital ethical committee and all subjects gave informed consent . forty eight ( 48 ) women , all premenopausal , aged between 21 and 49 years , participated in our study . twenty two had type 2 diabetes ( dm2 ) , and twenty six were healthy controls with normal glucose tolerance ( ngt ) . both groups were further divided into two groups : those with bmi < 27 and those with bmi > 27 ( table 1 ) . all women have had a stable weight , and they were on a normal eucaloric diet and normal physical activity , not participating in any specific exercise program for the last three months preceding the test . of the dm2 women , 15 were on diet alone , 4 on oral hypoglycaemic agents , 2 on insulin , and 1 on combined insulin and oral hypoglycaemic agents . all control subjects had no history of endocrine abnormality and were on no medications . body mass index ( bmi ) was calculated as the ratio of body weight ( kg ) per height ( m ) . insulin resistance was calculated by the homeostasis model assessment index ( homa - ir ) as [ fasting insulin ( u / ml ) fasting glucose ( mmol / l)]/22.5 . insulin sensitivity was calculated by the matsuda index as ( 10 000/sqare root of [ fasting glucose fasting insulin ] [ mean glucose during ogtt mean insulin during ogtt ] ) . fasting glucose , triglycerides , and free fatty acid ( ffa ) levels were analyzed on a falcor 300 chemical analyser ( menarini diagnostics , italy ) and fasting plasma insulin levels were measured by irma ( insi - ctk , diasorin , italy ) , in all individuals . peripheral blood was collected from all women after an overnight fast and the mononuclear cells were separated using a histopaque-1077 gradient ( sigma - aldrich , usa ) . at the same time , plasma was kept at 80c for subsequent measurement of adipokines . the mononuclear cells were then left to adhere for 1 hour for the selective isolation of the primary monocyte - derived macrophages , and total rna was extracted from cells as previously described . briefly , after the rna isolation , cdna synthesis was performed in all samples using oligo random primers ( neb , mass , usa ) and mmlv reverse transcriptase ( promega corp . , wis , usa ) . we developed a quantitative real - time pcr using fluorescently labeled hybridization probes to detect resistin and proinflammatory ( il-1 , tnf- , and il-6 ) mrna expression in human mononuclear cells , in a spectrofluorometric thermal cycler ( lightcycler , roche , mannheim , germany ) . hybridization - specific primers and hybridization - specific probes for all genes were designed and manufactured by the ( tib molbiol berlin , germany ) . hybridization primers and probes for all target and reference genes were showen in table 2 . primers were chosen to lie between exons whenever possible and the analysis was performed basically with the program oligo 6.0 ( molecular biology insights , cascade , colo , usa ) . relative quantification is the method that determines the changes in steady - state mrna levels of a gene across various samples and expresses them relative to the levels of an internal reference control gene , usually a housekeeping gene . in order to quantify resistin , il-1 , tnf- , and il-6 relative mrna levels , we used the calibrator normalized standard curve and the lightcycler relative quantification 1.0.1 software ( roche , mannheim , germany ) , as previously described . briefly , using this quantification method , results are expressed as the target / reference ratio of the sample divided by the target / reference ratio of the calibrator , and it does not require a standard curve in each run ( roche applied science , technical note no . the human housekeeping genes of -actin and porphobilinogen deaminase ( pbgd ) were used as standards for normalization . selection of either -actin or pbgd as housekeeping genes was necessary because the copy number of the housekeeping gene should be in a similar range with that of the target gene to make comparative quantification possible ( roche applied science , technical note no . thus , the pbgd house keeping gene was chosen for the relative quantification of the il-6 mrna expression because of the very low level of il-6 expression in the calibrator ( hl60 induced with phorbol myristyl acetate ( pma ) ) mrna . standard curves describing the pcr efficiencies of the target ( resistin , il-1 , tnf- , or il-6 ) and the reference genes ( -actin or pbgd ) were created from a dilution series of the calibrator cdna , using the second derivative maximum method with the arithmetic baseline adjustment . the second derivative maximum method was used for the determination of the various crossing points ( cps ) of the target and the reference gene in each sample . finally , a corresponding coefficient file was created , which determines the efficiency for the target and the reference gene . amplification of each sample was performed in triplicate , in different runs , and fluorescence resulting from the close proximity of the annealed hybridization probes was detected at the end of the annealing step of each pcr cycle . the results from different pcr runs were run randomly in ethidium bromide - stained agarose gels and photographed in an image analyser vds system ( amersham - pharmacia biotech , sweden ) . immunoassayscirculating levels of resistin ( biovender , canal zone , usa ) , tnf- ( r&d systems , uk ) , and il-6 ( r&d systems , uk ) were measured in the plasma of all women , using standard commercial enzyme - linked immunosorbent assays according to the manufacturers'-recommended protocols . il-1 circulating protein levels were measured in the plasma of all women , using a high sensitivity multiplex assay ( xmap technology ) and the fluorescently labeled microsphere beads ( linco - millipore corp . , we could not detect il-1 in the majority of our subjects ( in 57% of controls and 55% of dm2 ) , making the analysis of circulating il-1 problematic.the sensitivities of the assays were 0.2 ng / ml ( resistin ) , 0.12 pg / ml ( tnf- ) , < 0.094 pg / ml ( il-6 ) , 0.06 pg / ml ( il-1 ) , the intra- and the interassay coefficients of variation were , respectively , 3.6% and 6.7% ( resistin ) , 6.6% and 13.4% ( tnf- ) , 6.9% and 14.1% ( il-6 ) , 3.11% and 2.16% ( il-1 ) . circulating levels of resistin ( biovender , canal zone , usa ) , tnf- ( r&d systems , uk ) , and il-6 ( r&d systems , uk ) were measured in the plasma of all women , using standard commercial enzyme - linked immunosorbent assays according to the manufacturers'-recommended protocols . il-1 circulating protein levels were measured in the plasma of all women , using a high sensitivity multiplex assay ( xmap technology ) and the fluorescently labeled microsphere beads ( linco - millipore corp . , we could not detect il-1 in the majority of our subjects ( in 57% of controls and 55% of dm2 ) , making the analysis of circulating il-1 problematic . the sensitivities of the assays were 0.2 ng / ml ( resistin ) , 0.12 pg / ml ( tnf- ) , < 0.094 pg / ml ( il-6 ) , 0.06 pg / ml ( il-1 ) , the intra- and the interassay coefficients of variation were , respectively , 3.6% and 6.7% ( resistin ) , 6.6% and 13.4% ( tnf- ) , 6.9% and 14.1% ( il-6 ) , 3.11% and 2.16% ( il-1 ) . statistical analysis was performed using the spss version 14.0.1 software ( chicago , ill , usa ) . nonparametric statistical analyses were applied ( spearman 's correlation test and nonparametric mann - whitney test ) , where appropriate . table 1 shows the clinical characteristics of the dm2 and healthy ngt women , who were further divided into two subgroups according to their bmi , one with bmi < 27 and one with bmi > 27 . dm2 women , however , had significantly higher waist circumference , triglyceride and ffa levels , and lower hdl cholesterol levels compared to the bmi - matched controls . resistin , tnf- , il-6 , il-1 mrna expression from human mononuclear - monocytic cells is elevated in dm2 women . resistin mrna expression was easily detected in human mononuclear - monocytic cells in both dm2 and ngt women , and was significantly higher ( 0.57 0.1 versus 0.52 0.1 arbitrary units , p = .05 ) in the former group ( figure 1(a ) ) , while no significant differences were observed between the two bmi subgroups ( table 3 ) . tnf- , il-6 mrna and il-1 mrna levels were significantly higher ( 10- , 15- , and 10-fold , resp . ) in dm2 women compared to ngt women [ 0.34 0.1 versus 0.03 0.01 ( p < .001 ) , 342.98 127.9 versus 21.64 12.6 ( p < .001 ) , and 139.36 40.2 versus 13.21 7.8 arbitrary units ( p = .001 ) , resp . ] resistin mrna levels correlated positively with mononuclear il-1 , and tnf- , mrna levels ( r = 0.324 , p < .05 and r = 0.447 , p < .01 , resp . ) . furthermore , tnf- and il-6 mrna levels correlated positively between them ( r = 0.687 , p < .001 ) and also each one of them with the il-1 mrna levels ( r = 0.767 , p < .001 and r = 0.765 , p < .001 , resp . ) , the 2-hour ogtt glucose levels ( r = 0.462 , p = .005 and r = 0.486 , p = .004 , resp . ) and negatively with hdl cholesterol levels ( r = 0.367 , p < .02 and r = 0.372 , p < .02 , resp . ) , while il-1 mrna levels correlated positively with bmi ( r = 0.374 , p = .017 ) , waist circumference ( r = 0.414 , p = .019 ) , fasting glucose levels ( r = 0.365 , p = .022 ) , homa - ir ( r = 0.325 , p = .043 ) , glycosylated hemoglobin ( r = 0.542 , p = .001 ) , ffa levels ( r = 0.335 , p = .043 ) , and negatively with hdl cholesterol levels ( r = 0.395 , p = .015 ) . plasma resistin levels tended to be overall significantly higher in dm2 women compared to ngt control women ( p = .051 ) ( figure 2 ) , but no significant differences were observed between the two bmi subgroups . circulating tnf- , il-6 , and il-1 protein levels did not differentiate between the dm2 and the ngt - control women ( figure 2 ) , although in the subgroup of dm2 with bmi > 27 , tnf- and il-6 plasma levels were significantly higher compared to ngt women ( table 4 ) . circulating resistin correlated positively with mononuclear resistin , il-6 and il-1 mrna expression ( r = 0.526 , p = .001 and r = 0.354 , p = .029 and r = 0.425 , p = .009 , resp . ) , bmi and waist circumference ( r = 0.406 , p = .010 and r = 0.516 , p = .003 ) , but not with homa - ir , fasting insulin , or glucose plasma levels . tnf- and il-6 plasma levels also correlated with bmi and waist circumference ( r = 0.361 , p = .013 and r = 0.651 , p < .001 and r = 0.379 , p = .023 and r = 0.676 , p < .001 , resp . ) as well as with fasting glucose , insulin and triglyceride levels , the homa - ir , while they correlated negatively with the matsuda index and hdl cholesterol levels . we have demonstrated that resistin mrna expression from human peripheral monocyte - enriched mononuclear cells is elevated in type 2 diabetic women , compared to healthy control women , in parallel with significant increases in mononuclear il-1 , tnf- , and il-6 mrna expression . resistin , a 12.5-kda cysteine - rich adipokine , which was shown to cause insulin resistance and to impair glucose tolerance in rodents , is also highly expressed in human mononuclear cells and macrophages . while in rodents , adipocyte - produced resistin is mainly involved in glucose metabolism and insulin resistance : in humans , monocyte - produced resistin seems to have potent proinflammatory properties . various inflammatory stimuli could result in hyperresistinemia in humans [ 9 , 10 ] , while human resistin can stimulate the expression of the inflammatory cytokines tnf- and il-6 in both human and murine macrophages , via an nf-b - dependent pathway [ 11 , 14 ] . the proinflammatory nature of resistin was further documented by the findings that serum resistin concentrations were significantly elevated in patients with severe inflammatory disease and in patients with previous myocardial infarction , in whom they positively correlated with markers of inflammation , independent of crp [ 11 , 1820 ] . in the present study , we have demonstrated that resistin mrna levels from human peripheral monocyte - enriched mononuclear cells were higher in the dm2 compared to healthy women , which was independent of bmi . it should be noted that the difference did not reach significance between the individuals groups , but only when all patients were grouped together in ngt and dm2 . this is obviously due to the small number of individuals used in our study , which is a weakness of our data . the above overall difference may suggest that , unlike inflammatory cytokine mrna expression from human mononuclear cells , mononuclear resistin mrna is not appreciably increased in vivo with chronic hyperinsulinemia that characterizes obesity . in support of this , in vitro studies demonstrated that insulin decreases resistin mrna from human monocytes , while in the 3t3-l1 adipocytes , insulin downregulated resistin mrna and protein secretion possibly through the pi 3-kinase , erk , or p38 map - kinase pathways. recently , however , another study demonstrated that acute hyperinsulinemia is associated with increased resistin mrna expression in human subcutaneous adipose tissue and a similar increase in plasma resistin levels . macrophages and adipocytes share an overlapping biology and function , and infiltration of the adipose tissue with the macrophages is characteristic of obesity . several recent studies suggest that obesity is associated with an increase in adipose tissue macrophages , which also participate in the inflammatory process through the elaboration of cytokines [ 3 , 4 , 34 ] . proinflammatory cytokines ( tnf- and il-6 ) and adipokines ( resistin and visfatin ) , produced by the peripheral macrophages , can also be expressed in increased amounts in the fat tissue of obese individuals acting in parallel as important regulators of inflammation , atherosclerosis , and insulin sensitivity in both tissues . in a recent study , the expression of two adipokines , resistin , and visfatin was detected predominantly in the macrophages populating the human visceral fat tissue , suggesting that these adipokines might have an important role in the inflammatory load of obesity . the putative role of resistin in obesity - induced insulin resistance was confirmed by studies , in rodents , demonstrating that administration of recombinant resistin impairs hepatic insulin sensitivity and glucose metabolism , while possibly playing a role in maintaining fasting blood glucose levels . this was debated by various human studies demonstrating that there is no relationship of circulating resistin levels with bmi , insulin sensitivity , or other metabolic parameters [ 2426 ] . however , other studies found increased serum resistin levels in individuals with obesity and/or type 2 diabetes [ 2729 ] . the above conflicting data lead to the ascertainment that despite the initial belief that resistin could be the factor linking obesity and type 2 diabetes , its biologic significance in the pathogenesis of insulin resistance , at least in humans , is under controversy . in fact , while in rodents adipose tissue is the only source of resistin production , in humans resistin is barely detectable in adipocytes , and actual resistin mrna levels are much higher in monocytes and macrophages than in adipocytes . in the present study , we have additionally demonstrated that plasma resistin levels are marginally increased in type 2 diabetic women compared to healthy women , and this increase was detected in both subgroups , those with bmi < 27 and those with bmi > 27 . thus , our data are inline with previous reports that found elevated serum resistin levels in type 2 diabetic subjects compared with nondiabetic subjects [ 28 , 29 , 36 ] . however , the observed positive correlation of circulating resistin with bmi and waist circumference might support some association of resistin with obesity - induced insulin resistance . therefore , the observed increased plasma resistin levels in the dm2 women could be due to the increased visceral obesity , as it reveals the higher waist circumference of these patients . circulating il-1 did not differentiate between the healthy control and the dm2 women , a result that lines with previous studies in which il-1 alone can not associate with the risk of developing type 2 diabetes , while il-1 was even decreased in an impaired glucose tolerance ( igt ) group compared to ngt subjects [ 37 , 38 ] . it should be emphasized that due to the large number of subjects in whom plasma il-1 levels were undetectable , they should be interpreted with caution . tnf- and il-6 plasma levels were also similar between the ngt and the dm2 women of our study , although their levels are significantly increased in the subgroup of dm2 women with bmi > 27 , compared to ngt with bmi < 27 women ; moreover , they were significantly correlated with the obesity and insulin resistance indices . while in the epic - potsdam study the elevated levels of tnf- and il-6 were found to be strongly associated with future type 2 diabetes , other studies failed to demonstrate increased tnf- and il-6 plasma levels in dm2 patients [ 37 , 39 ] . the above discrepancy could be due to the small number of subjects used in the later study , as it was also in our case , and due to ethnic differences between the various studies . obesity - induced insulin resistance is characterized by a chronic , systemic low - grade state of inflammation and is strongly associated with inflammatory markers , while inflammation may contribute to insulin resistance and atherosclerosis [ 2 , 5 , 40 ] . therefore , with expanding obesity and increased macrophage infiltration of the adipose tissue , there is an increased cytokine production by both cell populations , with all the detrimental metabolic consequences on insulin resistance and on the inflammatory status [ 4 , 6 , 41 ] . in the present study , we have demonstrated that in type 2 diabetes , there is an increased mrna expression of resistin , along with significant increases in tnf- , il-6 , and il-1 mrna expression by the peripheral circulating mononuclear cells . the results from this study , together with previous data , showing higher mononuclear visfatin mrna expression in the dm2 women , suggest that an overload of mrna expression of inflammatory cytokines and adipokines occurs in the mononuclear cells from type 2 diabetic patients , aggravating the proinflammatory status of this condition . these data further support the notion that the peripheral circulating mononuclear cells can indeed be an important source of resistin production in humans . thus , in type 2 diabetes , macrophages that infiltrate the stroma of the adipose tissue and/or the vascular endothelium can locally produce and secrete increased resistin and cytokine ( il-1 , tnf- , and il-6 ) levels , which may enhance the inflammatory load and the associated insulin resistance and vascular dysfunction observed in these patients .

resistin has been shown to cause insulin resistance and to impair glucose tolerance in rodents , but in humans its physiological role still remains elusive . the aim of this study was to examine whether resistin mrna expression in human peripheral mononuclear cells ( pbmcs ) and its corresponding plasma levels are altered in type 2 diabetes . resistin mrna levels were easily detectable in human pbmc , and found to be higher in dm2 compared to healthy women ( p = .05 ) . similarly , mononuclear mrna levels of the proinflammatory cytokines il-1 , tnf- , and il-6 were all significantly higher in dm2 compared to control women ( p < .001 ) . the corresponding plasma resistin levels were slightly , but not significantly , increased in dm2 women ( p = .051 ) , and overall , they correlated significantly with bmi ( r = 0.406 , p = .010 ) and waist circumference ( r = 0.516 , p = .003 ) , but not with fasting insulin levels or homa - ir . resistin mrna expression is increased in pbmc from dm2 women , together with increased expression of the inflammatory cytokines il-1 , tnf- , and il-6 , independent of obesity . these results suggest that resistin and cytokines might contribute to the low - grade inflammation and the increased atherogenic risk observed in these patients .

1. INTRODUCTION 2. SUBJECTS AND METHODS 3. RESULTS 4. DISCUSSION

resistin and other molecules commonly called adipokines , such as tnf- , il-6 , and visfatin , are produced and secreted by both adipocytes and macrophages , and have been suggested to be important players in the pathogenesis of insulin resistance and atherosclerosis [ 1 , 2 ] . indeed , the common origin of macrophages and adipocytes has been well documented as well as their overlapping biology and function ( accumulation of lipids , expression and secretion of similar cytokines , such as tnf- , il-6 , resistin , visfatin , and expression of genes involved in lipid metabolism ) [ 4 , 5 ] . several studies have also demonstrated increased expression and release of the proinflammatory cytokines , tnf- , il-1 , and il-6 , from the adipocytes or the macrophages infiltrating the adipose tissue [ 3 , 7 ] . notably , resistin belongs to a family of molecules called found in inflammatory zone ( fizz ) , since a member of this family was found to be expressed in bronchial epithelial cells during allergic pulmonary inflammation in mice . in human mononuclear cells , where resistin is highly expressed , its expression is markedly upregulated by lipopolysaccharide ( lps ) and the proinflammatory cytokines ( il-6 , tnf- , and il-1 ) , acting via the nf-b - dependent pathway . consistent with the proinflammatory nature of resistin , human resistin powerfully stimulates tnf- , il-1 , il-6 , and il-12 expression in human pbmc and murine macrophages , an effect that can be abrogated by an nf-b inhibitor , indicating the importance of this signalling pathway in the resistin - mediated inflammation . resistin also acts on endothelial cells and promotes their activation , by inducing the expression and release of monocyte chemoattractant protein ( mcp)-1 , endothelin-1 ( et-1 ) , and adhesion molecules , such as vascular cell adhesion molecule -1 ( vcam-1 ) and intracellular adhesion molecule-1 ( icam-1 ) , while at the same time adiponectin abrogates this effect . indeed , patients with coronary artery disease ( cad ) have higher serum resistin levels that correlate with markers of inflammation , and are predictive of coronary atherosclerosis in humans [ 18 , 19 ] . while resistin 's role as an inflammatory marker in human 's and rodent 's physiology has been well documented , its role in obesity and insulin resistance in humans is still under debate . in the present study , we examined whether resistin tnf- , il-6 , and il-1 mrna levels from human peripheral mononuclear cells are altered in type 2 diabetes and whether they correlate with circulating resistin levels and with indices of obesity or insulin resistance . twenty two had type 2 diabetes ( dm2 ) , and twenty six were healthy controls with normal glucose tolerance ( ngt ) . of the dm2 women , 15 were on diet alone , 4 on oral hypoglycaemic agents , 2 on insulin , and 1 on combined insulin and oral hypoglycaemic agents . insulin resistance was calculated by the homeostasis model assessment index ( homa - ir ) as [ fasting insulin ( u / ml ) fasting glucose ( mmol / l)]/22.5 . fasting glucose , triglycerides , and free fatty acid ( ffa ) levels were analyzed on a falcor 300 chemical analyser ( menarini diagnostics , italy ) and fasting plasma insulin levels were measured by irma ( insi - ctk , diasorin , italy ) , in all individuals . the mononuclear cells were then left to adhere for 1 hour for the selective isolation of the primary monocyte - derived macrophages , and total rna was extracted from cells as previously described . we developed a quantitative real - time pcr using fluorescently labeled hybridization probes to detect resistin and proinflammatory ( il-1 , tnf- , and il-6 ) mrna expression in human mononuclear cells , in a spectrofluorometric thermal cycler ( lightcycler , roche , mannheim , germany ) . in order to quantify resistin , il-1 , tnf- , and il-6 relative mrna levels , we used the calibrator normalized standard curve and the lightcycler relative quantification 1.0.1 software ( roche , mannheim , germany ) , as previously described . thus , the pbgd house keeping gene was chosen for the relative quantification of the il-6 mrna expression because of the very low level of il-6 expression in the calibrator ( hl60 induced with phorbol myristyl acetate ( pma ) ) mrna . standard curves describing the pcr efficiencies of the target ( resistin , il-1 , tnf- , or il-6 ) and the reference genes ( -actin or pbgd ) were created from a dilution series of the calibrator cdna , using the second derivative maximum method with the arithmetic baseline adjustment . immunoassayscirculating levels of resistin ( biovender , canal zone , usa ) , tnf- ( r&d systems , uk ) , and il-6 ( r&d systems , uk ) were measured in the plasma of all women , using standard commercial enzyme - linked immunosorbent assays according to the manufacturers'-recommended protocols . il-1 circulating protein levels were measured in the plasma of all women , using a high sensitivity multiplex assay ( xmap technology ) and the fluorescently labeled microsphere beads ( linco - millipore corp . circulating levels of resistin ( biovender , canal zone , usa ) , tnf- ( r&d systems , uk ) , and il-6 ( r&d systems , uk ) were measured in the plasma of all women , using standard commercial enzyme - linked immunosorbent assays according to the manufacturers'-recommended protocols . il-1 circulating protein levels were measured in the plasma of all women , using a high sensitivity multiplex assay ( xmap technology ) and the fluorescently labeled microsphere beads ( linco - millipore corp . table 1 shows the clinical characteristics of the dm2 and healthy ngt women , who were further divided into two subgroups according to their bmi , one with bmi < 27 and one with bmi > 27 . dm2 women , however , had significantly higher waist circumference , triglyceride and ffa levels , and lower hdl cholesterol levels compared to the bmi - matched controls . resistin , tnf- , il-6 , il-1 mrna expression from human mononuclear - monocytic cells is elevated in dm2 women . resistin mrna expression was easily detected in human mononuclear - monocytic cells in both dm2 and ngt women , and was significantly higher ( 0.57 0.1 versus 0.52 0.1 arbitrary units , p = .05 ) in the former group ( figure 1(a ) ) , while no significant differences were observed between the two bmi subgroups ( table 3 ) . tnf- , il-6 mrna and il-1 mrna levels were significantly higher ( 10- , 15- , and 10-fold , resp . ) in dm2 women compared to ngt women [ 0.34 0.1 versus 0.03 0.01 ( p < .001 ) , 342.98 127.9 versus 21.64 12.6 ( p < .001 ) , and 139.36 40.2 versus 13.21 7.8 arbitrary units ( p = .001 ) , resp . ] resistin mrna levels correlated positively with mononuclear il-1 , and tnf- , mrna levels ( r = 0.324 , p < .05 and r = 0.447 , p < .01 , resp . ) furthermore , tnf- and il-6 mrna levels correlated positively between them ( r = 0.687 , p < .001 ) and also each one of them with the il-1 mrna levels ( r = 0.767 , p < .001 and r = 0.765 , p < .001 , resp . ) , the 2-hour ogtt glucose levels ( r = 0.462 , p = .005 and r = 0.486 , p = .004 , resp . ) and negatively with hdl cholesterol levels ( r = 0.367 , p < .02 and r = 0.372 , p < .02 , resp . ) , while il-1 mrna levels correlated positively with bmi ( r = 0.374 , p = .017 ) , waist circumference ( r = 0.414 , p = .019 ) , fasting glucose levels ( r = 0.365 , p = .022 ) , homa - ir ( r = 0.325 , p = .043 ) , glycosylated hemoglobin ( r = 0.542 , p = .001 ) , ffa levels ( r = 0.335 , p = .043 ) , and negatively with hdl cholesterol levels ( r = 0.395 , p = .015 ) . plasma resistin levels tended to be overall significantly higher in dm2 women compared to ngt control women ( p = .051 ) ( figure 2 ) , but no significant differences were observed between the two bmi subgroups . circulating tnf- , il-6 , and il-1 protein levels did not differentiate between the dm2 and the ngt - control women ( figure 2 ) , although in the subgroup of dm2 with bmi > 27 , tnf- and il-6 plasma levels were significantly higher compared to ngt women ( table 4 ) . circulating resistin correlated positively with mononuclear resistin , il-6 and il-1 mrna expression ( r = 0.526 , p = .001 and r = 0.354 , p = .029 and r = 0.425 , p = .009 , resp . ) , bmi and waist circumference ( r = 0.406 , p = .010 and r = 0.516 , p = .003 ) , but not with homa - ir , fasting insulin , or glucose plasma levels . tnf- and il-6 plasma levels also correlated with bmi and waist circumference ( r = 0.361 , p = .013 and r = 0.651 , p < .001 and r = 0.379 , p = .023 and r = 0.676 , p < .001 , resp . ) as well as with fasting glucose , insulin and triglyceride levels , the homa - ir , while they correlated negatively with the matsuda index and hdl cholesterol levels . we have demonstrated that resistin mrna expression from human peripheral monocyte - enriched mononuclear cells is elevated in type 2 diabetic women , compared to healthy control women , in parallel with significant increases in mononuclear il-1 , tnf- , and il-6 mrna expression . resistin , a 12.5-kda cysteine - rich adipokine , which was shown to cause insulin resistance and to impair glucose tolerance in rodents , is also highly expressed in human mononuclear cells and macrophages . while in rodents , adipocyte - produced resistin is mainly involved in glucose metabolism and insulin resistance : in humans , monocyte - produced resistin seems to have potent proinflammatory properties . various inflammatory stimuli could result in hyperresistinemia in humans [ 9 , 10 ] , while human resistin can stimulate the expression of the inflammatory cytokines tnf- and il-6 in both human and murine macrophages , via an nf-b - dependent pathway [ 11 , 14 ] . the proinflammatory nature of resistin was further documented by the findings that serum resistin concentrations were significantly elevated in patients with severe inflammatory disease and in patients with previous myocardial infarction , in whom they positively correlated with markers of inflammation , independent of crp [ 11 , 1820 ] . in the present study , we have demonstrated that resistin mrna levels from human peripheral monocyte - enriched mononuclear cells were higher in the dm2 compared to healthy women , which was independent of bmi . the above overall difference may suggest that , unlike inflammatory cytokine mrna expression from human mononuclear cells , mononuclear resistin mrna is not appreciably increased in vivo with chronic hyperinsulinemia that characterizes obesity . recently , however , another study demonstrated that acute hyperinsulinemia is associated with increased resistin mrna expression in human subcutaneous adipose tissue and a similar increase in plasma resistin levels . proinflammatory cytokines ( tnf- and il-6 ) and adipokines ( resistin and visfatin ) , produced by the peripheral macrophages , can also be expressed in increased amounts in the fat tissue of obese individuals acting in parallel as important regulators of inflammation , atherosclerosis , and insulin sensitivity in both tissues . in a recent study , the expression of two adipokines , resistin , and visfatin was detected predominantly in the macrophages populating the human visceral fat tissue , suggesting that these adipokines might have an important role in the inflammatory load of obesity . the putative role of resistin in obesity - induced insulin resistance was confirmed by studies , in rodents , demonstrating that administration of recombinant resistin impairs hepatic insulin sensitivity and glucose metabolism , while possibly playing a role in maintaining fasting blood glucose levels . the above conflicting data lead to the ascertainment that despite the initial belief that resistin could be the factor linking obesity and type 2 diabetes , its biologic significance in the pathogenesis of insulin resistance , at least in humans , is under controversy . in fact , while in rodents adipose tissue is the only source of resistin production , in humans resistin is barely detectable in adipocytes , and actual resistin mrna levels are much higher in monocytes and macrophages than in adipocytes . in the present study , we have additionally demonstrated that plasma resistin levels are marginally increased in type 2 diabetic women compared to healthy women , and this increase was detected in both subgroups , those with bmi < 27 and those with bmi > 27 . however , the observed positive correlation of circulating resistin with bmi and waist circumference might support some association of resistin with obesity - induced insulin resistance . therefore , the observed increased plasma resistin levels in the dm2 women could be due to the increased visceral obesity , as it reveals the higher waist circumference of these patients . circulating il-1 did not differentiate between the healthy control and the dm2 women , a result that lines with previous studies in which il-1 alone can not associate with the risk of developing type 2 diabetes , while il-1 was even decreased in an impaired glucose tolerance ( igt ) group compared to ngt subjects [ 37 , 38 ] . tnf- and il-6 plasma levels were also similar between the ngt and the dm2 women of our study , although their levels are significantly increased in the subgroup of dm2 women with bmi > 27 , compared to ngt with bmi < 27 women ; moreover , they were significantly correlated with the obesity and insulin resistance indices . while in the epic - potsdam study the elevated levels of tnf- and il-6 were found to be strongly associated with future type 2 diabetes , other studies failed to demonstrate increased tnf- and il-6 plasma levels in dm2 patients [ 37 , 39 ] . obesity - induced insulin resistance is characterized by a chronic , systemic low - grade state of inflammation and is strongly associated with inflammatory markers , while inflammation may contribute to insulin resistance and atherosclerosis [ 2 , 5 , 40 ] . therefore , with expanding obesity and increased macrophage infiltration of the adipose tissue , there is an increased cytokine production by both cell populations , with all the detrimental metabolic consequences on insulin resistance and on the inflammatory status [ 4 , 6 , 41 ] . in the present study , we have demonstrated that in type 2 diabetes , there is an increased mrna expression of resistin , along with significant increases in tnf- , il-6 , and il-1 mrna expression by the peripheral circulating mononuclear cells . the results from this study , together with previous data , showing higher mononuclear visfatin mrna expression in the dm2 women , suggest that an overload of mrna expression of inflammatory cytokines and adipokines occurs in the mononuclear cells from type 2 diabetic patients , aggravating the proinflammatory status of this condition . thus , in type 2 diabetes , macrophages that infiltrate the stroma of the adipose tissue and/or the vascular endothelium can locally produce and secrete increased resistin and cytokine ( il-1 , tnf- , and il-6 ) levels , which may enhance the inflammatory load and the associated insulin resistance and vascular dysfunction observed in these patients .

atherosclerosis is thought to be the result of chronic inflammation of the artery wall although the pathways and factors that initiate and modulate the inflammatory response in atherosclerosis have yet to be completely resolved . the mouse 5-lo gene , alox5 , has been shown to contribute to the development of atherosclerosis . variants in the human homologue ( alox5 ) are associated with carotid artery intima - media thickness ( imt ) . flap ( 5-lipoxygenase activating protein ) , encoded by the alox5ap gene , likely acts as an arachidonic acid - binding and transfer protein to facilitate 5lo activity . single snps and haplotypes of alox5ap have been associated with myocardial infarction in multiple populations [ 57 ] . human 12-lipoxygenase ( encoded by alox12 ) and 15-lipoxygenase ( encoded by alox15 ) have been localized to atherosclerotic plaques , suggesting that 12/15lo activity is involved in the development of atherosclerosis [ 810 ] . human aortic endothelial cells cultured in chronically high glucose levels results in elevated levels of 12s - hete , suggesting activation of lipoxygenase pathways [ 12 , 13 ] . the current research was motivated by the role of lipoxygenase pathway gene products in inflammation , initiation / progression of atherosclerosis , and their modulation of expression by glucose . the alox12 , alox15 , alox5 , and alox5ap genes represent strong candidates for atherosclerosis risk , especially in the context of type 2 diabetes . we have assessed genetic variants ( snps ) in lipoxygenase pathway genes for evidence of association with markers of subclinical atherosclerosis ( intima - media wall thickness [ imt ] , carotid artery calcified plaque [ carcp ] , coronary artery calcified plaque [ corcp ] , and aortic calcified plaque [ aorcp ] ) and biomarkers ( e.g. , icam-1 , e - selectin , crp ) in participants of the diabetes heart study ( dhs ) . recruitment and phenotyping of diabetes heart study ( dhs ) participants have been previously described [ 1416 ] . siblings concordant for type 2 diabetes were recruited if they had no evidence of renal insufficiency , as were all available nondiabetic siblings . participant examinations were conducted in the general clinical research center of the wake forest university school of medicine , and included interviews for medical history , medication use and health behaviors , anthropometry , resting blood pressure , a fasting blood sampling , and a spot urine collection . only caucasian participants were included in this report , due to small sample size and limited statistical power in the african - american cohort . all study protocols were approved by the institutional review board of wake forest university school of medicine . all participants provided written informed consent . intima - media thickness ( imt ) of the common carotid artery was measured by high - resolution b - mode ultrasonography with a 7.5-mhz transducer and a biosound esaote ( au5 ) ultrasound machine . the mean of up to 20 common carotid imt estimates was used as the phenotypic . calcified plaque was measured in the carotid and coronary arteries and the aorta using single and multidetector ct systems that employ a standardized protocol which includes ct scanner phantom testing based on those currently implemented in the national heart lung and blood institute 's ( nhlbi ) multiethnic study of atherosclerosis ( mesa ) studies [ 15 , 17 ] . total genomic dna was purified from whole blood samples obtained from subjects using puregene dna isolation kit ( gentra , inc . , dna was quantitated using standardized fluorometric readings on a hoefer dyna quant 200 fluorometer ( hoefer pharmacia biotech inc . single nucleotide polymorphisms ( snps ) were identified for alox12 ( 17p13.1 ) , alox15 ( 17p13.3 ) , alox5 ( 10q11.2 ) , and alox5ap ( 13q12 ) and were chosen for genotyping in order to provide coverage of linkage disequilibrium ( ld ) blocks using the program in genome applications ( pga , university of washington ) with r < 0.8 and minor allele frequency ( maf ) greater than 5% . as much of the hapmap data were not available at the time of the study , relatively few snps were available for each locus that passed the selection criteria and were suitable for the genotyping platform . all snp genotypes were determined using a massarray snp genotyping system ( sequenom , inc . , san diego , ca ) . the sample means , standard deviations ( sd ) , and medians were computed on continuous variables , while proportions were determined for discrete variables in those dhs participants who contributed to the genetic analyses . a series of generalized estimating equations ( gee ) assuming exchangeable correlation structure and using the empirical estimate of the variance ( to adjust for familial correlation induced by selection of related individuals in sibships ) was used for assessment of the effects of diabetes status on clinical variables . allele and genotype frequencies were determined with unrelated subjects ( a single member from each family ) and tested for departure from hardy - weinberg proportions using a chi - square test ( deviations with p < .001 were considered significant ) . association between each individual snp and each phenotype was determined using the quantitative pedigree disequilibrium test ( qpdt ) . the qpdt method reduces the effect of undetected stratification bias as transmission , rather than frequency of specific snp alleles , is used in estimating the association between snp and phenotype . all association analyses were conducted with adjustment for known epidemiologic risk factors of atherosclerosis ( age , gender , diabetes status , smoking , bmi , use of aspirin , estrogen ( women only ) , and lipid - lowering and hypertension medications ) . for any snp that exhibited a significant association with an atherosclerosis phenotype , additional snps within the ld block qpdt analyses were applied to these haplotypes , adjusting for the same risk factors as in the single snp analyses . genotype - specific means , medians , and number of subjects within each genotypic class and for each measure of atherosclerosis were computed under a specific genetic model . analyses of biomarkers ( e.g. , icam-1 , e - selectin , crp ) were performed using the same analytic strategy . the age at ascertainment of diabetic cases ( 62.0 9.3 years ) was slightly older than the nondiabetic siblings ( 59.5 10.0 years ) . duration of diabetes was 10.4 7.1 years , reflecting a relatively early age at onset of type 2 diabetes ( ~52 years ) . bmi was higher in diabetic siblings ( 32.4 6.7 kg / m ) than in nondiabetic siblings ( 28.9 5.2 kg / m ) . the nondiabetic subjects were , on average , overweight ( as defined by a bmi between 2529.9 kg / m ) and approaching obese . there were few differences in lipid ( total cholesterol , hdl , ldl ) profiles between diabetic and nondiabetic subjects , although lipid lowering agents were used more commonly in the diabetic subjects ( 45.4% ) than in the nondiabetic subjects ( 27.8% ) . current and past smoking was highly prevalent in both diabetic ( 59.4% ) and nondiabetic ( 57.4% ) participants . prevalence of cvd and extent of atherosclerosis ( corcp , carcp , aorcp , imt ) varied by location ( vascular bed ) and diabetes . diabetic participants had greater prevalence of all events / procedures ( heart attack , angina , stroke , cabg , angioplasty ) than nondiabetic siblings . diabetic participants had significantly more calcified plaque in the coronary ( 1425 29 versus 551 13 , mean stderr ) , carotid ( 374 25 versus 168 35 ) , and aorta ( 3995 166 versus 2342 309 ) than nondiabetic siblings ; however , there was no significant difference in imt with respect to diabetes status ( 0.68 0.01 in diabetics , 0.64 0.01 in nondiabetics ) . allele and genotype frequencies of snps in alox12 , alox15 , alox5 , and alox5ap were assessed for deviation from hardy - weinberg expectations in unrelated probands ( see supplementary table 1 in supplementary material available online at doi:10.1155/2010/170153 ) . the distribution of snps provided coverage within the block structure of the genes , as well as providing additional coverage ( either 3 or 5 ) of the regions adjacent to the lipoxygenase pathway genes . none of the 17 tagging snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with corcp ( table 3 ) . in exploratory analyses , four snps exhibited suggestive association ( p < .10 ) with corcp , one in alox12 ( rs2271316 , p = .061 , 3 of the gene ) , one in alox5 ( rs2115819 , p = .090 , intron 3 ) , and two in alox5ap ( rs9506352 , p = .097 , intron 2 ; rs4769060 , p = .073 , intron 4 ) . in alox12 , the association of rs1042357 with variation in corcp was not significant ( p = .211 ) and the two - snp haplotype was also not significant ( p = .157 ) . two additional snps were identified ( rs1369214 , intron 3 ; rs11239524 , intron 4 ) and were genotyped . these two snps bound the alox5 marginally associated snp ( rs2115819 , intron 3 ) . the alox5 snp rs1369214 is only 360 bp from rs2115819 , while the alox5 snp rs11239524 is ~11 kb from alox5 snp rs2115819 . the association between corcp with rs1369214 was similar in magnitude ( p = .098 ) , while the association between corcp with rs11239524 was much stronger ( p = .027 ) . two- and three - snp haplotypes were not more strongly associated than either the original snp ( rs2115819 in intron 3 ) or the snp in intron 4 ( rs11239524 ) , suggesting that there may be an effect of alox5 on variation of corcp in intron 4 worthy of further examination . in alox5ap , two snps were identified as having a marginal effect on variation in corcp ( rs9506352 in intron 2 and rs4769060 in intron 4 ) . since the distance between these two snps is ~17 kb , three additional snps ( rs4769874 in intron 3 , rs9315048 in intron 3 , and rs12019512 in intron 4 ) were genotyped in these samples . the three snps were not significantly associated with corcp , and analyses of two - snp haplotypes failed to increase evidence of association . thus , the exploratory genotyping and data analyses for alox5ap snp did not increase evidence of association with corcp . no snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with carcp ( table 3 ) . in exploratory analyses , one snp in alox5ap ( rs10507391 , p = .085 , intron 1 ) exhibited suggestive association ( p < .10 ) . two tagging snps adjacent to rs10507391 ( rs4769055 and rs9551960 ) span a 7 kb region in intron 1 of alox5ap . two - snp haplotype analyses identified the rs10507391-rs9551960 haplotype as significantly associated with variation in carcp ( p = .002 ) . the rs10507391-rs9551960-rs9506352 three - snp haplotype was also strongly associated with carcp ( p = .003 ) , while the rs4769055-rs10507391-rs9551960 haplotype was not associated ( p = 374 ) . these data suggest that the alox5ap effect on carcp may reside between rs10507391 ( intron 1 ) and rs9506352 ( intron 2 ) . no tagging snp in the four lipoxygenase pathway genes were significantly associated ( p < .05 ) with aorcp ( table 3 ) . in exploratory analyses , one alox5 snp ( rs2115819 in intron 3 ) exhibited the strongest ( p = .108 ) association . genotyping these two adjacent snps in alox5 ( rs1369214 , intron 3 ; rs11239524 , intron 4 ) failed to provide evidence of association , either in analyses of single snps or two- and three - snp haplotypes . none of the tagging snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with imt ( table 3 ) . in exploratory analyses , one alox5 snp ( rs3780906 in intron 6 ) two additional alox5 snps ( rs3780901 in intron 4 and rs1059696 in intron 7 ) were identified that were near the alox5 rs3780901 snp . neither bordering snp was significantly associated with imt ( rs3780901 , p = .871 ; rs1059696 , p = .746 ) . the two - snp haplotype formed by rs3780901-rs3780906 ( 12 kb between intron 4 and intron 6 ) was significantly associated with imt ( p = .047 ) . further , the three - snp haplotype ( rs3780901-rs3780906-rs1059696 ) was strongly associated with imt ( p = .019 ) . thus , this region in alox5 may be worthy of further examination for association with imt . genotype - specific means , medians , and number of subjects within each genotypic class and for each measure of subclinical atherosclerosis are presented in table 4 . although no statistically significant effects were observed based upon comparisons of genotypic means ( due to large variances of measured phenotypes and long - tailed phenotypic distributions ) , there were interesting trends in comparison of genotypic medians . for the alox5 rs2115819 snp with corcp and aorcp , the genotype - specific means suggested an inheritance pattern consistent with a dominant effect of allele 1 , with the group mean ( se ) for the combined ( 1/1 and 2/1 ) genotypes having significantly less corcp ( 1127 95 ) and aorcp ( 3026 183 ) than the 2/2 genotype ( 1540 135 and 5318 540 , resp . ) . pattern is also seen for the alox12 rs2271316 snp [ 1/1 and 2/1 genotypes having significantly less corcp ( 1097 97 ) than the 2/2 genotype ( 1423 216 ) ] and for the alox5ap rs9506352 snp [ 1/1 and 2/1 genotypes having significantly less corcp ( 1145 87 ) than the 2/2 genotype ( 1672 465 ) ] . however , for the alox5ap rs4769060 snp , the effect on corcp appears more consistent with additivity , with the 1/1 genotype having least corcp ( 996 114 ) , the 2/1 genotype having medium corcp ( 1244 132 ) , and the 2/2 genotype having the greatest corcp ( 1404 247 ) . the 1/1 genotype had greatest carcp ( 360 37 ) , the 2/1 genotype had intermediate carcp ( 313 36 ) , and the 2/2 genotype had the least carcp ( 216 43 ) . the effect of the alox5 rs3780906 snp on imt was also additive , with the 1/1 genotype having the least imt ( 0.666 0.006 ) , the 2/1 genotype having intermediate imt ( 0.675 0.007 ) , and the 2/2 genotype having the greatest imt ( 0.687 0.016 ) . the associations of lipoxygenase pathway gene variants ( alox snps ) with measures of atherosclerosis are indirect . the potential roles of these snps on mechanisms of atherosclerosis were explored by estimating the effect of each associated snp in a select panel of biomarkers . the biomarkers evaluated in this population include adiponectin , leptin , icam-1 , vcam-1 , e - selectin il-6 , crp , mgp , and mcp-1 ( table 5 ) . one in alox5 ( rs2115819 ) , one in alox12 ( rs2271316 ) , and two in alox5ap ( rs9506352 and rs4769060 ) . the two alox5ap snps exhibited significant associations with levels of adiponectin ( rs9506352 , p = .044 ; rs4769060 , p = .007 ) , crp ( rs9506352 , p = .028 ; rs4769060 , p = .002 ) ; and mgp ( rs9506352 , p = .003 ; rs4769060 , p = .037 ) . levels of mgp varied in a manner consistent with an additive gene effect , based upon the observed genotypic means [ rs9506352 gg ( 9.06 ) , ga ( 8.08 ) , aa ( 7.64 ) ; rs4769060 aa ( 8.89 ) , ag ( 8.41 ) , gg ( 7.88 ) ] . the alox12 snp also was associated with crp level ( p = .036 ) , but also significantly associated with icam-1 level ( p = .032 ) in a recessive pattern [ rs2271316 gg ( 295.7 ) , gc ( 268.3 ) , cc ( 266.0 ) ] . this snp was also associated with icam-1 ( p = .037 ) and e - selectin ( p = .0001 ) ; however , the genotypic means did not fit a classical single gene model , as the levels of both icam-1 and e - selectin for the heterozygote ( t / c ) class were greater than the means for the homozygote classes . nonetheless , these data suggest that lipoxygenase pathway variants that are associated with corcp affect markers of inflammation ( crp , icam-1 ) as well as arterial calcification ( mgp ) . the alox5ap snp identified a three - snp haplotype ( rs10507391-rs9551960-rs9506352 ) that was highly associated with carcp . each of these three individual snps was significantly ( p < .05 ) associated with mgp level [ rs10507391 aa ( 7.87 ) , at ( 8.63 ) , tt ( 9.83 ) ; rs9551960 gg ( 7.69 ) , ga ( 8.56 ) , aa ( 9.64 ) ; rs9506352 gg ( 9.06 ) , ga ( 8.08 ) , aa ( 7.64 ) ] as was the haplotype . a single snp in alox5 ( rs3780906 ) was associated with variation in imt , with a three - snp haplotype ( rs3780901-rs3780906-rs1059696 ) providing strongest evidence of association . no consistent pattern of association was evident with any biomarker for the single snp or the three - snp haplotype . type 2 diabetes is a major risk factor for cardiovascular disease ( cvd ) , whose clinical outcomes include myocardial infarction and ischemic stroke . the principle etiologic factor for cvd is atherosclerosis which is thought to be the result of a chronic inflammatory process within a vessel wall that precipitates a cascade of events , from establishment of a fatty streak lesion to plaque formation . the inflammatory process is triggered , in part , by oxidized lipids , including those of the lipoxygenase pathway . recent evidence has demonstrated that lipoxygenases have two basic functions , ( a ) membrane modification by peroxidation and ( b ) lipid mediator signaling by g protein - coupled receptors [ 21 , 22 ] . in the mouse , regulation of 12/15-lo and its pathway components ( 12s - hete and 13s - hode ) in the vessel wall modulates aortic monocyte / endothelial cell interactions [ 23 , 24 ] , which are key early events in vascular inflammation . an alternative pathway involves biosynthesis of proinflammatory leukotrienes ( e.g. , ltb4 ) by 5-lo ( and 5-lo - activating protein , flap , which transfers arachidonate to 5-lo ) , providing access to downstream leukotrienes binding to g protein - coupled receptors . recently , it has been shown that 5-lo pathway components are highly expressed in arterial walls in patients with atherosclerosis of the carotid and coronary arteries and the aorta . leukocyte - specific expression of human 15-lo has also been shown to reduce inflammation and atherosclerosis in rabbits [ 27 , 28 ] , most likely due to the generation of lipoxins from the dual action of 5-lo and 15-lo on arachidonic acid substrate in the leukocyte [ 2931 ] . studies in mice in which 15-lo was selectively expressed in endothelium indicated that endothelial - specific overexpression of 15-lo accelerated progression of atherosclerosis . one plausible explanation for these differences is that endothelial cells do not possess 5-lo and are unable to directly generate lipoxins or related anti - inflammatory eicosanoids . the cellular source of lipoxygenase may be a critical determinant of atherosclerosis ; hence , lipoxygenase pathways , their components , and their genetic determinants may be central to understanding the development of human atherosclerosis and cvd risk . genetic factors have long been known to modulate risk of atherosclerosis and cvd [ 32 , 33 ] . in studies enriched with diabetic individuals or those with cvd , the genetic contribution to variation in carotid artery imt ranges from 42%92% [ 14 , 34 , 35 ] . significant genetic contribution to calcified plaque has also been observed , whether in the coronary arteries , the carotid arteries [ 37 , 38 ] , or the aorta . in the diabetes heart study ( dhs ) , variation in quantitative measures of subclinical atherosclerosis ( corcp , carcp , aorcp , and imt ) appears to be differentially influenced by variants in genes of the lipoxygenase pathway . snps in alox5ap are associated with variation in corcp and carcp , while snps in alox12 are associated with variation in corcp . these results are consistent with findings that have been emerging from cellular and mouse models of atherosclerosis . enhanced ldl oxidation , il12 production , and endothelial / monocyte interaction have been observed through manipulation of 12/15-lo [ 11 , 25 , 40 , 41 ] . in mouse genetic studies , a region on mouse chromosome 6 ( the site of 5-lo ) was shown to be linked to atherosclerosis susceptibility [ 42 , 43 ] . later , it was demonstrated that the disruption of only one 5-lo allele significantly reduced the extent of atherosclerotic lesions at the aortic root in ldlr/ mice . previously , an alox5 variant , defined by the number of sp1 binding motifs in the promoter , was shown to be associated with variation in imt and crp level ( a marker of chronic inflammation ) in a healthy population . the promoter variant was also shown to interact with dietary intake of 5-lo substrates . the study population ( los angeles atherosclerosis study ) , in addition to being healthy , was composed of several ethnic groups ( hispanic subjects and smokers were oversampled ) . the dhs , on the other hand , consists of european - american and african - american families with at least two diabetic siblings , making direct comparisons difficult . in addition , characterization of variation in the alox5 gene was different ( number of promoter sp1 binding motifs versus snps within ld blocks across the entire alox5 gene ) . despite these differences in design and genetic evaluation , both studies observed that polymorphisms in alox5 were associated with variation in measures of atherosclerosis ( imt in both studies ; corcp and aorcp in dhs ) . unlike the los angeles atherosclerosis study , we did not detect an association between snps in ld blocks of alox5 on ultrasensitive crp level ( data not shown ) . the mechanism for associations between genes of the lipoxygenase pathway and subclinical atherosclerosis is not clear , although it may involve chemotaxis and proliferation that is induced by the effects of leukotrienes b4 ltb4 has been detected in human carotid artery , atherosclerotic plaques and is derived from the 5-lo metabolism ( via alox5ap and through g - coupled protein receptors ) of arachidonic acid [ 21 , 44 ] . recently , a variant of the gene encoding ltb4 hydrolase ( lta4h ) , a protein in the same biological pathway as alox5ap has been shown to be associated with risk of myocardial infarction , further strengthening the case for a role of the lipoxygenase pathway on cvd risk . these data suggest that there may be differential effects of genes in a common pathway on several vascular beds through diverse inflammatory mechanisms ( based upon the effects of lipoxygenase pathway snps on subclinical atherosclerosis and on markers of inflammation ( crp , e - selectin , icam-1 ) and aspects of calcification . recently , serum mgp levels were determined in 2 independent populations free of clinically apparent cardiovascular disease and an association of circulating mgp with increasing framingham chd risk score was observed , as were associations of circulating mgp with hdl and other individual chd risk factors . further characterization of genes in the lipoxygenase pathway may provide important clues to prediction of cvd . although variation in these genes may be associated with risk of atherosclerosis , they may also modulate the impact of other atherosclerotic risk factors ( e.g. , lipid levels ) or factors that are independent of traditional risk factors . the current data suggest that knowledge of the genetic profile of a pathway ( and , by extension , the interaction of components of the pathway ) may improve the prediction of risk . the extent of improvement should be greater once the multilocus examination of the pathway components becomes feasible . in this manner , the genetic biological network of atherosclerosis

aims . genes of the 5-lipoxygenase pathway are compelling candidates for atherosclerosis . we hypothesize that polymorphisms in alox12 , alox15 , alox5 , and alox5ap genes are associated with subclinical atherosclerosis in multiple vascular beds . methods . families with two or more siblings with type 2 diabetes and their nondiabetic siblings were studied as part of the diabetes heart study ( dhs ) . european american diabetic ( n = 828 ) and nondiabetic ( n = 170 ) siblings were genotyped for snps in the alox12 , alox15 , alox5 , and alox5ap genes . subclinical measures of atherosclerosis ( imt , coronary ( corcp ) , carotid ( carcp ) and aortic ( aorcp ) calcified plaque ) were obtained . results . associations were observed between alox12 with corcp , alox5 with corcp , aorcp , and imt , and alox5ap with corcp and carcp , independent of known epidemiologic risk factors . further , lipoxygenase pathway snps that were associated with measures of atherosclerosis were associated with markers of inflammation ( crp , icam-1 ) and calcification ( mgp ) . conclusions . polymorphisms within alox12 , alox5 , and alox5ap are genetically associated with subclinical atherosclerosis and with biomarkers of disease in families with type 2 diabetes . these results suggest that variants in lipoxygenase pathway genes may have pleiotropic effects on multiple components that determine risk of cardiovascular disease .

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

the mouse 5-lo gene , alox5 , has been shown to contribute to the development of atherosclerosis . variants in the human homologue ( alox5 ) are associated with carotid artery intima - media thickness ( imt ) . single snps and haplotypes of alox5ap have been associated with myocardial infarction in multiple populations [ 57 ] . human 12-lipoxygenase ( encoded by alox12 ) and 15-lipoxygenase ( encoded by alox15 ) have been localized to atherosclerotic plaques , suggesting that 12/15lo activity is involved in the development of atherosclerosis [ 810 ] . the current research was motivated by the role of lipoxygenase pathway gene products in inflammation , initiation / progression of atherosclerosis , and their modulation of expression by glucose . the alox12 , alox15 , alox5 , and alox5ap genes represent strong candidates for atherosclerosis risk , especially in the context of type 2 diabetes . we have assessed genetic variants ( snps ) in lipoxygenase pathway genes for evidence of association with markers of subclinical atherosclerosis ( intima - media wall thickness [ imt ] , carotid artery calcified plaque [ carcp ] , coronary artery calcified plaque [ corcp ] , and aortic calcified plaque [ aorcp ] ) and biomarkers ( e.g. , icam-1 , e - selectin , crp ) in participants of the diabetes heart study ( dhs ) . recruitment and phenotyping of diabetes heart study ( dhs ) participants have been previously described [ 1416 ] . siblings concordant for type 2 diabetes were recruited if they had no evidence of renal insufficiency , as were all available nondiabetic siblings . participant examinations were conducted in the general clinical research center of the wake forest university school of medicine , and included interviews for medical history , medication use and health behaviors , anthropometry , resting blood pressure , a fasting blood sampling , and a spot urine collection . only caucasian participants were included in this report , due to small sample size and limited statistical power in the african - american cohort . intima - media thickness ( imt ) of the common carotid artery was measured by high - resolution b - mode ultrasonography with a 7.5-mhz transducer and a biosound esaote ( au5 ) ultrasound machine . calcified plaque was measured in the carotid and coronary arteries and the aorta using single and multidetector ct systems that employ a standardized protocol which includes ct scanner phantom testing based on those currently implemented in the national heart lung and blood institute 's ( nhlbi ) multiethnic study of atherosclerosis ( mesa ) studies [ 15 , 17 ] . single nucleotide polymorphisms ( snps ) were identified for alox12 ( 17p13.1 ) , alox15 ( 17p13.3 ) , alox5 ( 10q11.2 ) , and alox5ap ( 13q12 ) and were chosen for genotyping in order to provide coverage of linkage disequilibrium ( ld ) blocks using the program in genome applications ( pga , university of washington ) with r < 0.8 and minor allele frequency ( maf ) greater than 5% . as much of the hapmap data were not available at the time of the study , relatively few snps were available for each locus that passed the selection criteria and were suitable for the genotyping platform . the sample means , standard deviations ( sd ) , and medians were computed on continuous variables , while proportions were determined for discrete variables in those dhs participants who contributed to the genetic analyses . all association analyses were conducted with adjustment for known epidemiologic risk factors of atherosclerosis ( age , gender , diabetes status , smoking , bmi , use of aspirin , estrogen ( women only ) , and lipid - lowering and hypertension medications ) . for any snp that exhibited a significant association with an atherosclerosis phenotype , additional snps within the ld block qpdt analyses were applied to these haplotypes , adjusting for the same risk factors as in the single snp analyses . genotype - specific means , medians , and number of subjects within each genotypic class and for each measure of atherosclerosis were computed under a specific genetic model . , icam-1 , e - selectin , crp ) were performed using the same analytic strategy . duration of diabetes was 10.4 7.1 years , reflecting a relatively early age at onset of type 2 diabetes ( ~52 years ) . bmi was higher in diabetic siblings ( 32.4 6.7 kg / m ) than in nondiabetic siblings ( 28.9 5.2 kg / m ) . the nondiabetic subjects were , on average , overweight ( as defined by a bmi between 2529.9 kg / m ) and approaching obese . there were few differences in lipid ( total cholesterol , hdl , ldl ) profiles between diabetic and nondiabetic subjects , although lipid lowering agents were used more commonly in the diabetic subjects ( 45.4% ) than in the nondiabetic subjects ( 27.8% ) . current and past smoking was highly prevalent in both diabetic ( 59.4% ) and nondiabetic ( 57.4% ) participants . prevalence of cvd and extent of atherosclerosis ( corcp , carcp , aorcp , imt ) varied by location ( vascular bed ) and diabetes . diabetic participants had significantly more calcified plaque in the coronary ( 1425 29 versus 551 13 , mean stderr ) , carotid ( 374 25 versus 168 35 ) , and aorta ( 3995 166 versus 2342 309 ) than nondiabetic siblings ; however , there was no significant difference in imt with respect to diabetes status ( 0.68 0.01 in diabetics , 0.64 0.01 in nondiabetics ) . allele and genotype frequencies of snps in alox12 , alox15 , alox5 , and alox5ap were assessed for deviation from hardy - weinberg expectations in unrelated probands ( see supplementary table 1 in supplementary material available online at doi:10.1155/2010/170153 ) . the distribution of snps provided coverage within the block structure of the genes , as well as providing additional coverage ( either 3 or 5 ) of the regions adjacent to the lipoxygenase pathway genes . none of the 17 tagging snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with corcp ( table 3 ) . in exploratory analyses , four snps exhibited suggestive association ( p < .10 ) with corcp , one in alox12 ( rs2271316 , p = .061 , 3 of the gene ) , one in alox5 ( rs2115819 , p = .090 , intron 3 ) , and two in alox5ap ( rs9506352 , p = .097 , intron 2 ; rs4769060 , p = .073 , intron 4 ) . in alox12 , the association of rs1042357 with variation in corcp was not significant ( p = .211 ) and the two - snp haplotype was also not significant ( p = .157 ) . two additional snps were identified ( rs1369214 , intron 3 ; rs11239524 , intron 4 ) and were genotyped . two- and three - snp haplotypes were not more strongly associated than either the original snp ( rs2115819 in intron 3 ) or the snp in intron 4 ( rs11239524 ) , suggesting that there may be an effect of alox5 on variation of corcp in intron 4 worthy of further examination . since the distance between these two snps is ~17 kb , three additional snps ( rs4769874 in intron 3 , rs9315048 in intron 3 , and rs12019512 in intron 4 ) were genotyped in these samples . the three snps were not significantly associated with corcp , and analyses of two - snp haplotypes failed to increase evidence of association . thus , the exploratory genotyping and data analyses for alox5ap snp did not increase evidence of association with corcp . no snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with carcp ( table 3 ) . the rs10507391-rs9551960-rs9506352 three - snp haplotype was also strongly associated with carcp ( p = .003 ) , while the rs4769055-rs10507391-rs9551960 haplotype was not associated ( p = 374 ) . these data suggest that the alox5ap effect on carcp may reside between rs10507391 ( intron 1 ) and rs9506352 ( intron 2 ) . no tagging snp in the four lipoxygenase pathway genes were significantly associated ( p < .05 ) with aorcp ( table 3 ) . genotyping these two adjacent snps in alox5 ( rs1369214 , intron 3 ; rs11239524 , intron 4 ) failed to provide evidence of association , either in analyses of single snps or two- and three - snp haplotypes . none of the tagging snps in the lipoxygenase pathway genes were significantly ( p < .05 ) associated with imt ( table 3 ) . in exploratory analyses , one alox5 snp ( rs3780906 in intron 6 ) two additional alox5 snps ( rs3780901 in intron 4 and rs1059696 in intron 7 ) were identified that were near the alox5 rs3780901 snp . neither bordering snp was significantly associated with imt ( rs3780901 , p = .871 ; rs1059696 , p = .746 ) . further , the three - snp haplotype ( rs3780901-rs3780906-rs1059696 ) was strongly associated with imt ( p = .019 ) . genotype - specific means , medians , and number of subjects within each genotypic class and for each measure of subclinical atherosclerosis are presented in table 4 . although no statistically significant effects were observed based upon comparisons of genotypic means ( due to large variances of measured phenotypes and long - tailed phenotypic distributions ) , there were interesting trends in comparison of genotypic medians . for the alox5 rs2115819 snp with corcp and aorcp , the genotype - specific means suggested an inheritance pattern consistent with a dominant effect of allele 1 , with the group mean ( se ) for the combined ( 1/1 and 2/1 ) genotypes having significantly less corcp ( 1127 95 ) and aorcp ( 3026 183 ) than the 2/2 genotype ( 1540 135 and 5318 540 , resp . ) however , for the alox5ap rs4769060 snp , the effect on corcp appears more consistent with additivity , with the 1/1 genotype having least corcp ( 996 114 ) , the 2/1 genotype having medium corcp ( 1244 132 ) , and the 2/2 genotype having the greatest corcp ( 1404 247 ) . the 1/1 genotype had greatest carcp ( 360 37 ) , the 2/1 genotype had intermediate carcp ( 313 36 ) , and the 2/2 genotype had the least carcp ( 216 43 ) . the effect of the alox5 rs3780906 snp on imt was also additive , with the 1/1 genotype having the least imt ( 0.666 0.006 ) , the 2/1 genotype having intermediate imt ( 0.675 0.007 ) , and the 2/2 genotype having the greatest imt ( 0.687 0.016 ) . the associations of lipoxygenase pathway gene variants ( alox snps ) with measures of atherosclerosis are indirect . the potential roles of these snps on mechanisms of atherosclerosis were explored by estimating the effect of each associated snp in a select panel of biomarkers . the biomarkers evaluated in this population include adiponectin , leptin , icam-1 , vcam-1 , e - selectin il-6 , crp , mgp , and mcp-1 ( table 5 ) . one in alox5 ( rs2115819 ) , one in alox12 ( rs2271316 ) , and two in alox5ap ( rs9506352 and rs4769060 ) . the two alox5ap snps exhibited significant associations with levels of adiponectin ( rs9506352 , p = .044 ; rs4769060 , p = .007 ) , crp ( rs9506352 , p = .028 ; rs4769060 , p = .002 ) ; and mgp ( rs9506352 , p = .003 ; rs4769060 , p = .037 ) . the alox12 snp also was associated with crp level ( p = .036 ) , but also significantly associated with icam-1 level ( p = .032 ) in a recessive pattern [ rs2271316 gg ( 295.7 ) , gc ( 268.3 ) , cc ( 266.0 ) ] . this snp was also associated with icam-1 ( p = .037 ) and e - selectin ( p = .0001 ) ; however , the genotypic means did not fit a classical single gene model , as the levels of both icam-1 and e - selectin for the heterozygote ( t / c ) class were greater than the means for the homozygote classes . nonetheless , these data suggest that lipoxygenase pathway variants that are associated with corcp affect markers of inflammation ( crp , icam-1 ) as well as arterial calcification ( mgp ) . each of these three individual snps was significantly ( p < .05 ) associated with mgp level [ rs10507391 aa ( 7.87 ) , at ( 8.63 ) , tt ( 9.83 ) ; rs9551960 gg ( 7.69 ) , ga ( 8.56 ) , aa ( 9.64 ) ; rs9506352 gg ( 9.06 ) , ga ( 8.08 ) , aa ( 7.64 ) ] as was the haplotype . a single snp in alox5 ( rs3780906 ) was associated with variation in imt , with a three - snp haplotype ( rs3780901-rs3780906-rs1059696 ) providing strongest evidence of association . type 2 diabetes is a major risk factor for cardiovascular disease ( cvd ) , whose clinical outcomes include myocardial infarction and ischemic stroke . the inflammatory process is triggered , in part , by oxidized lipids , including those of the lipoxygenase pathway . , ltb4 ) by 5-lo ( and 5-lo - activating protein , flap , which transfers arachidonate to 5-lo ) , providing access to downstream leukotrienes binding to g protein - coupled receptors . recently , it has been shown that 5-lo pathway components are highly expressed in arterial walls in patients with atherosclerosis of the carotid and coronary arteries and the aorta . leukocyte - specific expression of human 15-lo has also been shown to reduce inflammation and atherosclerosis in rabbits [ 27 , 28 ] , most likely due to the generation of lipoxins from the dual action of 5-lo and 15-lo on arachidonic acid substrate in the leukocyte [ 2931 ] . the cellular source of lipoxygenase may be a critical determinant of atherosclerosis ; hence , lipoxygenase pathways , their components , and their genetic determinants may be central to understanding the development of human atherosclerosis and cvd risk . genetic factors have long been known to modulate risk of atherosclerosis and cvd [ 32 , 33 ] . significant genetic contribution to calcified plaque has also been observed , whether in the coronary arteries , the carotid arteries [ 37 , 38 ] , or the aorta . in the diabetes heart study ( dhs ) , variation in quantitative measures of subclinical atherosclerosis ( corcp , carcp , aorcp , and imt ) appears to be differentially influenced by variants in genes of the lipoxygenase pathway . snps in alox5ap are associated with variation in corcp and carcp , while snps in alox12 are associated with variation in corcp . these results are consistent with findings that have been emerging from cellular and mouse models of atherosclerosis . previously , an alox5 variant , defined by the number of sp1 binding motifs in the promoter , was shown to be associated with variation in imt and crp level ( a marker of chronic inflammation ) in a healthy population . the study population ( los angeles atherosclerosis study ) , in addition to being healthy , was composed of several ethnic groups ( hispanic subjects and smokers were oversampled ) . despite these differences in design and genetic evaluation , both studies observed that polymorphisms in alox5 were associated with variation in measures of atherosclerosis ( imt in both studies ; corcp and aorcp in dhs ) . the mechanism for associations between genes of the lipoxygenase pathway and subclinical atherosclerosis is not clear , although it may involve chemotaxis and proliferation that is induced by the effects of leukotrienes b4 ltb4 has been detected in human carotid artery , atherosclerotic plaques and is derived from the 5-lo metabolism ( via alox5ap and through g - coupled protein receptors ) of arachidonic acid [ 21 , 44 ] . recently , a variant of the gene encoding ltb4 hydrolase ( lta4h ) , a protein in the same biological pathway as alox5ap has been shown to be associated with risk of myocardial infarction , further strengthening the case for a role of the lipoxygenase pathway on cvd risk . these data suggest that there may be differential effects of genes in a common pathway on several vascular beds through diverse inflammatory mechanisms ( based upon the effects of lipoxygenase pathway snps on subclinical atherosclerosis and on markers of inflammation ( crp , e - selectin , icam-1 ) and aspects of calcification . recently , serum mgp levels were determined in 2 independent populations free of clinically apparent cardiovascular disease and an association of circulating mgp with increasing framingham chd risk score was observed , as were associations of circulating mgp with hdl and other individual chd risk factors . further characterization of genes in the lipoxygenase pathway may provide important clues to prediction of cvd . although variation in these genes may be associated with risk of atherosclerosis , they may also modulate the impact of other atherosclerotic risk factors ( e.g. , lipid levels ) or factors that are independent of traditional risk factors . the current data suggest that knowledge of the genetic profile of a pathway ( and , by extension , the interaction of components of the pathway ) may improve the prediction of risk .

we sought to ascertain the validity of two screening scales for obstructive sleep apnea ( osa ) in pregnancy and to establish the prevalence of osa in pregnancy . screen positive subjects were referred for diagnostic polysomnography ( psg ) ; if admitted for antepartum care , screen positive subjects underwent a modified study with a type 3 device ( t3d ) . neither measure was a reliable diagnostic tool for osa as determined by t3d or psg ( detection rates of 10.3% and 18.0% , respectively ) . among screen positive subjects undergoing psg or t3d testing , in this prospective trial , screening positive on the bq or ess was poorly predictive of osa among gravidae and was associated with a high false referral rate . obstructive sleep apnea ( osa ) is characterized by recurrent cessation of respiratory airflow resulting from upper airway collapse during sleep and is accompanied by oxygen desaturation or arousalsz(1,2 ) . the diagnosis of osa is established by polysomnography ( psg ) ( 3 ) . given the time and expense associated with making this diagnosis , several questionnaire - based scales have been developed and validated as screening tools in the nonpregnant population , namely the berlin questionnaire ( bq ) and epworth sleepiness scale ( ess)(47 ) . delineating the relationship between osa screening and diagnosis is paramount given the proposed relationship of osa with adverse pregnancy outcomes , such as preeclampsia and fetal growth restriction(814 ) . furthermore , the majority of studies to date have failed to adjust for osa risk factors which also serve as potential outcome confounders , most notably obesity(13,15 ) . there are both epidemiological and physiological data to suggest that pregnant women may be predisposed to osa(1618 ) . diagnosing this condition in pregnancy our prior published research suggests that the bq performs poorly in this population with sensitivity and specificity of only 35% and 63.8% , respectively ( 8) . the goal of this study was to enroll a large , prospective , population - based cohort in order to establish the prevalence of osa in pregnancy and to ascertain the validity of the bq and the ess as screening tools for osa in pregnancy . based upon our pilot study of 100 patients undergoing bq screening followed by a modified four - channel sleep screening test(8 ) , we hypothesized that bq and ess would perform poorly as screening measures for osa in pregnancy . to accomplish these objectives , subsequent univariate and multivariate logistic regression analysis determined the point prevalence of osa in pregnancy , as well as established relative estimates of false referral rates . this study was performed in the harris county hospital district ( now harris health system ) between may 2010 and september 2012 . the study was approved by the institutional review board at baylor college of medicine and harris county hospital district and written informed consent was obtained from all enrolled subjects . gravidae presenting to the casa de amigos health center , people s health center ( now vallbona health center ) , and ben taub high - risk obstetrics clinic were approached for enrollment ; a combination of low and high obstetric - risk patients ( as defined by maternal and fetal comorbidities , but not risk of osa per se ) were specifically recruited to assess the validity of screening questionnaires in a " real world " heterogeneous clinical setting . consenting subjects were administered the standard screening measure questionnaire ( in english or spanish ) which comprised the ess , the bq , and questions collecting basic health and sleep information . exclusion criteria were subjects with known sleep - disordered breathing , multifetal gestation , fatal fetal anomalies , and subjects with significant underlying pulmonary or cardiac comorbidities due to likely confounding that could not be adequately controlled for . resource constraints only allowed diagnostic testing to be performed on subjects who were suspected of having osa based on ess or bq as described below . given the poor sensitivity of the bq , to capture as many cases as possible , enrollees who screened positive based upon either the ess or berlin measures were referred for diagnostic testing(8 ) . the ess and bq are screening measures that are clinically employed to assess the risk of being diagnosed with osa . the ess consists of eight questions that evaluate the tendency to fall asleep in certain situations and is a subjective measure of sleepiness(5,19 ) . in nonpregnant subjects , ess has a sensitivity of 6693.5% and specificity from 48100%(6,19 ) . for this study , we considered a patient to screen positive for osa with an ess of 10 or higher . the bq questions were developed to elicit factors or behaviors that consistently predicted the presence of sleep disordered breathing(4 ) . in nonpregnant adults , it has a sensitivity of 86% and specificity of 77%(4 ) . it is divided into three categories that focus on the presence and severity of snoring , witnessed hypopneas , daytime sleepiness , and presence of hypertension or obesity(4 ) . a high risk or screen positive individual is reported to have at least two out of the three symptom categories positive in the questionnaire(4 ) . screen positive subjects were referred for attended psg at the harris county hospital district sleep disorders center . subjects were scheduled for psg during their 26th28th week of gestation or as soon thereafter as feasible . the rationale for using this gestational age for testing was twofold : first , we did not have adequate resources to test women twice during pregnancy to assess for pre - existing osa versus new osa , so we could only perform diagnostic testing once ; second , we wanted to capture as many cases of osa as possible to allow accurate cohort assignment . we chose 2628 weeks based upon the prior finding that cpap requirement increased around 2426 weeks in gravidae with osa , suggesting that the course of the disease would have worsened or manifested itself by that gestational age(20 ) . subjects recruited after 28 weeks gestation were scheduled as soon after screening positive as feasible . screen positive subjects were contacted by mail and by designated phone contact ( k.m.a.1 ) in their native language on at least two occasions . once a psg was scheduled , the subject was contacted in their native language by two means on two separate occasions ( k.m.a.1 and the sleep center ) . given subjects and the sleep center s constraints , the protocol was subsequently amended to allow inpatient t3d testing as a surrogate for psg . for subjects undergoing t3d testing , attended psg testing was performed using a carefusion somnostar polysomnography system ( carefusion , yorba linda , california ) . via this multi - channel system , the following variables were monitored continuously : electroencephalography , electrocardiography , electro - oculography , submental and leg electromyography , and electrocardiography . continuous heart rate and pulse oximetry were monitored using a finger probe , and airflow was determined via nasal cannula - pressure transducer and thermistor . additional variables measured included body position sensors , snore microphones , and thoracic and abdominal piezoelectric bands . attended psg tests were administered by a certified technologist , and studies were reviewed by a physician , board certified in sleep medicine . osa was diagnosed with respiratory disturbance index ( rdi ) > 5 using attended psg . attended psg was scored using the aasm manual for the scoring of sleep and associated events , 2007(3 ) . the resmed apnealink plus ( resmed corp , map medizin technologie gmbh , poway , ca ) is a type 3 unattended home sleep testing device with a minimum of four channels used to test for osa . it consists of a pulse oximeter for oximetry data , heart rate recording , respiratory effort belt , and a nasal sensor for detection of flow limitation , apnea hypopnea index ( ahi ) , and snoring . the device generates a report with spo2 information and the ahi , among other variables . an ahi 5 was considered diagnostic of osa . at the time this study was performed , no home sleep testing had been validated in pregnancy , but it had been used in prior obstetric studies as a diagnostic tool(8,21 ) . for the purposes of this study , if a t3d was performed , the referral for psg testing was not cancelled as psg remains the standard for diagnosis . had a sufficient number of subjects undergone both t3d and psg testing , it would have additionally allowed for calculations of the predictive value of t3d in pregnancy . the diagnostic criteria for osa differ between psg and t3d ; psg uses rdi and t3d uses ahi . these measures have been previously compared in a non - gravid population and shown to correlate well ; thus , while they measure different components of the sleep study , the precedent exists to utilize the index specific to the study type(22 ) . covariates considered in these analyses were maternal age , race / ethnicity , smoking , parity , gestational age , prepregnancy body mass index ( where appropriate ) , pregestational diabetes , and chronic hypertension . bmi categories were defined according to the international obesity task force classification : normal weight 19 to 24.9 , overweight 25 to 29.9 , and obese > 30 kg / m . bmi was calculated using height and weight data ( kg / m2 ) that were collected during initial assessment . when prepregnancy weight was not known , the earliest available weight was used . as there is no sample size estimate calculation to assess the validity of a questionnaire , our sample size estimation was based on having adequate power to examine the relationship between osa and various maternal and neonatal outcomes , adjusted for the possible confounding influence of obesity(23 ) . in our previous analysis of n=100 subjects , the relationship between standard berlin measures and each of its components ( snoring questions , sleepiness questions ) and the t3d test that we used as criteria for diagnosis were analyzed via linear and logistic regression analyses(8 ) . based upon the screen positive rate for osa of 36% and overall osa diagnosis positive rate of 20% , we initially estimated that a minimum of 1100 subjects would need to be screened to detect a 20% difference in our primary perinatal outcomes . after 500 subjects were enrolled the protocol was amended ; enrollment was increased to 1600 as only 4 diagnostic psgs had been completed to that point . based upon the same pilot study which found a screen positive rate of osa of 36% , we anticipated performing psg on 40% of subjects(8 ) . descriptive findings of study sample characteristics by osa screening results from the bq and ess and osa diagnosis based on t3d or psg were reported . chi - squared testing or fisher s exact test was performed to assess differences between osa groups by study sample characteristics . scale reliability for the bq and ess were assessed by computing cronbach 's , which measures internal consistency , and item - test correlation , which is the correlation of each item with the overall scale . regarding unanswered test items , as no numerical value could be assigned to contribute to the overall sum on the ess or bq , they were treated as a " zero " for summation purposes . however , in all other item analyses , they were treated as missing data . multivariate logistic regression analysis was used to estimate associations between screening positive on the bq scale and its items and the ess scale and its items adjusting for confounders . in item analyses of bq and ess results were reported as adjusted odd ratios with 95% confidence intervals . due to resource constraints , overall screen negative subjects were not referred for diagnostic testing . as screen negative subjects did not undergo diagnostic testing , these terms represent the proportion of screened subjects who screened positive and had the disease and the proportion of subjects who screened positive who did not have the disease , respectively . stata 10.0 ( stata corporation , college station , tx ) and spss 13.0 ( spss incorporated , chicago , il ) were used for analyses . 79 women had enrolled on more than one occasion during their pregnancy ; in these cases data from the questionnaire completed closest to 2628 weeks was used for analysis ; the duplicate questionnaires these women completed were not included as doing so would introduce co - linearity . twenty - one subjects were excluded for multi - fetal gestation and eight were excluded for fatal fetal anomalies . one subject participated in the study during two separate pregnancies ; her data was retained as the data were unique for each pregnancy . 75 subjects only fully completed one screening measure or the other , but not both ; their available data was analyzed . five subjects did not fully complete either screening measure ( bq or ess ) , thus the bq or ess measure could not adequately be characterized , but the available individual items of each questionnaire were used for analysis . the majority of gravid subjects were hispanic ( 87.8% ) and non - smokers ( 96.5% ) , which is consistent with the obstetric population of harris health system ( figure 1 ) . over three - fourths of the subjects were overweight ( 35.1% ) or obese ( 40.4% ) . as shown in figure 1 , after excluding the five subjects that did not complete either screening measure , of the 1504 remaining subjects , 456 ( 30.3% ) screened positive on either the bq or ess and 1048 ( 69.4% ) screened negative on at least one measure . table 1 demonstrates the demographics of subjects who screened positive or negative on the bq or ess and shows the characteristics of subjects who screened positive or negative for osa ; the prevalence of subjects who screened positive varied by screening method . among subjects who adequately completed the respective screening method , the prevalence of those screening positive was 15.1% by bq , 20.4% by ess , and 31.9% by either bq or ess . the screen positive rate by either bq or ess differs in the table as a result of excluding 75 subjects with inadequate completion of the screening measures . multivariate logistic regression analyses adjusting for age , ethnicity , smoking status , parity , gestational age at survey , bmi , and pregestational diabetes showed increased odds of screening positive on either the bq or the ess among black subjects ( aor 3.59 , 95% ci 2.126.07 ) , subjects who smoked during pregnancy ( aor 2.60 , 95% ci 1.225.54 ) , and subjects with bmi over 30 ( aor 2.72 , 95% ci1.893.93 ) . figure 2 demonstrates the percentage distribution of bq and ess items . among subjects who screened positive on the bq scale , 40.4% of the sample had a bmi>30 , 21.4% reported snoring , and 24.9% felt tired during waking time . cronbach 's for the bq items was 0.62 and item - test correlations ranged from 0.30 to 0.66 . among subjects who screened positive on the ess , cronbach 's for the ess items was 0.82 and item - test correlations ranged from 0.55 to 0.74 . as shown in table 2 , screening positive on the bq scale was not associated with screening positive on the ess after controlling for covariates . specific bq items positively associated with at least two ess items after adjusting for covariates were items about snoring , feeling tired , and falling asleep while driving . 58 women were tested for osa by t3d ( n 52 ( 50 results used ) ) or psg ( n 8) . two women underwent both t3d testing and psg ; one was negative on both the t3d and psg and one was negative on t3d and positive on psg ; the psg results ( rather than the t3d results ) were used for both women in these analyses . for the purposes of this analysis , psg and t3d fischer 's exact test showed no differences between osa diagnosis by study sample characteristics , including age , race / ethnicity , smoking , bmi , gestational age at testing , and pregestational diabetes . ( data not shown . ) . as shown in table 3 , no items on the bq were significantly associated with testing positive on psg or t3d similarly , no items on the ess were positively associated with testing positive on psg or t3d , however , the item concerning the likelihood of falling asleep " as a passenger in a car for an hour without a break " was negatively associated with testing positive on psg or t3d . the overall detection rate rate for screening positive on bq or ess was 15.5 , and the false referral rate was 84.5 . the detection rate for bq was 10.3 with a false referral rate of 89.7 whereas the detection rate for ess was 18.0 with a false referral rate of 82.1 . using the psg , however , very few subjects completed psg . as 31.9% of women screened positive on either bq or ess and 15.5% of those women tested positive on t3d or psg , the overall prevalence of osa in this study would be 4.9% , if the test positive rate among untested women is the same as tested women . among subjects undergoing diagnostic testing , 8.0% ( 4/50 ) undergoing thus , if the testing modes were analyzed separately , using t3d the prevalence of osa would be 2.6% and by psg the prevalence would be 19.9% . as we have demonstrated here , the prevalence of osa in our studied obstetric population remains difficult to determine . our 4.9% prevalence rate is based on all positive cases having occurred among screen positive women . we have observed that screening by either bq or ess is poorly predictive of osa among gravidae . the detection rate of bq and ess were both poor , and the false referral rates were high , particularly when using t3d as the diagnostic test . no individual item on either questionnaire was positively associated with testing positive by psg or t3d ; one item was negatively associated with screening positive on psg or t3d , which was the item about falling asleep as a passenger in a car . it is not clear why there was a negative association ; it may be that women with true osa were too tired to go on trips over an hour , even as a passenger , but this is speculation . by study design we were not able to calculate the true prevalence or the sensitivity and specificity , but the poor predictive value of bq and ess during pregnancy has been previously suggested by ourselves and others(9,12 ) . after this study was initiated , facco , et al . , demonstrated that the bq has a sensitivity and specificity of 39% and 68% , respectively , while the ess has a sensitivity and specificity of 36% and 77% , respectively(9 ) . one recent study found a much higher sensitivity and specificity of the bq among gravidae , but only performed diagnostic testing on women who either did not score as " high risk " in any of the three categories of the bq or scored as " high risk " in all three categories(10 ) . thus the sensitivity and specificity in their study may have limited clinical utility among a population with more intermediate scores , or prone to confounding by virtue of inclusion of obesity on the bq . regarding the questionnaires themselves , after adjusting for confounding variables , screening positive on bq overall , the association between bmi>30 and screening positive on bq was expected as obesity is a weighted component of the bq . the cronbach for bq items of 0.62 reflects that it has relatively low internal consistency as a measure . the cronbach for ess was 0.82 , which reflects a good amount of internal consistency ; however , its ability to predict osa in pregnancy is poor , as noted here and elsewhere(9,12 ) . there are several factors which may contribute to the bq and ess poor performance in pregnancy . both questionnaires query daytime sleepiness , which is a common complaint during pregnancy(2527 ) ; thus this symptom may not distinguish women with and without osa . the frequency of snoring increases during pregnancy , with 37% reporting snoring often or every night in the last week ( 27 ) , so it also may not serve as a very discriminating symptom . as shown in table 3 , no items were positively associated with testing positive for osa by psg or t3d . strengths of our study include its robust subject accrual , and dedicated means to minimize barriers to diagnosis and follow - up . real - world setting , it reveals the challenges of employing a broad - based screening program for this condition which has garnered increased attention in recent years . as we have shown here , questionnaire - based diagnosis of osa in pregnancy is prone to disease misclassification and suggest that among graviade ess and bq should only be regarded as screening measures . this study was designed with the limited availability of psg in mind , thus only screen positive subjects were referred for psg . as a prevalence study should refer all subjects for diagnostic testing , we attempted to refer as many subjects as possible and thus referred subjects screening positive on either the bq or ess . limitations of this study include the relatively small number of subjects who underwent attended psg testing and the use of often used but as of yet unvalidated t3d for diagnosis of osa in pregnancy : 456 subjects were referred for psg , and 58 completed diagnostic testing . although our ratio of screen positive to completion of diagnostic testing was poor , it is still the largest population based study performed to date and thus imparts valuable data . there were several challenges to completing diagnostic testing in our population based cohort , and all are reflective of the realities of clinical practice among those at greatest risk for undiagnosed osa . notably , the psgs themselves were technically feasible when performed , which is consistent with prior findings(15,2831 ) . psg completion was solely hindered by the systematic availability of this limited resource and also by subject - based impediments inherent to any gravid population . there were a number of factors impeding the completion of psgs , all of which are worthy of consideration for investigators and clinicians alike . first , despite collaboration with the department of pulmonary , critical care , and sleep medicine at the onset of the study design and throughout the study period , there were significant obstacles to completing psg . the waiting period for psgs often exceeded subjects gestation ; thus , we coordinated with sleep center staff and when appointments became available due to cancellations , study subjects were prioritized . second , mid - way through the study a co - pay was initiated for attended psgs . in order to relieve this financial burden , all co - pays were covered for enrolled study subjects . third , while attended psgs were covered by a county - based health care plan , they were not covered by emergency pregnancy medicaid . fourth , when appointments at the sleep disorders center were made , subjects often lacked transportation and childcare which led to cancelations or truancy in over 95% of appointments . despite these recognized obstacles , either t3d or psg diagnostic testing was completed in 12.7% of subjects ; this represents the largest population - based outpatient validation testing undertaken and reported to date ( pubmed and ovid , 1986-present ) . an additional limitation is the fact that the unattended t3d testing device used in this study had not been validated in pregnancy . t3d testing is known to have a high false negative rate , especially among patients with low ahi(32)(26 ) , and women have a lower ahi than men(33)(27 ) . in this study , among screen positive subjects undergoing sleep studies , the number of subjects completing both t3d and psg was insufficient to draw conclusions regarding the accuracy of t3d . however , the percentage of patients tested who were diagnosed as positive via each method was discrepant ( 8.0% via t3d versus 62.5% via psg ) , which may suggest non - equivalence . since the completion of this study , two ambulatory sleep testing devices have demonstrated validity in pregnancy ( 34,35)(28 , 29 ) . further studies of the validity of other t3d are needed , especially as they are commonly used as a surrogate for psg in the literature . regarding bias , as the majority of subjects were tested during antepartum admissions for non - study related indications , it is unlikely that subjects ' concern regarding osa introduced bias in subjects undergoing t3d testing . however , while all screen positive women were referred for psg , women who were themselves concerned about a sleep disorder may have had higher motivation to undergo attended psg , resulting in a higher rate of positive diagnosis . a final limitation is its limited external validity given that the majority of subjects were hispanic . in sum , because of the inherent challenges in diagnosis and lack of pregnancy - specific screening measures , the prevalence of obstructive sleep apnea in pregnancy remains poorly characterized . our current estimate of 4.9% in the general obstetric population likely represents the most robust point prevalence data to date . this analysis suggests that the bq and ess as they are currently administered and scored are poorly predictive of osa in pregnancy . this study also elucidated possibly pervasive barriers to psg . based on our data presented herein , in the interval until screening and alternate diagnostic measures are fully developed and validated in pregnancy , cautious use of these screening tools in clinical obstetrical practice is warranted .

objectivewe sought to ascertain the validity of two screening scales for obstructive sleep apnea ( osa ) in pregnancy and to establish the prevalence of osa in pregnancy.study designin this prospective observational study , two screening scales were administered . screen positive subjects were referred for diagnostic polysomnography ( psg ) ; if admitted for antepartum care , screen positive subjects underwent a modified study with a type 3 device ( t3d).result1509 subjects underwent osa screening ; 58 completed diagnostic testing . neither measure was a reliable diagnostic tool for osa as determined by t3d or psg ( detection rates of 10.3% and 18.0% , respectively ) . among screen positive subjects undergoing psg or t3d testing , 15.5% ultimately met gold standard osa diagnostic criteria for an estimated point prevalence of 4.9%.conclusionin this prospective trial , screening positive on the bq or ess was poorly predictive of osa among gravidae and was associated with a high false referral rate .

Objective Study Design Result Conclusion INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

we sought to ascertain the validity of two screening scales for obstructive sleep apnea ( osa ) in pregnancy and to establish the prevalence of osa in pregnancy . screen positive subjects were referred for diagnostic polysomnography ( psg ) ; if admitted for antepartum care , screen positive subjects underwent a modified study with a type 3 device ( t3d ) . neither measure was a reliable diagnostic tool for osa as determined by t3d or psg ( detection rates of 10.3% and 18.0% , respectively ) . among screen positive subjects undergoing psg or t3d testing , in this prospective trial , screening positive on the bq or ess was poorly predictive of osa among gravidae and was associated with a high false referral rate . obstructive sleep apnea ( osa ) is characterized by recurrent cessation of respiratory airflow resulting from upper airway collapse during sleep and is accompanied by oxygen desaturation or arousalsz(1,2 ) . the diagnosis of osa is established by polysomnography ( psg ) ( 3 ) . given the time and expense associated with making this diagnosis , several questionnaire - based scales have been developed and validated as screening tools in the nonpregnant population , namely the berlin questionnaire ( bq ) and epworth sleepiness scale ( ess)(47 ) . delineating the relationship between osa screening and diagnosis is paramount given the proposed relationship of osa with adverse pregnancy outcomes , such as preeclampsia and fetal growth restriction(814 ) . furthermore , the majority of studies to date have failed to adjust for osa risk factors which also serve as potential outcome confounders , most notably obesity(13,15 ) . diagnosing this condition in pregnancy our prior published research suggests that the bq performs poorly in this population with sensitivity and specificity of only 35% and 63.8% , respectively ( 8) . the goal of this study was to enroll a large , prospective , population - based cohort in order to establish the prevalence of osa in pregnancy and to ascertain the validity of the bq and the ess as screening tools for osa in pregnancy . based upon our pilot study of 100 patients undergoing bq screening followed by a modified four - channel sleep screening test(8 ) , we hypothesized that bq and ess would perform poorly as screening measures for osa in pregnancy . to accomplish these objectives , subsequent univariate and multivariate logistic regression analysis determined the point prevalence of osa in pregnancy , as well as established relative estimates of false referral rates . gravidae presenting to the casa de amigos health center , people s health center ( now vallbona health center ) , and ben taub high - risk obstetrics clinic were approached for enrollment ; a combination of low and high obstetric - risk patients ( as defined by maternal and fetal comorbidities , but not risk of osa per se ) were specifically recruited to assess the validity of screening questionnaires in a " real world " heterogeneous clinical setting . consenting subjects were administered the standard screening measure questionnaire ( in english or spanish ) which comprised the ess , the bq , and questions collecting basic health and sleep information . resource constraints only allowed diagnostic testing to be performed on subjects who were suspected of having osa based on ess or bq as described below . given the poor sensitivity of the bq , to capture as many cases as possible , enrollees who screened positive based upon either the ess or berlin measures were referred for diagnostic testing(8 ) . for this study , we considered a patient to screen positive for osa with an ess of 10 or higher . the bq questions were developed to elicit factors or behaviors that consistently predicted the presence of sleep disordered breathing(4 ) . it is divided into three categories that focus on the presence and severity of snoring , witnessed hypopneas , daytime sleepiness , and presence of hypertension or obesity(4 ) . a high risk or screen positive individual is reported to have at least two out of the three symptom categories positive in the questionnaire(4 ) . screen positive subjects were referred for attended psg at the harris county hospital district sleep disorders center . subjects were scheduled for psg during their 26th28th week of gestation or as soon thereafter as feasible . the rationale for using this gestational age for testing was twofold : first , we did not have adequate resources to test women twice during pregnancy to assess for pre - existing osa versus new osa , so we could only perform diagnostic testing once ; second , we wanted to capture as many cases of osa as possible to allow accurate cohort assignment . subjects recruited after 28 weeks gestation were scheduled as soon after screening positive as feasible . screen positive subjects were contacted by mail and by designated phone contact ( k.m.a.1 ) in their native language on at least two occasions . given subjects and the sleep center s constraints , the protocol was subsequently amended to allow inpatient t3d testing as a surrogate for psg . for subjects undergoing t3d testing , attended psg testing was performed using a carefusion somnostar polysomnography system ( carefusion , yorba linda , california ) . attended psg tests were administered by a certified technologist , and studies were reviewed by a physician , board certified in sleep medicine . the resmed apnealink plus ( resmed corp , map medizin technologie gmbh , poway , ca ) is a type 3 unattended home sleep testing device with a minimum of four channels used to test for osa . an ahi 5 was considered diagnostic of osa . at the time this study was performed , no home sleep testing had been validated in pregnancy , but it had been used in prior obstetric studies as a diagnostic tool(8,21 ) . for the purposes of this study , if a t3d was performed , the referral for psg testing was not cancelled as psg remains the standard for diagnosis . had a sufficient number of subjects undergone both t3d and psg testing , it would have additionally allowed for calculations of the predictive value of t3d in pregnancy . the diagnostic criteria for osa differ between psg and t3d ; psg uses rdi and t3d uses ahi . these measures have been previously compared in a non - gravid population and shown to correlate well ; thus , while they measure different components of the sleep study , the precedent exists to utilize the index specific to the study type(22 ) . as there is no sample size estimate calculation to assess the validity of a questionnaire , our sample size estimation was based on having adequate power to examine the relationship between osa and various maternal and neonatal outcomes , adjusted for the possible confounding influence of obesity(23 ) . in our previous analysis of n=100 subjects , the relationship between standard berlin measures and each of its components ( snoring questions , sleepiness questions ) and the t3d test that we used as criteria for diagnosis were analyzed via linear and logistic regression analyses(8 ) . based upon the screen positive rate for osa of 36% and overall osa diagnosis positive rate of 20% , we initially estimated that a minimum of 1100 subjects would need to be screened to detect a 20% difference in our primary perinatal outcomes . after 500 subjects were enrolled the protocol was amended ; enrollment was increased to 1600 as only 4 diagnostic psgs had been completed to that point . based upon the same pilot study which found a screen positive rate of osa of 36% , we anticipated performing psg on 40% of subjects(8 ) . descriptive findings of study sample characteristics by osa screening results from the bq and ess and osa diagnosis based on t3d or psg were reported . scale reliability for the bq and ess were assessed by computing cronbach 's , which measures internal consistency , and item - test correlation , which is the correlation of each item with the overall scale . regarding unanswered test items , as no numerical value could be assigned to contribute to the overall sum on the ess or bq , they were treated as a " zero " for summation purposes . multivariate logistic regression analysis was used to estimate associations between screening positive on the bq scale and its items and the ess scale and its items adjusting for confounders . due to resource constraints , overall screen negative subjects were not referred for diagnostic testing . as screen negative subjects did not undergo diagnostic testing , these terms represent the proportion of screened subjects who screened positive and had the disease and the proportion of subjects who screened positive who did not have the disease , respectively . twenty - one subjects were excluded for multi - fetal gestation and eight were excluded for fatal fetal anomalies . five subjects did not fully complete either screening measure ( bq or ess ) , thus the bq or ess measure could not adequately be characterized , but the available individual items of each questionnaire were used for analysis . the majority of gravid subjects were hispanic ( 87.8% ) and non - smokers ( 96.5% ) , which is consistent with the obstetric population of harris health system ( figure 1 ) . over three - fourths of the subjects were overweight ( 35.1% ) or obese ( 40.4% ) . as shown in figure 1 , after excluding the five subjects that did not complete either screening measure , of the 1504 remaining subjects , 456 ( 30.3% ) screened positive on either the bq or ess and 1048 ( 69.4% ) screened negative on at least one measure . table 1 demonstrates the demographics of subjects who screened positive or negative on the bq or ess and shows the characteristics of subjects who screened positive or negative for osa ; the prevalence of subjects who screened positive varied by screening method . among subjects who adequately completed the respective screening method , the prevalence of those screening positive was 15.1% by bq , 20.4% by ess , and 31.9% by either bq or ess . the screen positive rate by either bq or ess differs in the table as a result of excluding 75 subjects with inadequate completion of the screening measures . multivariate logistic regression analyses adjusting for age , ethnicity , smoking status , parity , gestational age at survey , bmi , and pregestational diabetes showed increased odds of screening positive on either the bq or the ess among black subjects ( aor 3.59 , 95% ci 2.126.07 ) , subjects who smoked during pregnancy ( aor 2.60 , 95% ci 1.225.54 ) , and subjects with bmi over 30 ( aor 2.72 , 95% ci1.893.93 ) . among subjects who screened positive on the bq scale , 40.4% of the sample had a bmi>30 , 21.4% reported snoring , and 24.9% felt tired during waking time . cronbach 's for the bq items was 0.62 and item - test correlations ranged from 0.30 to 0.66 . among subjects who screened positive on the ess , cronbach 's for the ess items was 0.82 and item - test correlations ranged from 0.55 to 0.74 . as shown in table 2 , screening positive on the bq scale was not associated with screening positive on the ess after controlling for covariates . specific bq items positively associated with at least two ess items after adjusting for covariates were items about snoring , feeling tired , and falling asleep while driving . 58 women were tested for osa by t3d ( n 52 ( 50 results used ) ) or psg ( n 8) . two women underwent both t3d testing and psg ; one was negative on both the t3d and psg and one was negative on t3d and positive on psg ; the psg results ( rather than the t3d results ) were used for both women in these analyses . for the purposes of this analysis , psg and t3d fischer 's exact test showed no differences between osa diagnosis by study sample characteristics , including age , race / ethnicity , smoking , bmi , gestational age at testing , and pregestational diabetes . as shown in table 3 , no items on the bq were significantly associated with testing positive on psg or t3d similarly , no items on the ess were positively associated with testing positive on psg or t3d , however , the item concerning the likelihood of falling asleep " as a passenger in a car for an hour without a break " was negatively associated with testing positive on psg or t3d . the overall detection rate rate for screening positive on bq or ess was 15.5 , and the false referral rate was 84.5 . the detection rate for bq was 10.3 with a false referral rate of 89.7 whereas the detection rate for ess was 18.0 with a false referral rate of 82.1 . as 31.9% of women screened positive on either bq or ess and 15.5% of those women tested positive on t3d or psg , the overall prevalence of osa in this study would be 4.9% , if the test positive rate among untested women is the same as tested women . among subjects undergoing diagnostic testing , 8.0% ( 4/50 ) undergoing thus , if the testing modes were analyzed separately , using t3d the prevalence of osa would be 2.6% and by psg the prevalence would be 19.9% . as we have demonstrated here , the prevalence of osa in our studied obstetric population remains difficult to determine . our 4.9% prevalence rate is based on all positive cases having occurred among screen positive women . we have observed that screening by either bq or ess is poorly predictive of osa among gravidae . the detection rate of bq and ess were both poor , and the false referral rates were high , particularly when using t3d as the diagnostic test . no individual item on either questionnaire was positively associated with testing positive by psg or t3d ; one item was negatively associated with screening positive on psg or t3d , which was the item about falling asleep as a passenger in a car . it is not clear why there was a negative association ; it may be that women with true osa were too tired to go on trips over an hour , even as a passenger , but this is speculation . by study design we were not able to calculate the true prevalence or the sensitivity and specificity , but the poor predictive value of bq and ess during pregnancy has been previously suggested by ourselves and others(9,12 ) . , demonstrated that the bq has a sensitivity and specificity of 39% and 68% , respectively , while the ess has a sensitivity and specificity of 36% and 77% , respectively(9 ) . one recent study found a much higher sensitivity and specificity of the bq among gravidae , but only performed diagnostic testing on women who either did not score as " high risk " in any of the three categories of the bq or scored as " high risk " in all three categories(10 ) . thus the sensitivity and specificity in their study may have limited clinical utility among a population with more intermediate scores , or prone to confounding by virtue of inclusion of obesity on the bq . regarding the questionnaires themselves , after adjusting for confounding variables , screening positive on bq overall , the association between bmi>30 and screening positive on bq was expected as obesity is a weighted component of the bq . the cronbach for ess was 0.82 , which reflects a good amount of internal consistency ; however , its ability to predict osa in pregnancy is poor , as noted here and elsewhere(9,12 ) . there are several factors which may contribute to the bq and ess poor performance in pregnancy . both questionnaires query daytime sleepiness , which is a common complaint during pregnancy(2527 ) ; thus this symptom may not distinguish women with and without osa . as shown in table 3 , no items were positively associated with testing positive for osa by psg or t3d . as we have shown here , questionnaire - based diagnosis of osa in pregnancy is prone to disease misclassification and suggest that among graviade ess and bq should only be regarded as screening measures . this study was designed with the limited availability of psg in mind , thus only screen positive subjects were referred for psg . as a prevalence study should refer all subjects for diagnostic testing , we attempted to refer as many subjects as possible and thus referred subjects screening positive on either the bq or ess . limitations of this study include the relatively small number of subjects who underwent attended psg testing and the use of often used but as of yet unvalidated t3d for diagnosis of osa in pregnancy : 456 subjects were referred for psg , and 58 completed diagnostic testing . although our ratio of screen positive to completion of diagnostic testing was poor , it is still the largest population based study performed to date and thus imparts valuable data . there were several challenges to completing diagnostic testing in our population based cohort , and all are reflective of the realities of clinical practice among those at greatest risk for undiagnosed osa . first , despite collaboration with the department of pulmonary , critical care , and sleep medicine at the onset of the study design and throughout the study period , there were significant obstacles to completing psg . the waiting period for psgs often exceeded subjects gestation ; thus , we coordinated with sleep center staff and when appointments became available due to cancellations , study subjects were prioritized . despite these recognized obstacles , either t3d or psg diagnostic testing was completed in 12.7% of subjects ; this represents the largest population - based outpatient validation testing undertaken and reported to date ( pubmed and ovid , 1986-present ) . an additional limitation is the fact that the unattended t3d testing device used in this study had not been validated in pregnancy . t3d testing is known to have a high false negative rate , especially among patients with low ahi(32)(26 ) , and women have a lower ahi than men(33)(27 ) . in this study , among screen positive subjects undergoing sleep studies , the number of subjects completing both t3d and psg was insufficient to draw conclusions regarding the accuracy of t3d . however , the percentage of patients tested who were diagnosed as positive via each method was discrepant ( 8.0% via t3d versus 62.5% via psg ) , which may suggest non - equivalence . since the completion of this study , two ambulatory sleep testing devices have demonstrated validity in pregnancy ( 34,35)(28 , 29 ) . further studies of the validity of other t3d are needed , especially as they are commonly used as a surrogate for psg in the literature . regarding bias , as the majority of subjects were tested during antepartum admissions for non - study related indications , it is unlikely that subjects ' concern regarding osa introduced bias in subjects undergoing t3d testing . however , while all screen positive women were referred for psg , women who were themselves concerned about a sleep disorder may have had higher motivation to undergo attended psg , resulting in a higher rate of positive diagnosis . a final limitation is its limited external validity given that the majority of subjects were hispanic . in sum , because of the inherent challenges in diagnosis and lack of pregnancy - specific screening measures , the prevalence of obstructive sleep apnea in pregnancy remains poorly characterized . our current estimate of 4.9% in the general obstetric population likely represents the most robust point prevalence data to date . this analysis suggests that the bq and ess as they are currently administered and scored are poorly predictive of osa in pregnancy . based on our data presented herein , in the interval until screening and alternate diagnostic measures are fully developed and validated in pregnancy , cautious use of these screening tools in clinical obstetrical practice is warranted .

the majority of behavioral risk factors of non - communicable chronic diseases ( ncds ) originate in childhood and adolescence . sedentary life - style has been linked to an undesirable cardiovascular disease risk profile including obesity , insulin resistance , and high blood pressure . on the other hand , tobacco use at a young age has been associated with emotional and psychological problems , risky behaviors such as violence and sexual activity and an increased risk of cancers later in life . in addition , childhood obesity has been linked to increased risk of dyslipidemia , hyperinsulinemia , hypertension and a number of psychosocial problems . the global school - based health survey ( gshs ) in students has been developed by world health organization ( who ) and centers for disease control and prevention in cooperation with united nations unicef , unesco , unaids . the gshs was designed to help countries measure and assess the behavioral risk factors and protective factors among children and adolescents aged 13 - 17 years . this surveillance system is entitled childhood and adolescence surveillance and prevention of adult non - communicable disease it is a national school - based surveillance of the risk behaviors and risk factors of chronic diseases using gshs among children and adolescents in iran . so far , four surveys of caspian studies have been conducted , beginning from 2003 to 2004 . the surveys are repeated every 2 years , with blood sampling for biochemical measurements every 4 years . this paper presents the methodological aspects and early findings of the 4 survey of caspian study . the caspian - iv study was performed in 2011 - 2012 in urban and rural areas of the central counties of 31 provinces in iran . data was checked at the district level by academic supervisors ( expert of school health ) and controlled by national supervisors and operators . the study population consisted of students from elementary , intermediate and high schools of urban and rural areas . they were selected by multistage , cluster sampling method from 31 provinces of the country ( 48 clusters of 10 students in each province ) . stratification was performed in each province according to the residence area ( urban / rural ) and school grade ( elementary / intermediate / high school ) . the sampling was proportional to size with equal sex ratio ; i.e. , equal number of boys and girls were selected from each province and the ratios in urban and rural areas were proportionate to the population of urban and rural students . in this way , the number of samples in rural / urban areas and in each school grade was divided proportionally to the population of students in each grade . cluster sampling with equal clusters was used in each province to reach the necessary sample size . clusters were determined at the level of schools , including 10 sample units ( students and their parents ) in each cluster . the sample size was determined according to the cluster sampling method and to achieve a good estimate of the main risk factors of interest such as dietary behaviors , overweight and obesity and physical inactivity . the maximum sample size that could give a good estimate of all risk factors of interest was selected . a total of 48 clusters of 10 subjects in each of the provinces and a total of 14,880 students and an equal number of their parents were selected from 31 provinces . the face validity was approved by the expert panel and in the phase of content validity assessment , questions got a score of more than 0.75 which affirm the content validity . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . trained personnel completed the questionnaires in a calm atmosphere inside the schools ; the whole process was supervised and controlled by a team of health care professionals . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . body mass index ( bmi ) was calculated by dividing weight ( kg ) to height squared ( m ) . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . wrist circumference was measured before measuring blood pressure . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . study protocols were reviewed and approved by ethical committees and other relevant national regulatory organizations . after complete explanation of the study objectives and protocols , written informed consent and verbal consent was obtained from the parents and students , respectively . mean of continuous variables was reported with 95% of the confidence interval ( ci ) , categorical variables are expressed as a percentage . comparison of mean of continuous variables across genders was investigated by student t - test . the study population consisted of students from elementary , intermediate and high schools of urban and rural areas . they were selected by multistage , cluster sampling method from 31 provinces of the country ( 48 clusters of 10 students in each province ) . stratification was performed in each province according to the residence area ( urban / rural ) and school grade ( elementary / intermediate / high school ) . the sampling was proportional to size with equal sex ratio ; i.e. , equal number of boys and girls were selected from each province and the ratios in urban and rural areas were proportionate to the population of urban and rural students . in this way , the number of samples in rural / urban areas and in each school grade was divided proportionally to the population of students in each grade . cluster sampling with equal clusters was used in each province to reach the necessary sample size . clusters were determined at the level of schools , including 10 sample units ( students and their parents ) in each cluster . the sample size was determined according to the cluster sampling method and to achieve a good estimate of the main risk factors of interest such as dietary behaviors , overweight and obesity and physical inactivity . the maximum sample size that could give a good estimate of all risk factors of interest was selected . a total of 48 clusters of 10 subjects in each of the provinces and a total of 14,880 students and an equal number of their parents were selected from 31 provinces . the face validity was approved by the expert panel and in the phase of content validity assessment , questions got a score of more than 0.75 which affirm the content validity . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . trained personnel completed the questionnaires in a calm atmosphere inside the schools ; the whole process was supervised and controlled by a team of health care professionals . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . body mass index ( bmi ) was calculated by dividing weight ( kg ) to height squared ( m ) . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . the face validity was approved by the expert panel and in the phase of content validity assessment , questions got a score of more than 0.75 which affirm the content validity . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . trained personnel completed the questionnaires in a calm atmosphere inside the schools ; the whole process was supervised and controlled by a team of health care professionals . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . body mass index ( bmi ) was calculated by dividing weight ( kg ) to height squared ( m ) . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . study protocols were reviewed and approved by ethical committees and other relevant national regulatory organizations . after complete explanation of the study objectives and protocols , written informed consent and verbal consent was obtained from the parents and students , respectively . mean of continuous variables was reported with 95% of the confidence interval ( ci ) , categorical variables are expressed as a percentage . comparison of mean of continuous variables across genders was investigated by student t - test . the population of this survey consisted of 13,486 children and adolescents out of 14,880 invited subjects ( participation rate of 90.6% ) and one of their parents . the whole data of one of the provinces was not available ; therefore analysis was performed on data of 30 provinces . the students were 6640 girls and 6846 boys with a mean age of 12.5 years ( 12.3 - 12.6 , 95% ci ) . a total of 75.6% of students were from urban and nearly 24.4% were from rural areas . tables 1 and 2 present the characteristics of students by sex and living area , respectively . characteristics of participants according to gender : the caspian - iv study characteristics of participants according to living area : the caspian - iv study overall , 12.2% ( 11.3% of girls and 13.0% of boys ) were underweight , 9.7% ( 10.1% of girls and 9.3% of boys ) were overweight and 11.9% ( 10.1% of girls and 13.6% of boys ) were obese . abdominal obesity was documented in 19.1% of students ( 17.8% of girls and 20.4% of boys ; 21.2% in urban and 12.8% in rural residents ) . table 3 presents some health and dietary behaviors of students or their families . according to reports by students , 42% of urban participants and 34% of rural participants brush their teeth once a day , 34% of girls and 20% of boys reported to brush their teeth more than once a day . overall , 37% of families in both urban and rural areas consumed whole - wheat breads . most of the rural families used hydrogenated solid fat ( 53% ) and the most prevalent types of cooking oil in urban families were liquid oil and hydrogenated solid fat ( 39% and 32% , respectively ) . more rural students reported to always add salt to table food than urban participants ( 36% vs. 29% , respectively , p < 0.05 ) . overall , 36% of students ate junk foods every week , 31% consumed soft drinks every week and 24% ate fast foods every week . more urban students reported to use fresh fruit daily than rural students ( 59% vs. 45% , respectively , p < 0.05 ) . some health and dietary behaviors in iranian children and adolescents : the caspian - iv study according to reports by students , 18% had at least 30-min a day leisure - time physical activity all days during the week prior to the study ; however , 9% reported not to have any leisure time physical activity . of students participated , 41% used computer in weekdays and 44% in weekends ; these frequencies were higher in urban areas and among boys [ table 4 ] . physical activity and leisure time activity in iranian children and adolescents : the caspian - iv study regarding passive smoking , 34.5% of students reported to have at least one cigarette smoker and 21.5% reported to have waterpipe smoker in their relatives , totally 43.9% of students were passive smoker . overall , 5.9% of students ( 7.5% in boys and 4.2% in girls ) were ever smoker ; this prevalence was higher in adolescents than in younger age group . the mean age of the first attempt to smoke was 11.9 years . a total of 27.4% and 17.5% of students asserted that in the 3 months before the study they bullied others or experienced getting bullied which was significantly more frequent among boys than girls . regarding injuries , 20.3% of students ( 19.4% elementary , 23.1% middle , 18.9% in high school ; 25.7% in boys and 14.6% in girls ) reported that in the 12 months prior to the study , they had suffered an injury needing the help of school health providers . the prevalence of these injuries was not statistically different according to living area ( 20.1% in urban and 20.7% in rural areas ) . in most of the cases , the injury occurred in home ( 39.9% ) when they were playing and/or exercising . the average duration of breastfeeding of students during the two 1 years of life was 15.39 months . in the fourth phase of caspian survey , different aspects of the general health status were assessed among a large number of iranian children and adolescents . according to our findings , there is a need for reinforcement of the current health regulations and for setting practical policies , education and intervention strategies for both health promotion and primordial / primary prevention of chronic diseases . in the current survey , both features of weight disorders , namely overweight and underweight were documented in the study participants and this confirms the nutrition and epidemiologic transition in iran . compared with the caspian - iii study , the increment in the prevalence of abdominal obesity was observed in the current survey ( 16.3% vs. 19% ) . the frequency of underweight had decreased from 13.9% in the caspian - iii study to 12% and it may be a confirmatory evidence for improvement in health care system . the aforementioned changes are consistent with the results reported from other studies conducted in the population of most developing countries in the east - mediterranean region , which are experiencing double burden of nutritional disorders due to the rapid socio - economic alterations . shifting away from healthy eating habits to fast foods , high - calorie low - nutrient diets combined with sedentary activities made children and adolescents more prone for development of overweight and its associated comorbidities as type two diabetes , cardiovascular and fatty liver diseases . assessment of physical activity showed that most of the students were physically active for only 1 day / week ; this finding is in line with the previous surveys of the caspian study . overall , the frequency and intensity of leisure time physical activity is low in iranian children and adolescents . the exception was for elementary school students and rural districts devoting all weekdays for exercise . having more free time , less homework and type of activities of elementary students , reinforcement of the ministerial regulations for physical activity in elementary schools , as well as more active life - styles in rural areas may explain such differences . feeding pattern in the 1 year of life was breast milk , which can be a protective factor against many risk factors of chronic diseases , as obesity and hypertension . however , conflicting results exist and longitudinal studies are necessary to document such long - term effects . regarding dietary habits ; refined grains ( 63% ) and liquid oil ( 39% ) were mostly consumed among iranian families . however , more than half of rural inhabitants ( 57% ) reported consumption of solid hydrogenated fats . added salt after the food preparation was mentioned by most of the students , except for elementary children . type of dairy consumed was pasteurized - regular fat , eaten daily except for high - school students ( weekly ) . although the consumption frequency of unhealthy foods such as processed foods and low - nutrient snacks was low , parents declared that they made no significant changes in the child 's diet in the year prior to the study . given the high prevalence of overweight and metabolic disorders in the country , it seems necessary to make practical policies to modify children 's diet . as an example , dairy products , containing high amounts of calcium , with preventive effects against osteoporosis , overweight and insulin resistance , were not consumed daily among high - school students . self - reported smoking was not prevalent among iranian students compared to their peers in other countries , however the first attempt to smoke was around 11 years , which was about 2 years earlier than the first caspian study in 2003 - 2004 . assessment of the students health status showed that most of the participants were in suitable health condition , rarely felt sadness , dizziness and so on . violence , bullying and other aggressive behaviors , were not frequent , similar to reports from other countries . the main limitation of this study was its cross - sectional design by which causality can not be determined . despite this limitation , the major strengths of this survey are the large sample size and the nationwide design of the study , which ensures the representativeness of the findings and providing the possibility of making comparisons with previous phases of the caspian study . besides , data of wrist circumference was collected for the 1 time during all caspians ; a measure which has been recently proposed as an important predictor of diabetes and pre - diabetes in adults and correlates with cardio metabolic risk factors . finally , a high quality control of data collection was the other strength of this study . the main limitation of this study was its cross - sectional design by which causality can not be determined . despite this limitation , the major strengths of this survey are the large sample size and the nationwide design of the study , which ensures the representativeness of the findings and providing the possibility of making comparisons with previous phases of the caspian study . besides , data of wrist circumference was collected for the 1 time during all caspians ; a measure which has been recently proposed as an important predictor of diabetes and pre - diabetes in adults and correlates with cardio metabolic risk factors . finally , a high quality control of data collection was the other strength of this study . the findings of this survey and the changes of variables studied in this surveillance system provide useful information for health policy makers and stakeholders to implement action - oriented interventions . the priorities of the health system for health promotion of children and adolescents are assumed to be development of effective intersectoral intervention programs promoting physical activity , healthy eating and improved life - style behaviors involving cooperative and immediate efforts of individuals , families , schools , community and government for primordial and primary prevention of ncds .

background : the fourth survey of the surveillance system named childhood and adolescence surveillance and prevention of adult non - communicable disease ( caspian - iv study ) , was conducted among a national representative sample of iranian students . this paper describes the methods and early findings of this survey.methods:this nationwide school - based study was conducted in 2011 - 2012 in 30 provinces of iran among 13,486 students , 6 - 18 years ( 6640 girls , 75.6% from urban areas ) and one of their parents.results:mean age of students was 12.5 years . based on the world health organization growth curves , 12.2% were underweight , 9.7% overweight and 11.9% were obese . abdominal obesity was observed in 19.1% of students . the dominant type of cooking oil in urban families was liquid oil and hydrogenated fat ( 39% and 32% ) , most rural families used hydrogenated fat ( 53% ) , respectively . a total of 18% of students had at least 30 min of daily physical activity ; 41% of students used computer in weekdays and 44% used it in weekends . almost 34.5% of students reported to have at least one cigarette smoker and 21.5% reported to have a waterpipe smoker in their relatives . moreover , 20.3% of students reported that they had suffered an injury needing the help of school health providers during the year prior to the study.conclusions:current evidence on the health risky behaviors among iranian children and adolescents confirms the importance of conducting comprehensive surveillance surveys to identify health risk behaviors . data of this survey and the trend of variables provide necessary information for health policy makers to implement action - oriented interventions .

INTRODUCTION METHODS Study population and sampling framework Procedure and measurements Questionnaires Physical measurements Ethical concerns Statistical analysis RESULTS DISCUSSION Study limitations and strengths CONCLUSIONS

the majority of behavioral risk factors of non - communicable chronic diseases ( ncds ) originate in childhood and adolescence . on the other hand , tobacco use at a young age has been associated with emotional and psychological problems , risky behaviors such as violence and sexual activity and an increased risk of cancers later in life . in addition , childhood obesity has been linked to increased risk of dyslipidemia , hyperinsulinemia , hypertension and a number of psychosocial problems . the global school - based health survey ( gshs ) in students has been developed by world health organization ( who ) and centers for disease control and prevention in cooperation with united nations unicef , unesco , unaids . the gshs was designed to help countries measure and assess the behavioral risk factors and protective factors among children and adolescents aged 13 - 17 years . this surveillance system is entitled childhood and adolescence surveillance and prevention of adult non - communicable disease it is a national school - based surveillance of the risk behaviors and risk factors of chronic diseases using gshs among children and adolescents in iran . this paper presents the methodological aspects and early findings of the 4 survey of caspian study . the caspian - iv study was performed in 2011 - 2012 in urban and rural areas of the central counties of 31 provinces in iran . data was checked at the district level by academic supervisors ( expert of school health ) and controlled by national supervisors and operators . the study population consisted of students from elementary , intermediate and high schools of urban and rural areas . they were selected by multistage , cluster sampling method from 31 provinces of the country ( 48 clusters of 10 students in each province ) . stratification was performed in each province according to the residence area ( urban / rural ) and school grade ( elementary / intermediate / high school ) . , equal number of boys and girls were selected from each province and the ratios in urban and rural areas were proportionate to the population of urban and rural students . in this way , the number of samples in rural / urban areas and in each school grade was divided proportionally to the population of students in each grade . clusters were determined at the level of schools , including 10 sample units ( students and their parents ) in each cluster . the sample size was determined according to the cluster sampling method and to achieve a good estimate of the main risk factors of interest such as dietary behaviors , overweight and obesity and physical inactivity . the maximum sample size that could give a good estimate of all risk factors of interest was selected . a total of 48 clusters of 10 subjects in each of the provinces and a total of 14,880 students and an equal number of their parents were selected from 31 provinces . the face validity was approved by the expert panel and in the phase of content validity assessment , questions got a score of more than 0.75 which affirm the content validity . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . after complete explanation of the study objectives and protocols , written informed consent and verbal consent was obtained from the parents and students , respectively . mean of continuous variables was reported with 95% of the confidence interval ( ci ) , categorical variables are expressed as a percentage . the study population consisted of students from elementary , intermediate and high schools of urban and rural areas . they were selected by multistage , cluster sampling method from 31 provinces of the country ( 48 clusters of 10 students in each province ) . stratification was performed in each province according to the residence area ( urban / rural ) and school grade ( elementary / intermediate / high school ) . , equal number of boys and girls were selected from each province and the ratios in urban and rural areas were proportionate to the population of urban and rural students . in this way , the number of samples in rural / urban areas and in each school grade was divided proportionally to the population of students in each grade . cluster sampling with equal clusters was used in each province to reach the necessary sample size . clusters were determined at the level of schools , including 10 sample units ( students and their parents ) in each cluster . the sample size was determined according to the cluster sampling method and to achieve a good estimate of the main risk factors of interest such as dietary behaviors , overweight and obesity and physical inactivity . a total of 48 clusters of 10 subjects in each of the provinces and a total of 14,880 students and an equal number of their parents were selected from 31 provinces . the face validity was approved by the expert panel and in the phase of content validity assessment , questions got a score of more than 0.75 which affirm the content validity . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . cronbach 's alpha coefficient of the whole questionnaires was 0.97 and the pearson correlation coefficient of the test - retest phase was 0.94 , which confirmed the reliability of questionnaires . the student questionnaire included questions about the relationship with peers , body image , and psychosocial environment of school , dietary habits , life - style habits , physical activity pattern , unintentional injuries , violence behavior , active and passive smoking , as well as psychosocial relations with family . concerns such as family composition , socio - demographic factors and genetic determinants ( family history of hypertension , diabetes , obesity , sever osteoporosis , cancer ) , past history of student ( birth weight , breastfeeding , type of complementary food ) , family dietary habits and unintentional injuries to the student were included in the parent 's questionnaire . these questionnaires were also completed by trained interviewers and at least one of the parents was required to be present for answering the questions . weight was measured to the nearest 0.1 kg on a scale placed on a flat ground and subjects wearing light clothing and standing motionless and height were measured without shoes to the nearest 0.1 cm . waist circumference was measured using a non - elastic tape at a point midway between the lower border of the rib cage and the iliac crest at the end of normal expiration to the nearest 0.1 cm . hip circumference was measured at the widest part of the hip at the level of the greater trochanter to the nearest 0.1 cm . abdominal obesity was defined as waist - to - height ratio of more than 0.5 . wrist circumference was measured to the nearest 0.1 cm on the dominant arm using a tape meter . the superior border of the tape measure was placed just distal to the prominences of radial and ulnar bones . the wrist circumference was measured without the tape was too tight or too loose and with lying flat on the skin . blood pressure was measured in the sitting position on the right arm using a mercury sphygmomanometer with an appropriate cuff size . after complete explanation of the study objectives and protocols , written informed consent and verbal consent was obtained from the parents and students , respectively . mean of continuous variables was reported with 95% of the confidence interval ( ci ) , categorical variables are expressed as a percentage . the population of this survey consisted of 13,486 children and adolescents out of 14,880 invited subjects ( participation rate of 90.6% ) and one of their parents . the whole data of one of the provinces was not available ; therefore analysis was performed on data of 30 provinces . the students were 6640 girls and 6846 boys with a mean age of 12.5 years ( 12.3 - 12.6 , 95% ci ) . a total of 75.6% of students were from urban and nearly 24.4% were from rural areas . tables 1 and 2 present the characteristics of students by sex and living area , respectively . characteristics of participants according to gender : the caspian - iv study characteristics of participants according to living area : the caspian - iv study overall , 12.2% ( 11.3% of girls and 13.0% of boys ) were underweight , 9.7% ( 10.1% of girls and 9.3% of boys ) were overweight and 11.9% ( 10.1% of girls and 13.6% of boys ) were obese . abdominal obesity was documented in 19.1% of students ( 17.8% of girls and 20.4% of boys ; 21.2% in urban and 12.8% in rural residents ) . table 3 presents some health and dietary behaviors of students or their families . according to reports by students , 42% of urban participants and 34% of rural participants brush their teeth once a day , 34% of girls and 20% of boys reported to brush their teeth more than once a day . most of the rural families used hydrogenated solid fat ( 53% ) and the most prevalent types of cooking oil in urban families were liquid oil and hydrogenated solid fat ( 39% and 32% , respectively ) . more rural students reported to always add salt to table food than urban participants ( 36% vs. 29% , respectively , p < 0.05 ) . overall , 36% of students ate junk foods every week , 31% consumed soft drinks every week and 24% ate fast foods every week . more urban students reported to use fresh fruit daily than rural students ( 59% vs. 45% , respectively , p < 0.05 ) . some health and dietary behaviors in iranian children and adolescents : the caspian - iv study according to reports by students , 18% had at least 30-min a day leisure - time physical activity all days during the week prior to the study ; however , 9% reported not to have any leisure time physical activity . of students participated , 41% used computer in weekdays and 44% in weekends ; these frequencies were higher in urban areas and among boys [ table 4 ] . physical activity and leisure time activity in iranian children and adolescents : the caspian - iv study regarding passive smoking , 34.5% of students reported to have at least one cigarette smoker and 21.5% reported to have waterpipe smoker in their relatives , totally 43.9% of students were passive smoker . overall , 5.9% of students ( 7.5% in boys and 4.2% in girls ) were ever smoker ; this prevalence was higher in adolescents than in younger age group . the mean age of the first attempt to smoke was 11.9 years . a total of 27.4% and 17.5% of students asserted that in the 3 months before the study they bullied others or experienced getting bullied which was significantly more frequent among boys than girls . regarding injuries , 20.3% of students ( 19.4% elementary , 23.1% middle , 18.9% in high school ; 25.7% in boys and 14.6% in girls ) reported that in the 12 months prior to the study , they had suffered an injury needing the help of school health providers . the prevalence of these injuries was not statistically different according to living area ( 20.1% in urban and 20.7% in rural areas ) . in most of the cases , the injury occurred in home ( 39.9% ) when they were playing and/or exercising . the average duration of breastfeeding of students during the two 1 years of life was 15.39 months . in the fourth phase of caspian survey , different aspects of the general health status were assessed among a large number of iranian children and adolescents . according to our findings , there is a need for reinforcement of the current health regulations and for setting practical policies , education and intervention strategies for both health promotion and primordial / primary prevention of chronic diseases . in the current survey , both features of weight disorders , namely overweight and underweight were documented in the study participants and this confirms the nutrition and epidemiologic transition in iran . compared with the caspian - iii study , the increment in the prevalence of abdominal obesity was observed in the current survey ( 16.3% vs. 19% ) . the frequency of underweight had decreased from 13.9% in the caspian - iii study to 12% and it may be a confirmatory evidence for improvement in health care system . the aforementioned changes are consistent with the results reported from other studies conducted in the population of most developing countries in the east - mediterranean region , which are experiencing double burden of nutritional disorders due to the rapid socio - economic alterations . shifting away from healthy eating habits to fast foods , high - calorie low - nutrient diets combined with sedentary activities made children and adolescents more prone for development of overweight and its associated comorbidities as type two diabetes , cardiovascular and fatty liver diseases . assessment of physical activity showed that most of the students were physically active for only 1 day / week ; this finding is in line with the previous surveys of the caspian study . overall , the frequency and intensity of leisure time physical activity is low in iranian children and adolescents . having more free time , less homework and type of activities of elementary students , reinforcement of the ministerial regulations for physical activity in elementary schools , as well as more active life - styles in rural areas may explain such differences . regarding dietary habits ; refined grains ( 63% ) and liquid oil ( 39% ) were mostly consumed among iranian families . however , more than half of rural inhabitants ( 57% ) reported consumption of solid hydrogenated fats . added salt after the food preparation was mentioned by most of the students , except for elementary children . type of dairy consumed was pasteurized - regular fat , eaten daily except for high - school students ( weekly ) . although the consumption frequency of unhealthy foods such as processed foods and low - nutrient snacks was low , parents declared that they made no significant changes in the child 's diet in the year prior to the study . given the high prevalence of overweight and metabolic disorders in the country , it seems necessary to make practical policies to modify children 's diet . as an example , dairy products , containing high amounts of calcium , with preventive effects against osteoporosis , overweight and insulin resistance , were not consumed daily among high - school students . self - reported smoking was not prevalent among iranian students compared to their peers in other countries , however the first attempt to smoke was around 11 years , which was about 2 years earlier than the first caspian study in 2003 - 2004 . assessment of the students health status showed that most of the participants were in suitable health condition , rarely felt sadness , dizziness and so on . the main limitation of this study was its cross - sectional design by which causality can not be determined . despite this limitation , the major strengths of this survey are the large sample size and the nationwide design of the study , which ensures the representativeness of the findings and providing the possibility of making comparisons with previous phases of the caspian study . besides , data of wrist circumference was collected for the 1 time during all caspians ; a measure which has been recently proposed as an important predictor of diabetes and pre - diabetes in adults and correlates with cardio metabolic risk factors . finally , a high quality control of data collection was the other strength of this study . the main limitation of this study was its cross - sectional design by which causality can not be determined . despite this limitation , the major strengths of this survey are the large sample size and the nationwide design of the study , which ensures the representativeness of the findings and providing the possibility of making comparisons with previous phases of the caspian study . besides , data of wrist circumference was collected for the 1 time during all caspians ; a measure which has been recently proposed as an important predictor of diabetes and pre - diabetes in adults and correlates with cardio metabolic risk factors . finally , a high quality control of data collection was the other strength of this study . the findings of this survey and the changes of variables studied in this surveillance system provide useful information for health policy makers and stakeholders to implement action - oriented interventions . the priorities of the health system for health promotion of children and adolescents are assumed to be development of effective intersectoral intervention programs promoting physical activity , healthy eating and improved life - style behaviors involving cooperative and immediate efforts of individuals , families , schools , community and government for primordial and primary prevention of ncds .

sight loss from glaucoma can be avoided as early treatment of the condition reduces the risk of sight loss . however , in the uk around 3000 people are newly registered with sight impairment due to glaucoma each year . delayed detection and thus access to early treatment is the main risk factor for sight loss and may be linked to areal or individual socioeconomic deprivation . there may be patient delay in terms of attendance for testing , process delay in terms of missed detection , or system delay leading to delayed referral for treatment . the public health importance of glaucoma could indicate that a screening programme might be warranted . before a screening policy is adopted evidence is required that the benefits of screening , namely reduced visual impairment , outweigh any harms , for example anxiety and cost . an earlier evaluation using economic modelling found that screening the uk population , selected on age alone , was unlikely to be cost - effective as the prevalence is too low in all age groups ( screening at age 40 or 65 or 75 ) . a surveillance programme targeted to higher risk groups ( a sibling with glaucoma ; ethnic minority groups ; diabetics ; or people with ocular risk factors such as raised intraocular pressure ( iop ) and myopia ) or those who do not normally use eye care might be worthwhile . the modelling evaluation , hereafter referred to as the glaucoma screening model , used the best data available but still had some uncertainties : how best to screen ( tests and location ) ; likely uptake of any screening programme ; and the effectiveness and coverage of current eye care services . the most robust way to evaluate any proposed screening programme there are no rcts evaluating glaucoma screening and any trial would need to be large and thus costly . in a recent trial platform study , we undertook a multicomponent mixed - methods approach to provide evidence to inform the optimal design for a trial . we followed the medical research council guidance for the development and evaluation of complex interventions . our initial work consisted of addressing the development of the screening test schedule and the factors associated with motivation of the public to attend for screening . we took a systematic , theory - based approach to intervention development ( identifying the evidence , modelling process and developing outcomes ) and explored the feasibility ( for service providers ) , acceptability ( for providers and users ) , and cost - effectiveness ( for health and social services ) of trial components . , we report the integration of the findings ( revised screening test schedule , likely uptake of screening and uptake of usual care ) into the glaucoma screening economic model to inform whether a glaucoma screening trial would be worthwhile . we used a markov model to assess how worthwhile a glaucoma screening trial would be . we took the perspective that interventions compared within the model could be delivered ( technical feasibility ) and were acceptable ( likely uptake by providers and the public ) in the context of the nhs . we revised the structure of the existing glaucoma screening model with the most likely cost - effective glaucoma testing schedules that might be brought to trial based on a prior delphi survey and views of nhs providers . we updated parameter estimates for screening attendance based on our survey of the public to identify factors associated with their hypothetical intention ( motivation ) to attend an eye health test . we also used data collected in the survey on attendance by the public for an eye test within the last three years to estimate uptake of the comparator pathway within the model of opportunistic case finding within current eye care . we sought estimates of attendance at eye care services for several risk groups ( age over 50 , black ethnicity , diabetes , myopia , family history of glaucoma , and low socioeconomic status from the british household panel survey data to develop and fit the probability distributions around the mean uptake of current eye care for the general population and subgroups using the excel add - on oracle crystal ball . revised utility data were based on the eq-5d-3l responses from 640 participants with ocular hypertension and glaucoma sampled from a secondary glaucoma service ( prior , personal communication ) . the model allowed movement between health states every year , estimated costs from nhs and personal social services perspectives and used the eq-5d-3l quality of life weights to calculate quality adjusted life years ( qalys ) . the base case analysis conducted from nhs perspective , considered a cohort of 40-year old males with prevalence from 1% to 5% . sex - specific variables were not available for any of the model parameters except for mortality . we used male mortality in the base - case analysis , consistent with good modelling practice , as they are a conservative assumption for screening . alternative likelihoods of attending screening ( e.g. 30100% ) and estimated costs and qalys over their estimated lifetime with screening occurring every 10 years were included . all costs and qalys we conducted sensitivity analyses to identify plausible situations where screening might be considered worthwhile by varying screening start age and accuracy of glaucoma detection within current eye care services . we used alternative data for uptake of current eye care ( based on the survey of the public and british household panel survey ( bhps ) data ) for the whole population , for cohorts aged 50 and 60 as well as for higher risk subgroups of the adult population : those self - reported as having diabetes , sight problems in the family ( excluding using spectacles ) as a proxy for family history of glaucoma , and those at low household income ( below 10,000 a year ) . data were not available within the bhps to investigate the impact of ethnicity or myopia on the uptake rates of current eye care by these groupings . the effect of including personal social services costs was explored in the sensitivity analysis by incorporating an annual cost of sight impairment from 1000 to 40,000 . the upper value is in line with personal social services expenditure per person with sight impairment for england for 200910 . variations in cost of sight impairment were combined with variation in both prevalence rates and screening uptake to identify situations where screening might be worthwhile . we explored the impact of increasing screening uptake because of a behavioural intervention and incorporating an additional cost for its provision . without a behavioural intervention , glaucoma screening uptake was considered to be about 23% ( based on our survey where 45% of the sample had strong intention scores ( mean intention score of 7 on a 17 scale ) and the health behaviour literature that 50% of strong intenders will perform the intended behaviour . we varied screening uptake from 30% to 100% to explore the impact of a behavioural intervention . reflecting a simple behavioural intervention such as an invitation letter targeted to improve motivation by making it easier to attend a screening appointment to a fully tailored intervention , and thus more costly , to persuade those with low intentions to attend . the mean qalys were expressed in pounds sterling by multiplying them by the willingness to pay for a qaly threshold ( e.g. 30,000 , see figure 1 ) . figure 1.strategies with the highest net - benefit ( defined as 30,000 mean qalys minus mean costs ) for alternative values of annual cost of sight impairment and percentage of screening uptake for a 50-year - old cohort . willingness to pay is 30,000.(a ) 1% glaucoma prevalence and 17% uptake current eye care practice . for the range of values selected for the annual cost of sight impairment and uptake rate , only gps11 ( iop + vf) are potentially cost - effective when society is willing to pay 30,000 per qaly . the screening strategy gps11d ( iop + vf) has the highest net - benefit when the screening uptake is 30% and the annual cost of sight impairment is 30,000 . the vertical continuous line at 19,000 cost of sight impairment illustrates that screening is not cost - effective below this value , regardless of the screening uptake . ( b ) 5% glaucoma prevalence and 17% uptake of current eye care.the dashed line is illustrative . the screening strategy gps11d ( iop + vf) has the highest net - benefit for screening attendance of 40% and annual cost of sight impairment just above 4500 . the vertical continuous line at 3000 cost of sight impairment illustrates that screening by any pathway is not cost - effective below this value , regardless of screening uptake . the screening strategy gps11d ( iop + vf) having the highest net - benefit for screening attendance of 40% and annual cost of sight impairment above 18,000 . iop : intraocular pressure ; gps : glaucoma screening platform study ; qaly : quality adjusted life years ; vf : visual field . strategies with the highest net - benefit ( defined as 30,000 mean qalys minus mean costs ) for alternative values of annual cost of sight impairment and percentage of screening uptake for a 50-year - old cohort . ( a ) 1% glaucoma prevalence and 17% uptake current eye care practice . for the range of values selected for the annual cost of sight impairment and uptake rate , only gps11 ( iop + vf) are potentially cost - effective when society is willing to pay 30,000 per qaly . the screening strategy gps11d ( iop + vf) has the highest net - benefit when the screening uptake is 30% and the annual cost of sight impairment is 30,000 . the vertical continuous line at 19,000 cost of sight impairment illustrates that screening is not cost - effective below this value , regardless of the screening uptake . the dashed line is illustrative . the screening strategy gps11d ( iop + vf) has the highest net - benefit for screening attendance of 40% and annual cost of sight impairment just above 4500 . the vertical continuous line at 3000 cost of sight impairment illustrates that screening by any pathway is not cost - effective below this value , regardless of screening uptake . the dashed line is illustrative . the screening strategy gps11d ( iop + vf) having the highest net - benefit for screening attendance of 40% and annual cost of sight impairment above 18,000 . iop : intraocular pressure ; gps : glaucoma screening platform study ; qaly : quality adjusted life years ; vf : visual field . we also explored whether enhanced current eye care would be better than enrolling in a screening programme by modifying the model in order to compare two current practice strategies . two uptake rates of current eye care were compared using bhps data : 6.5% ( corresponding to the uptake rate of low income groups ) and 17% ( estimated uptake of eye care for people with diabetes ) . all analyses incorporated probabilistic sensitivity analyses where the statistical imprecision is allowed for by sampling ( e.g. 1000 times ) from probability distributions attached to model mean parameter values . the uncertainty in the model parameter values has cost implications as the correct strategy might not be chosen . in effect , if the analysis is run with alternative parameter values the choice of strategy might differ from the strategy finally adopted . the sum of the benefits forgone for not being able to make the right decision due to this uncertainty is the expected value of perfect information ( evpi ) , as perfect information would eliminate the possibility of making wrong decisions . the evpi can be compared with the cost of reducing uncertainty in the model by collecting further information . the expected value of parameter perfect information ( evppi ) is a similar concept but corresponds to a particular parameter or group of parameters in the model . evppi was calculated to identify model parameters that contribute the most to the overall model decision uncertainty . the total evpi and evppi were obtained by multiplying the evpi and evppi estimated at an individual level by the number of individuals who would benefit from the intervention in a given period of time . we assumed 380,000 individuals with undiagnosed glaucoma in the uk and an annual incidence of 11,000 new cases a time horizon of 10 years ( representing the lifespan of the technology ; in this case a specific screening strategy ) and a discount rate of 3.5% . the revised model , now named the glaucoma screening platform study ( gps ) model , considers four possible screening strategies against a current practice comparator ( no screening ) . the screening test schedule , based on the views of service providers in terms of practicality and equity , was screening an inception cohort age 40 . the screening strategies allow for a technician as first screening contact . however , they differ in the tests performed . glaucoma tests were either optic nerve photography ( onp ) or screening mode perimetry ( a measure of visual field ( vf ) sensitivity ) with or without tonometry ( a measure of iop ) . for those testing positive two pathways were explored , a diagnostic refinement step , using a specialized optometrist to examine screen positives as in the original model , or no referral refinement with screen test positives referred to a hospital based glaucoma service . table 1.prevalence , incidence and progression of glaucoma.probabilityvaluesourceprevalence of glaucoma15%assumption based on prevalence rates for general population and high prevalence subgroupsprogression to moderate glaucoma0.129progression data from gsm 2007progression to severe glaucoma0.048progression data from gsm 2007progression to visual impaired0.042progression data from gsm 2007annual probability of having an eye test in current practice ( not screening):general population ( adults over 40 years old)0.1728based on survey of the uk publicgeneral population ( adults over 40 years old)0.0741based on bhps data and alternative assumptionsindividuals with diabetes0.1693based on bhps dataindividuals with eye problems0.1192based on bhps dataindividuals within low income households0.0653based on bhps datavisual field based glaucoma stagingmean defect score ( db)mild glaucoma001 to 600 dbmoderate glaucoma601 to 1200 dbsevere glaucoma1201 to 2000 dbvisual impairment ( partial sight / blind)2001 db or worsegsm : glaucoma screening model ; bhps : british household panel survey.avisual field based glaucoma staging . table 2.data on screening tests and test performance.probabilityvaluesourcecurrent eye careoptometry testing , sensitivity0.32gsm 2007optometry testing , specificity0.99gsm 2007proportion of normal ( no glaucoma ) with iop < 26 mmhg0.96gsm 2007proportion of glaucoma with iop 26 mmhg0.35gsm 2007screening testsoptic nerve photography , sensitivity0.73gsm 2007optic nerve photography , specificity0.89gsm 2007perimetry ( frequency doubling technology - c-20 - 1 ) , sensitivity0.79gsm 2007perimetry ( frequency doubling technology - c-20 - 1 ) specificity0.94gsm 2007iop : intraocular pressure ; gsm : glaucoma screening model . gsm : glaucoma screening model ; bhps : british household panel survey . visual field based glaucoma staging . iop : intraocular pressure ; gsm : glaucoma screening model . on average , screening the general population selected on age alone was not cost - effective at a 30,000 threshold ( table 3 ) . it should be noted that as this is a population screening model we would expect a relatively small proportion of individuals to be identified with glaucoma and hence , on average for the population , only small gains in benefits . table 3.cost-effectiveness base case analysis results.screening acceptance ( % ) strategycostqalysicercurrent practice17619.253030%gps iid ( iop + vf)23919.253788,908gps i d ( iop + onp)23919.2536(dominated)gps ii ( iop + vf)26619.254097,136gps i ( iop + onp)27619.2539(dominated)current practice17619.253050%gps iid ( iop + vf)26119.253974,408gps 1d ( iop + onp)26119.2538(dominated)gps ii ( iop + vf)30419.2543103,985gps i ( iop + onp)32119.2541(dominated)current practice17619.253070%gps iid ( iop + vf)28219.254168,718gps 1d ( iop + onp)28319.2540(dominated)gps ii ( iop + vf)34219.2545111,427gps i ( iop + onp)36619.2544(dominated)qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field.aforty-year-old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs.bicers are related to the , on average , cheapest non - dominated strategy . box 1.description of the pathways compared within the economic model.glaucoma screening platform study ( gps)1 . ( tonometry ( measurement of intraocular pressure ( iop ) ) and optic nerve photography ( onp ) ) : the population to be screened are invited to a primary care setting to undergo tonometry and onp by a technician or nurse who has received some training . screen positives referred to hospital eye service.gps11 ( iop and visual field ( vf ) ) : as above but screening with tonometry and visual field test ( perimetry ) . screen positives referred to hospital eye service.gps1d ( iop + onp ) : screening with tonometry and optic nerve photography and screen positives examined by a specialized optometrist ( diagnosis ) . diagnostic test positives referred to hospital eye service.gps11d ( iop + vf ) : screening with tonometry and visual field test ( perimetry ) with further diagnostic refinement and screen positives examined by a specialized optometrist ( diagnosis ) . diagnostic test positives referred to hospital eye service.current practice ( uk nhs eye care ) : opportunistic sight test at community optometrist with referral of suspect glaucoma to the hospital eye service.iop 26 mmhg = screen positive.iop < 26 mmhg + second technology test positive = screen positive.iop < 26 mmhg + second technology test negative = return to current eye care and re - screen cycle . qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field . forty - year - old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs . description of the pathways compared within the economic model . for cohorts starting at 50 and 60 years old , icers were , regardless of the level of screening uptake , well above the typically accepted threshold value of 30,000 ( see supplementary material online , tables 4 and 5 ) . similar results were obtained when the sensitivity and specificity of case detection in current eye care were reduced to plausible minimum levels ( see supplementary material online , tables 6 and 7 ) . using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . figure 1 shows the two - way sensitivity analyses results for the annual cost of sight impairment versus the percentage of screening uptake . that is , for each combination of cost of sight impairment and proportion of screening uptake , the figure which shows the strategy with the highest net - benefit ( e.g. willingness to pay multiplied by the mean qalys minus mean cost ) , assuming a 30,000 willingness to pay for a qaly . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . for a higher risk subgroup ( assumed glaucoma prevalence of 5% ) a targeted screening programme ( surveillance ) could be worthwhile if the annual cost of sight impairment is above 3000 ( figure 1(b ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . this uncertainty was built into the probabilistic sensitivity analysis by investigating the impact of the annual cost of visual impairment being equally likely to have any value between 1000 and 40,000 ( i.e. assuming a uniform distribution ) and investigating the value of removing all the imprecision around parameter estimates within the model , i.e. the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. evppi . figure 2 shows the average evpi and evppi curves for a 50-year - old cohort with a 5% prevalence rate of glaucoma ( higher risk subgroup ) and when uptake of current practice is 7.4% ( described in table 1 ) . this situation was chosen as , based on the base case results , was the most likely scenario favouring screening . the value of removing the imprecision around the model parameter estimates is illustrated in figure 2 . figure 2.average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi).scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi ) . scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . the peak in the evpi curve corresponds to the willingness to pay for a qaly value where decision uncertainty is at its highest ( at around 20,000 wtp ) and is the decision point on whether to screen or not . for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. 14 million ) , and screening acceptance or current practice eye uptake rate ( e.g. 8 million each ) . on average , screening the general population selected on age alone was not cost - effective at a 30,000 threshold ( table 3 ) . it should be noted that as this is a population screening model we would expect a relatively small proportion of individuals to be identified with glaucoma and hence , on average for the population , only small gains in benefits . table 3.cost-effectiveness base case analysis results.screening acceptance ( % ) strategycostqalysicercurrent practice17619.253030%gps iid ( iop + vf)23919.253788,908gps i d ( iop + onp)23919.2536(dominated)gps ii ( iop + vf)26619.254097,136gps i ( iop + onp)27619.2539(dominated)current practice17619.253050%gps iid ( iop + vf)26119.253974,408gps 1d ( iop + onp)26119.2538(dominated)gps ii ( iop + vf)30419.2543103,985gps i ( iop + onp)32119.2541(dominated)current practice17619.253070%gps iid ( iop + vf)28219.254168,718gps 1d ( iop + onp)28319.2540(dominated)gps ii ( iop + vf)34219.2545111,427gps i ( iop + onp)36619.2544(dominated)qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field.aforty-year-old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs.bicers are related to the , on average , cheapest non - dominated strategy . box 1.description of the pathways compared within the economic model.glaucoma screening platform study ( gps)1 . ( tonometry ( measurement of intraocular pressure ( iop ) ) and optic nerve photography ( onp ) ) : the population to be screened are invited to a primary care setting to undergo tonometry and onp by a technician or nurse who has received some training . screen positives referred to hospital eye service.gps11 ( iop and visual field ( vf ) ) : as above but screening with tonometry and visual field test ( perimetry ) . screen positives referred to hospital eye service.gps1d ( iop + onp ) : screening with tonometry and optic nerve photography and screen positives examined by a specialized optometrist ( diagnosis ) . diagnostic test positives referred to hospital eye service.gps11d ( iop + vf ) : screening with tonometry and visual field test ( perimetry ) with further diagnostic refinement and screen positives examined by a specialized optometrist ( diagnosis ) . diagnostic test positives referred to hospital eye service.current practice ( uk nhs eye care ) : opportunistic sight test at community optometrist with referral of suspect glaucoma to the hospital eye service.iop 26 mmhg = screen positive.iop < 26 mmhg + second technology test positive = screen positive.iop < 26 mmhg + second technology test negative = return to current eye care and re - screen cycle . qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field . forty - year - old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs . for cohorts starting at 50 and 60 years old , icers were , regardless of the level of screening uptake , well above the typically accepted threshold value of 30,000 ( see supplementary material online , tables 4 and 5 ) . similar results were obtained when the sensitivity and specificity of case detection in current eye care were reduced to plausible minimum levels ( see supplementary material online , tables 6 and 7 ) . using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . figure 1 shows the two - way sensitivity analyses results for the annual cost of sight impairment versus the percentage of screening uptake . that is , for each combination of cost of sight impairment and proportion of screening uptake , the figure which shows the strategy with the highest net - benefit ( e.g. willingness to pay multiplied by the mean qalys minus mean cost ) , assuming a 30,000 willingness to pay for a qaly . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . for a higher risk subgroup ( assumed glaucoma prevalence of 5% ) a targeted screening programme ( surveillance ) could be worthwhile if the annual cost of sight impairment is above 3000 ( figure 1(b ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . this uncertainty was built into the probabilistic sensitivity analysis by investigating the impact of the annual cost of visual impairment being equally likely to have any value between 1000 and 40,000 ( i.e. assuming a uniform distribution ) and investigating the value of removing all the imprecision around parameter estimates within the model , i.e. the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. evppi . figure 2 shows the average evpi and evppi curves for a 50-year - old cohort with a 5% prevalence rate of glaucoma ( higher risk subgroup ) and when uptake of current practice is 7.4% ( described in table 1 ) . this situation was chosen as , based on the base case results , was the most likely scenario favouring screening . the value of removing the imprecision around the model parameter estimates is illustrated in figure 2 . figure 2.average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi).scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi ) . scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . the peak in the evpi curve corresponds to the willingness to pay for a qaly value where decision uncertainty is at its highest ( at around 20,000 wtp ) and is the decision point on whether to screen or not . for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. 14 million ) , and screening acceptance or current practice eye uptake rate ( e.g. 8 million each ) . for cohorts starting at 50 and 60 years old , icers were , regardless of the level of screening uptake , well above the typically accepted threshold value of 30,000 ( see supplementary material online , tables 4 and 5 ) . similar results were obtained when the sensitivity and specificity of case detection in current eye care were reduced to plausible minimum levels ( see supplementary material online , tables 6 and 7 ) . using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . figure 1 shows the two - way sensitivity analyses results for the annual cost of sight impairment versus the percentage of screening uptake . that is , for each combination of cost of sight impairment and proportion of screening uptake , the figure which shows the strategy with the highest net - benefit ( e.g. willingness to pay multiplied by the mean qalys minus mean cost ) , assuming a 30,000 willingness to pay for a qaly . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . for a higher risk subgroup ( assumed glaucoma prevalence of 5% ) a targeted screening programme ( surveillance ) could be worthwhile if the annual cost of sight impairment is above 3000 ( figure 1(b ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . increasing uptake rates would , however , be likely to incur costs . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . this uncertainty was built into the probabilistic sensitivity analysis by investigating the impact of the annual cost of visual impairment being equally likely to have any value between 1000 and 40,000 ( i.e. assuming a uniform distribution ) and investigating the value of removing all the imprecision around parameter estimates within the model , i.e. the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. evppi . figure 2 shows the average evpi and evppi curves for a 50-year - old cohort with a 5% prevalence rate of glaucoma ( higher risk subgroup ) and when uptake of current practice is 7.4% ( described in table 1 ) . this situation was chosen as , based on the base case results , was the most likely scenario favouring screening . the value of removing the imprecision around the model parameter estimates is illustrated in figure 2 . figure 2.average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi).scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi ) . scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . the upper and lower bounds limits for this distribution were informed by the literature , assuming that nhs treatment as well as pss cost were included . incremental cost - effectiveness ratio for moving to screening ( gps1d ( iop + vf ) = 21,720 ) . the peak in evpi corresponds to the uncertainty in the decision of changing from current practice ( opportunistic case finding ) to screening with a technician conducting tonometry and visual field test ( perimetry ) with screen positives examined by a specialized optometrist ( gps11d ( iop + vf ) ) . iop : intraocular pressure ; gps : glaucoma screening platform study ; pss : personal social services ; vf : visual field . the peak in the evpi curve corresponds to the willingness to pay for a qaly value where decision uncertainty is at its highest ( at around 20,000 wtp ) and is the decision point on whether to screen or not . for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. 14 million ) , and screening acceptance or current practice eye uptake rate ( e.g. 8 million each ) . findings from the original glaucoma screening model had suggested that glaucoma screening of a population selected on age is unlikely to be considered cost - effective at values for a qaly that society is typically willing to pay , but screening of high risk groups ( prevalence of around 4% ) might be . across all the scenarios , we modelled the conclusion from the original evaluation is unchanged . results were robust to all changes in the model parameter values except for the annual cost of sight impairment . when taking a wider perspective on the costs of sight impairment , findings from the two way sensitivity analysis , varying screening uptake and costs of sight impairment , suggest that assuming higher estimated costs of sight impairment screening the general population selected on age alone as opposed to surveillance of high risk groups might be worthwhile . the health costs of severe sight impairment due to glaucoma have been estimated as 800 per year ( updated to 20102011 prices ) . for adults with physical disabilities , these data include estimates from people with sight loss but data were not reported separately . the cost of sight impairment in the uk is reported by the royal national institute for the blind as 12,457 per person per year . however , these calculations include indirect costs ( productivity losses ) , cost due to lower employment , premature mortality , as well as 8782 corresponding to burden of disease cost the use of such figures entails some double counting as some of these effects are captured within the qaly estimates . estimates by mead and hyde suggest that the cost of blindness for another chronic eye condition , age related macular degeneration , range around 8000 ( updated to 20102011 prices ) for the first year of blindness but highlighted the limitations in the data to determine the true costs of failing eyesight . the purpose of updating the original glaucoma screening model was to determine whether , given the newly defined screening pathways and target population , a large glaucoma screening rct would be value for money in terms of informing policy decisions . the value of removing all uncertainties in the model was around 107 million , indicating that further research on glaucoma screening might be worthwhile . the uncertainty around cost of sight impairment contributed most followed by the uncertainty around screening test performance , and the uptake of screening or current eye care services . these findings suggest that before proceeding to a large rct evaluating a glaucoma screening or surveillance programme , further research to understand and quantify the cost of sight impairment is a priority . these data would be best collected within a prospective cohort study , following individuals through the spectrum of visual impairment and as needs change and adaptation to sight loss occurs . our findings suggest that behavioural intervention , such as a carefully worded invitation with a reminder or a more intensive intervention ( e.g. tailored message , sms reminder , buddy system ) may improve attendance . this study used a transparent iterative approach building on a robust evidence synthesis and sought views of all stakeholders to inform decisions regarding glaucoma screening . we used robust qualitative and quantitative methods to provide an evidence - based recommendation for future research . it was not within the scope of this study to estimate the accuracy of screening tests or to evaluate the accuracy of current eye care services in the detection of glaucoma . we used the same estimates of performance of community optometric detection of glaucoma ( current eye care ) as used in the original model , based on a survey in the late 1980s which may not represent current practice . to estimate screening test performance we used estimates from an evidence synthesis but . we had limited data on the uptake of current eye care services by higher risk groups , particularly by ethnic minority groups and those who might expect to have a higher uptake of current eye care , those with a family history of glaucoma . we did however use new primary data from our survey on eye testing within the last few years and used data from the bhps that is considered to be representative . the value for information analysis can only capture those uncertainties incorporated into the economic model and is dependent on the model structure . the model did not consider the potential harms due to screening ( e.g. anxiety for those with false positives ) or the side effects of treatment ( e.g. cataracts ) . while it is unlikely that these omissions would have a major impact on the model results , they are a limitation of the analysis . finally , while the evpi analysis constitutes a necessary condition in determining whether further research is worthwhile it is not in itself sufficient to determine whether research should be conducted ( although it can give a strong indication that further research is not worthwhile , if for example evpi is less than the costs of conducting further research ) . a positive expected net - benefit of sampling ( enbs ) constitutes a necessary and sufficient condition to conduct further research . enbs is the difference between the benefits of reducing uncertainty with a particular sample size study and the cost of obtaining that sample size . unfortunately , enbs can be obtained only under very restrictive assumptions and is often computationally prohibitive and there have been few examples where it has been used in practice to inform , for example , the sample size of a trial . in such circumstances , we believe the evpi establishes a first step to inform the judgment that further research is potentially worthwhile . findings from the original glaucoma screening model had suggested that glaucoma screening of a population selected on age is unlikely to be considered cost - effective at values for a qaly that society is typically willing to pay , but screening of high risk groups ( prevalence of around 4% ) might be . across all the scenarios , we modelled the conclusion from the original evaluation is unchanged . results were robust to all changes in the model parameter values except for the annual cost of sight impairment . when taking a wider perspective on the costs of sight impairment , findings from the two way sensitivity analysis , varying screening uptake and costs of sight impairment , suggest that assuming higher estimated costs of sight impairment screening the general population selected on age alone as opposed to surveillance of high risk groups might be worthwhile . the health costs of severe sight impairment due to glaucoma have been estimated as 800 per year ( updated to 20102011 prices ) . for adults with physical disabilities , these data include estimates from people with sight loss but data were not reported separately . the cost of sight impairment in the uk is reported by the royal national institute for the blind as 12,457 per person per year . however , these calculations include indirect costs ( productivity losses ) , cost due to lower employment , premature mortality , as well as 8782 corresponding to burden of disease cost the use of such figures entails some double counting as some of these effects are captured within the qaly estimates . estimates by mead and hyde suggest that the cost of blindness for another chronic eye condition , age related macular degeneration , range around 8000 ( updated to 20102011 prices ) for the first year of blindness but highlighted the limitations in the data to determine the true costs of failing eyesight . the purpose of updating the original glaucoma screening model was to determine whether , given the newly defined screening pathways and target population , a large glaucoma screening rct would be value for money in terms of informing policy decisions . the value of removing all uncertainties in the model was around 107 million , indicating that further research on glaucoma screening might be worthwhile . the uncertainty around cost of sight impairment contributed most followed by the uncertainty around screening test performance , and the uptake of screening or current eye care services . these findings suggest that before proceeding to a large rct evaluating a glaucoma screening or surveillance programme , further research to understand and quantify the cost of sight impairment is a priority . these data would be best collected within a prospective cohort study , following individuals through the spectrum of visual impairment and as needs change and adaptation to sight loss occurs . our findings suggest that behavioural intervention , such as a carefully worded invitation with a reminder or a more intensive intervention ( e.g. tailored message , sms reminder , buddy system ) may improve attendance . this study used a transparent iterative approach building on a robust evidence synthesis and sought views of all stakeholders to inform decisions regarding glaucoma screening . we used robust qualitative and quantitative methods to provide an evidence - based recommendation for future research . it was not within the scope of this study to estimate the accuracy of screening tests or to evaluate the accuracy of current eye care services in the detection of glaucoma . we used the same estimates of performance of community optometric detection of glaucoma ( current eye care ) as used in the original model , based on a survey in the late 1980s which may not represent current practice . to estimate screening test performance we used estimates from an evidence synthesis but these were based on evidence from studies with heterogeneous populations and high risk of bias . we had limited data on the uptake of current eye care services by higher risk groups , particularly by ethnic minority groups and those who might expect to have a higher uptake of current eye care , those with a family history of glaucoma . we did however use new primary data from our survey on eye testing within the last few years and used data from the bhps that is considered to be representative . the value for information analysis can only capture those uncertainties incorporated into the economic model and is dependent on the model structure . the model did not consider the potential harms due to screening ( e.g. anxiety for those with false positives ) or the side effects of treatment ( e.g. cataracts ) . while it is unlikely that these omissions would have a major impact on the model results , they are a limitation of the analysis . finally , while the evpi analysis constitutes a necessary condition in determining whether further research is worthwhile it is not in itself sufficient to determine whether research should be conducted ( although it can give a strong indication that further research is not worthwhile , if for example evpi is less than the costs of conducting further research ) . a positive expected net - benefit of sampling ( enbs ) constitutes a necessary and sufficient condition to conduct further research . enbs is the difference between the benefits of reducing uncertainty with a particular sample size study and the cost of obtaining that sample size . unfortunately , enbs can be obtained only under very restrictive assumptions and is often computationally prohibitive and there have been few examples where it has been used in practice to inform , for example , the sample size of a trial . in such circumstances , we believe the evpi establishes a first step to inform the judgment that further research is potentially worthwhile . a glaucoma screening trial is currently unlikely to be the best use of research resources to inform policy decisions on screening policy in the uk . further research to quantify the health and personal social services costs of sight impairment is recommended . further development of glaucoma tests and an evaluation of a behavioural intervention to improve attendance for those who do not use eye care services would be worthwhile . our findings are uk specific , but the methods used and the modelling framework can be adapted and populated with country specific parameters . this paper was developed from a strategic grant funded by the medical research council ( project reference g0701759 ) . the health services research unit and the health economics research unit are both core funded by the chief scientist office of the scottish government health directorates . the views expressed in this report are those of the authors and not necessarily those of the funders .

objectivesto assess the value of conducting a glaucoma screening randomized controlled trial in the uk.methodsdecision model based economic evaluation and value of information analysis . model derived from a previous health technology assessment . model updated in terms of structure and parameter estimates with data from surveys , interviews with members of the public and health care providers and routine sources.resultson average , across a range of ages of initiating screening ( 4060 years ) , glaucoma prevalence ( 15% ) , screening uptake ( 30100% ) , and the performance of current case finding , screening was not cost - effective at a 30,000 threshold per quality adjusted life year ( qaly ) from the perspective of the national health service ( nhs ) . the societal value of removing all uncertainty around glaucoma screening is 107 million at a threshold of 20,000 per qaly . for informing policy decisions on glaucoma screening , reducing uncertainty surrounding the nhs and personal social care cost of sight impairment ( 74 million ) was of most value , followed by reducing uncertainty in test performance ( 14 million ) and uptake of either screening or current eye care ( 8 million each).conclusionsa glaucoma screening trial in the uk is unlikely to be the best use of research resources . further research to quantify the costs of sight impairment falling on the nhs and personal social services is a priority . further development of glaucoma tests and research into strategies to promote the uptake of screening or current eye care such as through the use of a behavioural intervention would be worthwhile .

Introduction Methods Results Base case analysis (inception cohort aged 40 estimated 1% glaucoma prevalence, screening every 10 years and taking a NHS perspective) Sensitivity analysis Screening start age and performance of current eye care Varying uptake of screening and current practice Cost of sight impairment Enhancing current eye care Discussion Main findings Strengths and limitations Conclusions Supplementary Material Funding Supplementary Material

the modelling evaluation , hereafter referred to as the glaucoma screening model , used the best data available but still had some uncertainties : how best to screen ( tests and location ) ; likely uptake of any screening programme ; and the effectiveness and coverage of current eye care services . , we report the integration of the findings ( revised screening test schedule , likely uptake of screening and uptake of usual care ) into the glaucoma screening economic model to inform whether a glaucoma screening trial would be worthwhile . we took the perspective that interventions compared within the model could be delivered ( technical feasibility ) and were acceptable ( likely uptake by providers and the public ) in the context of the nhs . we also used data collected in the survey on attendance by the public for an eye test within the last three years to estimate uptake of the comparator pathway within the model of opportunistic case finding within current eye care . we sought estimates of attendance at eye care services for several risk groups ( age over 50 , black ethnicity , diabetes , myopia , family history of glaucoma , and low socioeconomic status from the british household panel survey data to develop and fit the probability distributions around the mean uptake of current eye care for the general population and subgroups using the excel add - on oracle crystal ball . the model allowed movement between health states every year , estimated costs from nhs and personal social services perspectives and used the eq-5d-3l quality of life weights to calculate quality adjusted life years ( qalys ) . we used alternative data for uptake of current eye care ( based on the survey of the public and british household panel survey ( bhps ) data ) for the whole population , for cohorts aged 50 and 60 as well as for higher risk subgroups of the adult population : those self - reported as having diabetes , sight problems in the family ( excluding using spectacles ) as a proxy for family history of glaucoma , and those at low household income ( below 10,000 a year ) . without a behavioural intervention , glaucoma screening uptake was considered to be about 23% ( based on our survey where 45% of the sample had strong intention scores ( mean intention score of 7 on a 17 scale ) and the health behaviour literature that 50% of strong intenders will perform the intended behaviour . for the range of values selected for the annual cost of sight impairment and uptake rate , only gps11 ( iop + vf) are potentially cost - effective when society is willing to pay 30,000 per qaly . the vertical continuous line at 19,000 cost of sight impairment illustrates that screening is not cost - effective below this value , regardless of the screening uptake . the vertical continuous line at 3000 cost of sight impairment illustrates that screening by any pathway is not cost - effective below this value , regardless of screening uptake . for the range of values selected for the annual cost of sight impairment and uptake rate , only gps11 ( iop + vf) are potentially cost - effective when society is willing to pay 30,000 per qaly . the vertical continuous line at 19,000 cost of sight impairment illustrates that screening is not cost - effective below this value , regardless of the screening uptake . the vertical continuous line at 3000 cost of sight impairment illustrates that screening by any pathway is not cost - effective below this value , regardless of screening uptake . two uptake rates of current eye care were compared using bhps data : 6.5% ( corresponding to the uptake rate of low income groups ) and 17% ( estimated uptake of eye care for people with diabetes ) . table 1.prevalence , incidence and progression of glaucoma.probabilityvaluesourceprevalence of glaucoma15%assumption based on prevalence rates for general population and high prevalence subgroupsprogression to moderate glaucoma0.129progression data from gsm 2007progression to severe glaucoma0.048progression data from gsm 2007progression to visual impaired0.042progression data from gsm 2007annual probability of having an eye test in current practice ( not screening):general population ( adults over 40 years old)0.1728based on survey of the uk publicgeneral population ( adults over 40 years old)0.0741based on bhps data and alternative assumptionsindividuals with diabetes0.1693based on bhps dataindividuals with eye problems0.1192based on bhps dataindividuals within low income households0.0653based on bhps datavisual field based glaucoma stagingmean defect score ( db)mild glaucoma001 to 600 dbmoderate glaucoma601 to 1200 dbsevere glaucoma1201 to 2000 dbvisual impairment ( partial sight / blind)2001 db or worsegsm : glaucoma screening model ; bhps : british household panel survey.avisual field based glaucoma staging . on average , screening the general population selected on age alone was not cost - effective at a 30,000 threshold ( table 3 ) . table 3.cost-effectiveness base case analysis results.screening acceptance ( % ) strategycostqalysicercurrent practice17619.253030%gps iid ( iop + vf)23919.253788,908gps i d ( iop + onp)23919.2536(dominated)gps ii ( iop + vf)26619.254097,136gps i ( iop + onp)27619.2539(dominated)current practice17619.253050%gps iid ( iop + vf)26119.253974,408gps 1d ( iop + onp)26119.2538(dominated)gps ii ( iop + vf)30419.2543103,985gps i ( iop + onp)32119.2541(dominated)current practice17619.253070%gps iid ( iop + vf)28219.254168,718gps 1d ( iop + onp)28319.2540(dominated)gps ii ( iop + vf)34219.2545111,427gps i ( iop + onp)36619.2544(dominated)qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field.aforty-year-old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs.bicers are related to the , on average , cheapest non - dominated strategy . using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . assuming a uniform distribution ) and investigating the value of removing all the imprecision around parameter estimates within the model , i.e. the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. on average , screening the general population selected on age alone was not cost - effective at a 30,000 threshold ( table 3 ) . table 3.cost-effectiveness base case analysis results.screening acceptance ( % ) strategycostqalysicercurrent practice17619.253030%gps iid ( iop + vf)23919.253788,908gps i d ( iop + onp)23919.2536(dominated)gps ii ( iop + vf)26619.254097,136gps i ( iop + onp)27619.2539(dominated)current practice17619.253050%gps iid ( iop + vf)26119.253974,408gps 1d ( iop + onp)26119.2538(dominated)gps ii ( iop + vf)30419.2543103,985gps i ( iop + onp)32119.2541(dominated)current practice17619.253070%gps iid ( iop + vf)28219.254168,718gps 1d ( iop + onp)28319.2540(dominated)gps ii ( iop + vf)34219.2545111,427gps i ( iop + onp)36619.2544(dominated)qaly : quality adjusted life years ; icer : incremental cost - effectiveness ratio ; iop : intraocular pressure ; gps : glaucoma screening platform study ; vf : visual field.aforty-year-old inception cohort , 17% uptake of current practice , 1% glaucoma prevalence , lifetime time horizon , nhs costs.bicers are related to the , on average , cheapest non - dominated strategy . using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. figure 2.average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi).scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. using alternative assumptions about the estimated uptake of current eye care services , varying from low annual uptake 6.5% ( estimated uptake of eye care for low income households ) to 17% ( estimated uptake of eye care for people with diabetes ) based upon bhps data , and varying the assumed uptake of screening from 50% to 100% , the cost - effectiveness of screening improved , although the icer remained well above a 30,000 threshold ( see supplementary material online , tables 8 and 9 ) . for the illustrated cohort ( inception cohort aged 50 , assumed 1% glaucoma prevalence and 17% annual uptake of current practice ) none of the screening pathways would have a higher net - benefit compared with current practice , unless the annual cost of sight impairment is above 19,000 . when the annual cost of sight impairment is above 30,000 screening is worthwhile if the uptake of screening is above 30% ( figure 1(a ) ) . assuming a low uptake of current eye care ( 6.5% per year ) , as might be expected within a low income subgroup screening could be considered cost - effective if the annual cost of visual impairment is above 18,000 per year with screening uptake of 40% for a cohort with an assumed glaucoma prevalence of 1% ( figure 1(c ) ) . increasing the uptake of current eye care for those in higher risk groups ( > 5% glaucoma prevalence ) , as opposed to a screening programme , is cost - effective when the annual cost of sight impairment is above 8000 and assuming no cost for a behavioural intervention to improve uptake . the cost per individual of increasing uptake would need to be < 20 with an annual cost of sight impairment per person of 20,000 for this strategy to approach cost - effectiveness . the evpi as well as the value of removing all uncertainty surrounding a particular ( group of ) parameter(s ) , i.e. figure 2.average expected value of perfect information ( evpi ) and expected value of parameter perfect information ( evppi).scenario : model start age ( and screening ) 50 years old , prevalence rate 5% , screening every 10 years , whole population , current practice annual eye test uptake rate 7.4% , average annual cost of sight impairment 20,500 . for a willingness to pay threshold of 20,000 for a qaly , the population evpi is around 107 million , with the costs of sight impairment contributing the most to this decision uncertainty ( evppi , 74 million ) , followed by the uncertainty due to test performance ( e.g. findings from the original glaucoma screening model had suggested that glaucoma screening of a population selected on age is unlikely to be considered cost - effective at values for a qaly that society is typically willing to pay , but screening of high risk groups ( prevalence of around 4% ) might be . when taking a wider perspective on the costs of sight impairment , findings from the two way sensitivity analysis , varying screening uptake and costs of sight impairment , suggest that assuming higher estimated costs of sight impairment screening the general population selected on age alone as opposed to surveillance of high risk groups might be worthwhile . the cost of sight impairment in the uk is reported by the royal national institute for the blind as 12,457 per person per year . the purpose of updating the original glaucoma screening model was to determine whether , given the newly defined screening pathways and target population , a large glaucoma screening rct would be value for money in terms of informing policy decisions . the value of removing all uncertainties in the model was around 107 million , indicating that further research on glaucoma screening might be worthwhile . the uncertainty around cost of sight impairment contributed most followed by the uncertainty around screening test performance , and the uptake of screening or current eye care services . these findings suggest that before proceeding to a large rct evaluating a glaucoma screening or surveillance programme , further research to understand and quantify the cost of sight impairment is a priority . it was not within the scope of this study to estimate the accuracy of screening tests or to evaluate the accuracy of current eye care services in the detection of glaucoma . we had limited data on the uptake of current eye care services by higher risk groups , particularly by ethnic minority groups and those who might expect to have a higher uptake of current eye care , those with a family history of glaucoma . findings from the original glaucoma screening model had suggested that glaucoma screening of a population selected on age is unlikely to be considered cost - effective at values for a qaly that society is typically willing to pay , but screening of high risk groups ( prevalence of around 4% ) might be . when taking a wider perspective on the costs of sight impairment , findings from the two way sensitivity analysis , varying screening uptake and costs of sight impairment , suggest that assuming higher estimated costs of sight impairment screening the general population selected on age alone as opposed to surveillance of high risk groups might be worthwhile . the purpose of updating the original glaucoma screening model was to determine whether , given the newly defined screening pathways and target population , a large glaucoma screening rct would be value for money in terms of informing policy decisions . the value of removing all uncertainties in the model was around 107 million , indicating that further research on glaucoma screening might be worthwhile . the uncertainty around cost of sight impairment contributed most followed by the uncertainty around screening test performance , and the uptake of screening or current eye care services . these findings suggest that before proceeding to a large rct evaluating a glaucoma screening or surveillance programme , further research to understand and quantify the cost of sight impairment is a priority . it was not within the scope of this study to estimate the accuracy of screening tests or to evaluate the accuracy of current eye care services in the detection of glaucoma . we had limited data on the uptake of current eye care services by higher risk groups , particularly by ethnic minority groups and those who might expect to have a higher uptake of current eye care , those with a family history of glaucoma . a glaucoma screening trial is currently unlikely to be the best use of research resources to inform policy decisions on screening policy in the uk . further research to quantify the health and personal social services costs of sight impairment is recommended . further development of glaucoma tests and an evaluation of a behavioural intervention to improve attendance for those who do not use eye care services would be worthwhile .

most patients with hypertension do not benefit from treatment.1 data from nhanes / nchs 19992004 showed that 71.8% of individuals were aware of their condition , 61.4% were under current treatment and 35.1% had it under control.2 although the percentage of individuals with controlled blood pressure in brazil is unknown , we estimate it to be low because the percentage of patients with their high blood pressure under control is around 30 - 35% at outpatient clinics that specialize in hypertension , and these clinics do not represent the national reality.3 many reasons exist for non - adherence to medical regimens , including adverse drug effects , poor instructions , poor provider - patient relationship , poor memory , a patient s disagreement with the need for treatment or inability to afford medication.4,5 one of the strategies employed to improve treatment compliance is the use of active telephone calls6 to patients through which they are given guidance and treatment questions are answered . using this strategy , several studies have shown an increase in treatment compliance , a higher percentage of blood pressure control6 - 8 and a decrease in mortality.9 - 11 another strategy that aims at improving treatment compliance is the employment of two low - dose medications . evidence suggests that the combination of two antihypertensive agents provides a higher percentage of blood pressure control due to complementary mechanisms of action . in addition , this results in better tolerability and consequently , better treatment compliance.12 - 14 the ascot study15 ( anglo - scandinavian cardiac outcomes trial ) has indicated a significant reduction in all causes of mortality with the use of current medications for treating hypertension : calcium channel antagonists and angiotensin - converting enzyme inhibitors . it has also been demonstrated that a high incidence of adverse events may decrease treatment compliance . however , in the life16 study , angiotensin receptor antagonists proved to be better at reducing the risks related to cardiovascular events and they resulted in a lower rate of treatment discontinuation when compared to a beta - blocker . in view of these data , we evaluated the importance of providing guidelines to patients via active telephone calls for blood pressure management and preventing treatment discontinuation in hypertensive patients . we used two treatment regimens with low - dose medications that were offered for free to avoid the influence of financial factors . we opted for one regimen called traditional based on diuretics and beta - blockers and another called current based on angiotensin ii antagonists and calcium channel blockers . the study participants were selected at the hypertension unit , university of so paulo general hospital , nephrology division , university of so paulo school of medicine . the patients had essential hypertension and were able to receive telephone calls to be reminded of their medical appointments and be given guidance about hypertension . patients were of both genders , from diverse ethnic backgrounds , over 18 years of age , and had body mass indices below 40 kg / m . the patients were enrolled in the study after signing a free and informed consent form . the study was approved by the ethics committee of the board of directors of the university of so paulo school of medicine . patients were excluded on several bases , including blood pressure < 140/90 mmhg without antihypertensive medication , secondary hypertension , white - coat hypertension with systolic pressures 140 mmhg and/or diastolic pressures 90 mmhg at the doctor s office and awake mean systolic pressures < 135 mmhg or awake mean diastolic pressures < 85 mmhg without antihypertensive medication , malignant hypertension and patients with a previous history of hypersensitivity reaction to the study medications . other exclusion criteria were as follows : pregnant women or nursing mothers , the presence of liver dysfunction evidenced by the patient s clinical history or by one of the liver function tests ( levels twice the normal values for alkaline phosphatase , total bilirubin , aspartate aminotransferase ) , patients with clinical conditions that might interfere with the total conformity with the study or those who might have an increased risk for participating in the study , patients with a history of alcoholism , drug abuse or mental disorders that might invalidate the free and informed consent or limit the patient s ability to meet the protocol rules and patients who had participated in any other studies involving investigational drugs or drugs already marketed within the previous month before enrollment in this study or concomitantly with this study . blood pressure and heart rate measurements were performed on the right upper limb on sitting patients five times by the nursing staff using an appropriately sized cuff with a validated automatic oscillometric device ( dixtal , dx2710 , so paulo , brazil).17 the mean of the last two measurements was calculated and recorded if the difference between these measurements was less than 4 mmhg . if after the five measurements the difference between the last two was greater than 4 mmhg , the measurement was repeated until the difference between the two measurements was less than 4 mmhg . during the study , blood pressure measurements were always performed in the afternoon by the same nursing staff using the same device . the diagnosis of hypertension was made when the mean values of the last two measurements were as follows : systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg with or without medication at the initial visit ( visit 0 ) . patients who were receiving antihypertensive medication at the initial visit and had a systolic pressure < 140 mmhg or a diastolic pressure < 90 mmhg were re - evaluated eight weeks after discontinuation of their medication and introduction of placebo ; these patients were included in the study when the mean values included a systolic pressure 140 mmhg and/or a diastolic pressure 90 mmhg . controlled blood pressure was defined in two levels as ( with the patient in the seated position ) a systolic / diastolic pressure less than 140/90 or 120/80 mmhg . all of the patients underwent ambulatory blood pressure monitoring ( abpm ) , which was performed with a validated oscillometric device ( spacelabs 90207 , spacelabs inc , richmond , wa , usa),18,19 five weeks after the start of the placebo to eliminate cases of patients with white - coat hypertension and , in patients who did not receive placebo , to identify the white - coat effect . according to clinical characteristics , uncomplicated hypertensive patients without complications and without other concurrent diseases and b ) complicated- patients with severe hypertension ( mean diastolic pressure 110 mmhg with or without medication ) or comorbidities such as diabetes mellitus , renal failure ( serum creatinine > 1.4 mg / dl ) , coronary insufficiency , congestive heart failure or prior history of cerebrovascular accident . all patients , from both the complicated and the uncomplicated groups , were open - block randomized to receive active telephone calls ( phone calls group ) or not to receive telephone calls ( no phone calls group ) and to follow two treatment regimens , traditional or thus , after the first visit and randomization , patients from the phone calls group were invited to enroll by telephone in a program called biosinttica assistance , supported by biosinttica laboratory . the patients who subscribed started receiving active telephone calls from appropriately trained operators and also started receiving magazines with health - related information , which were sent periodically by mail . the patient was reminded to attend the next visit , and he / she was educated about hypertension and any necessary clarifications about his / her treatment . phone calls group were invited to attend occasional informative lectures with the participation of a multidisciplinary team . at the initial stage of treatment ( following eight weeks of treatment with placebo ) , the uncomplicated group received one of the following treatment regimens : a ) traditional treatment with 6.25 mg 2x / day hydrochlorothiazide and 25 mg 2x / day atenolol ; b ) current treatment with 25 mg 2x / day losartan and 2.5 mg 2x / day amlodipine . if the blood pressure could not be controlled during the visits , the medication doses were doubled or another antihypertensive was added . complicated group did not undergo the treatment period with placebo and was randomized to receive either traditional or current drug regimens similar to the ones administered to the uncomplicated group , though the specifics of each condition were carefully considered . the addition of other antihypertensive agents in the uncomplicated group and the specificities of the patient regimens in the complicated group were performed according to the guidelines from the v brazilian guidelines on arterial hypertension.3 all patients were instructed to take their medication every day at 7:00 am and 7:00 pm , with a variation of up to one hour . a 12-month supply of the necessary medication was supplied to the patients by the physician at the end of the visit at no cost to eliminate the financial factor in this analysis ; patients were given enough medication to last between visits . patients were instructed to bring the remaining pills to their subsequent visits , at which time they were counted by the nursing staff without the patients knowledge of this procedure . doctors visits , preceded by the nursing staff visit , took place every eight weeks for 56 weeks and included measurements of blood pressure , heart rate and weight . the weight was checked with the patient wearing light clothes standing barefoot on a scale ( model 2096pp , toledo do brasil , so paulo , brazil ) . study withdrawal was characterized by non - attendance to appointments for up to three months after the scheduled date . patients who returned to the medical clinic within three months after the scheduled date were allowed to continue in the study and be evaluated in an unscheduled visit . the tests performed during the placebo treatment and after 40 weeks of active treatments included the following : fasting glucose , urea , creatinine , total cholesterol , fractions of cholesterol , triglycerides , uric acid , total bilirubin , creatine phosphokinase ( cpk ) , sodium ( na+ ) , potassium ( k+ ) , hemoglobin , thyroid - stimulating hormone ( tsh ) , alkaline phosphatase , aspartate aminotransferase ( ast ) , alanine aminotranferase ( alt ) and urinary excretion of sodium in 24 h. data were analyzed using an analysis of variance ( anova ) according to the factor of controlled or uncontrolled hypertension . blood pressure and heart rate measurements were performed on the right upper limb on sitting patients five times by the nursing staff using an appropriately sized cuff with a validated automatic oscillometric device ( dixtal , dx2710 , so paulo , brazil).17 the mean of the last two measurements was calculated and recorded if the difference between these measurements was less than 4 mmhg . if after the five measurements the difference between the last two was greater than 4 mmhg , the measurement was repeated until the difference between the two measurements was less than 4 mmhg . during the study , blood pressure measurements were always performed in the afternoon by the same nursing staff using the same device . the diagnosis of hypertension was made when the mean values of the last two measurements were as follows : systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg with or without medication at the initial visit ( visit 0 ) . patients who were receiving antihypertensive medication at the initial visit and had a systolic pressure < 140 mmhg or a diastolic pressure < 90 mmhg were re - evaluated eight weeks after discontinuation of their medication and introduction of placebo ; these patients were included in the study when the mean values included a systolic pressure 140 mmhg and/or a diastolic pressure 90 mmhg . controlled blood pressure was defined in two levels as ( with the patient in the seated position ) a systolic / diastolic pressure less than 140/90 or 120/80 mmhg . all of the patients underwent ambulatory blood pressure monitoring ( abpm ) , which was performed with a validated oscillometric device ( spacelabs 90207 , spacelabs inc , richmond , wa , usa),18,19 five weeks after the start of the placebo to eliminate cases of patients with white - coat hypertension and , in patients who did not receive placebo , to identify the white - coat effect . according to clinical characteristics , complicated- patients with severe hypertension ( mean diastolic pressure 110 mmhg with or without medication ) or comorbidities such as diabetes mellitus , renal failure ( serum creatinine > 1.4 mg / dl ) , coronary insufficiency , congestive heart failure or prior history of cerebrovascular accident . all patients , from both the complicated and the uncomplicated groups , were open - block randomized to receive active telephone calls ( phone calls group ) or not to receive telephone calls ( no phone calls group ) and to follow two treatment regimens , traditional or thus , after the first visit and randomization , patients from the phone calls group were invited to enroll by telephone in a program called biosinttica assistance , supported by biosinttica laboratory . the patients who subscribed started receiving active telephone calls from appropriately trained operators and also started receiving magazines with health - related information , which were sent periodically by mail . the patient was reminded to attend the next visit , and he / she was educated about hypertension and any necessary clarifications about his / her treatment . all patients randomized to the phone calls group were invited to attend occasional informative lectures with the participation of a multidisciplinary team . at the initial stage of treatment ( following eight weeks of treatment with placebo ) , the uncomplicated group received one of the following treatment regimens : a ) traditional treatment with 6.25 mg 2x / day hydrochlorothiazide and 25 mg 2x / day atenolol ; b ) current treatment with 25 mg 2x / day losartan and 2.5 mg 2x / day amlodipine . if the blood pressure could not be controlled during the visits , the medication doses were doubled or another antihypertensive was added . the complicated group did not undergo the treatment period with placebo and was randomized to receive either traditional or current drug regimens similar to the ones administered to the uncomplicated group , though the specifics of each condition were carefully considered . the addition of other antihypertensive agents in the uncomplicated group and the specificities of the patient regimens in the complicated group were performed according to the guidelines from the v brazilian guidelines on arterial hypertension.3 all patients were instructed to take their medication every day at 7:00 am and 7:00 pm , with a variation of up to one hour . a 12-month supply of the necessary medication was supplied to the patients by the physician at the end of the visit at no cost to eliminate the financial factor in this analysis ; patients were given enough medication to last between visits . patients were instructed to bring the remaining pills to their subsequent visits , at which time they were counted by the nursing staff without the patients knowledge of this procedure . doctors visits , preceded by the nursing staff visit , took place every eight weeks for 56 weeks and included measurements of blood pressure , heart rate and weight . the weight was checked with the patient wearing light clothes standing barefoot on a scale ( model 2096pp , toledo do brasil , so paulo , brazil ) . study withdrawal was characterized by non - attendance to appointments for up to three months after the scheduled date . patients who returned to the medical clinic within three months after the scheduled date were allowed to continue in the study and be evaluated in an unscheduled visit . the tests performed during the placebo treatment and after 40 weeks of active treatments included the following : fasting glucose , urea , creatinine , total cholesterol , fractions of cholesterol , triglycerides , uric acid , total bilirubin , creatine phosphokinase ( cpk ) , sodium ( na+ ) , potassium ( k+ ) , hemoglobin , thyroid - stimulating hormone ( tsh ) , alkaline phosphatase , aspartate aminotransferase ( ast ) , alanine aminotranferase ( alt ) and urinary excretion of sodium in 24 h. data were analyzed using an analysis of variance ( anova ) according to the factor of controlled or uncontrolled hypertension . 44 were excluded for the following reasons : a ) lack of enrollment in the biosinttica assistance program ( n = 17 ) ; b ) body mass index > 40 kg / m ( n = 6 ) ; c ) secondary hypertension ( n = 4 ) ; d ) white - coat hypertension ( n = 3 ) ; e ) white - coat normotension ( masked hypertension ) ( n = 3 ) ; f ) alcohol use ( n = 2 ) ; g ) classification error ( n = 4 ) ; h ) refusal to do abpm ( n = 2 ) ; i ) loss of blood pressure measurements from the first visit ( n = 1 ) ; j ) pregnancy ( n = 1 ) ; k ) death ( n = 1 ) . a total of 354 patients were evaluated . there were no statistically significant differences in age , gender , skin color , body mass index , blood pressure or heart rate between the groups classified as : complicated ( n = 175 ) and uncomplicated ( n = 179 ) ; traditional ( a marked reduction in blood pressure was seen in patients from both the complicated and uncomplicated groups ( table 2 ) . the blood pressure was reduced by 1910/2014 mmhg in the uncomplicated group and by 1814/2217 mmhg ( systolic / diastolic ) in the complicated group ( p>0.05 ; n = 354 ) . groups showed significantly lower blood pressures at the end of treatment ( p < 0.00001 ) . traditional and current groups showed significantly lower blood pressures at the end of treatment ( figure 1 ) . the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) was also high at the end of treatment ( 74% ) . on the other hand , the percentage of patients with blood pressure reduced to < 120/80 mmhg was only 29% . there was no difference in the percentage of patients with controlled blood pressure between the phone calls and no phone calls groups or in the reduction of blood pressure and the percentage of patients with controlled blood pressure among the however , patients in the phone calls group ( 80% ) had better blood pressure control than those in the no phone calls group ( 71% ) , though the difference was not statistically significant . uncomplicated and complicated groups had statistically significant differences in the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) ( 80% vs. 67% , respectively ; p<0.000001 ) . at the next to last study visit ( visit 7 ) , 90% and 66% of patients had blood pressure measurements < 140/90 mmhg in the uncomplicated and complicated groups , respectively . however , there was no difference in the percentage of patients with blood pressure < 120/80 mmhg ( 31% on the final visit in both groups , table 3 ) . among patients with a blood pressure < 140/90 mmhg at the final visit , only 3% had received only one type of antihypertensive medication ; most patients ( 56% ) received 2 ( 34% ) or 3 ( 22% ) types of antihypertensive medication ( table 4 ) . a significantly lower number of patients in the group quit the treatment compared to the no phone calls group ( 4 vs. 30 , respectively ; figure 2 ) . there was no difference in the percentage discontinuation in the complicated and uncomplicated groups or in the current and traditional treatment regimens ( p>0.05 ) . patients in the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone calls did not show statistically significant differences in medication intake , as verified by counting the tablets returned at the visits . however , patients in the current + uncomplicated + phone call group had the highest rate of compliance ( 93% ) . the lowest rate of compliance occurred in the traditional + uncomplicated + no phone call group ( 85% ) ( p>0.05 ) . blood pressure reductions occurred in the presence of increased body weight ( baseline visit : 7314 kg ; final visit : 7414 kg , p = 0.0008 , table 5 ) . most of the adverse events that occurred during the study were of light or moderate intensity . patients in the traditional group showed significantly more depression symptoms ( 5 vs. 0 ) and coughing ( 30 vs. 4 ) when compared with patients in the current treatment group . on the other hand , patients in the current group had a greater number of complaints of dizziness when compared to patients in the traditional treatment group ( 63 vs. 42 , respectively ) . compared to patients in the uncomplicated group , patients in the complicated group presented a larger number the following symptoms : abdominal pain ( 19 vs. 8) , sleepiness ( 32 vs. 10 ) , cough ( 23 vs. 11 ) , weakness ( 23 vs. 11 ) and blurred vision ( 6 vs. 0 ) . conversely , compared to patients in the complicated group , patients in the uncomplicated group presented a larger number of complaints such as pain ( 74 vs. 53 ) and headache ( 80 vs. 59 ) . group reported a 34% rate of symptoms , while a 66% rate of symptoms was reported by patients in the no phone calls group . the most frequently ( > 10% ) found adverse events were as follows : headache ( 62.1% ) , unspecific pain ( 58.2% ) , dizziness ( 45.8% ) , edema ( 40.1% ) , fatigue ( 15.3% ) , sleepiness ( 14.4% ) , cough ( 13.3% ) , precordial pain ( 13.3% ) , weakness ( 11.9% ) , tachycardia ( 11.3% ) , insomnia ( 10.5% ) and paresthesia ( 10.5% ) . there were 26 ( 8% ) severe adverse events : death ( 5 ) , unstable angina ( 4 ) , acute myocardial infarction ( 1 ) , transient ischemic attack ( 1 ) , hypertensive encephalopathy ( 1 ) , breast cancer ( 3 ) , bronchospastic crisis ( 1 ) , cholecystopathy ( 1 ) , diabetic decompensation ( 1 ) , syncope ( 1 ) , nephrectomy ( 2 ) , trauma ( 2 ) , peritoneal bypass ( 1 ) , diarrhea ( 1 ) and gastrectomy ( 1 ) . the results of the laboratory tests performed in the beginning and end of the study are shown in table 6 . there was an increase in the 24-hour urinary sodium excretion from the beginning to the end of the study ( 12046 vs. 12945 a marked reduction in blood pressure was seen in patients from both the complicated and uncomplicated groups ( table 2 ) . the blood pressure was reduced by 1910/2014 mmhg in the uncomplicated group and by 1814/2217 mmhg ( systolic / diastolic ) in the complicated group ( p>0.05 ; n = 354 ) . groups showed significantly lower blood pressures at the end of treatment ( p < 0.00001 ) . traditional and current groups showed significantly lower blood pressures at the end of treatment ( figure 1 ) . the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) was also high at the end of treatment ( 74% ) . on the other hand , the percentage of patients with blood pressure reduced to < 120/80 mmhg was only 29% . there was no difference in the percentage of patients with controlled blood pressure between the phone calls and no phone calls groups or in the reduction of blood pressure and the percentage of patients with controlled blood pressure among the however , patients in the phone calls group ( 80% ) had better blood pressure control than those in the no phone calls group ( 71% ) , though the difference was not statistically significant . uncomplicated and complicated groups had statistically significant differences in the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) ( 80% vs. 67% , respectively ; p<0.000001 ) . at the next to last study visit ( visit 7 ) , 90% and 66% of patients had blood pressure measurements < 140/90 mmhg in the uncomplicated and complicated groups , respectively . however , there was no difference in the percentage of patients with blood pressure < 120/80 mmhg ( 31% on the final visit in both groups , table 3 ) . among patients with a blood pressure < 140/90 mmhg at the final visit , only 3% had received only one type of antihypertensive medication ; most patients ( 56% ) received 2 ( 34% ) or 3 ( 22% ) types of antihypertensive medication ( table 4 ) . a significantly lower number of patients in the phone calls group quit the treatment compared to the no phone calls group ( 4 vs. 30 , respectively ; figure 2 ) . there was no difference in the percentage discontinuation in the complicated and uncomplicated groups or in the current and traditional treatment regimens ( p>0.05 ) . patients in the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone calls did not show statistically significant differences in medication intake , as verified by counting the tablets returned at the visits . however , patients in the current + uncomplicated + phone call group had the highest rate of compliance ( 93% ) . the lowest rate of compliance occurred in the traditional + uncomplicated + no phone call group ( 85% ) ( p>0.05 ) . blood pressure reductions occurred in the presence of increased body weight ( baseline visit : 7314 kg ; final visit : 7414 kg , p = 0.0008 , table 5 ) . most of the adverse events that occurred during the study were of light or moderate intensity . patients in the traditional group showed significantly more depression symptoms ( 5 vs. 0 ) and coughing ( 30 vs. 4 ) when compared with patients in the current treatment group . on the other hand , patients in the current group had a greater number of complaints of dizziness when compared to patients in the traditional treatment group ( 63 vs. 42 , respectively ) . compared to patients in the uncomplicated group , patients in the complicated group presented a larger number the following symptoms : abdominal pain ( 19 vs. 8) , sleepiness ( 32 vs. 10 ) , cough ( 23 vs. 11 ) , weakness ( 23 vs. 11 ) and blurred vision ( 6 vs. 0 ) . conversely , compared to patients in the complicated group , patients in the uncomplicated group presented a larger number of complaints such as pain ( 74 vs. 53 ) and headache ( 80 vs. 59 ) . patients in the phone calls group reported a 34% rate of symptoms , while a 66% rate of symptoms was reported by patients in the no phone calls group . the most frequently ( > 10% ) found adverse events were as follows : headache ( 62.1% ) , unspecific pain ( 58.2% ) , dizziness ( 45.8% ) , edema ( 40.1% ) , fatigue ( 15.3% ) , sleepiness ( 14.4% ) , cough ( 13.3% ) , precordial pain ( 13.3% ) , weakness ( 11.9% ) , tachycardia ( 11.3% ) , insomnia ( 10.5% ) and paresthesia ( 10.5% ) . there were 26 ( 8% ) severe adverse events : death ( 5 ) , unstable angina ( 4 ) , acute myocardial infarction ( 1 ) , transient ischemic attack ( 1 ) , hypertensive encephalopathy ( 1 ) , breast cancer ( 3 ) , bronchospastic crisis ( 1 ) , cholecystopathy ( 1 ) , diabetic decompensation ( 1 ) , syncope ( 1 ) , nephrectomy ( 2 ) , trauma ( 2 ) , peritoneal bypass ( 1 ) , diarrhea ( 1 ) and gastrectomy ( 1 ) . the results of the laboratory tests performed in the beginning and end of the study are shown in table 6 . there was an increase in the 24-hour urinary sodium excretion from the beginning to the end of the study ( 12046 vs. 12945 adherence to a medication regimen is generally defined as the extent to which patients take medications as prescribed by their health care providers.20 this can be classified into three aspects of the individual s behavior regarding his / her health : 1 ) take the medication correctly , 2 ) follow the professionals instructions related to diets and lifestyle changes and 3 ) attend medical visits.21 the discontinuation of medication is considered the most serious type of noncompliance with treatment . in our study , the phone calls group showed a significantly lower percentage of patients who discontinued treatment compared with the no phone calls group . a meta - analysis of randomized studies22 suggested that treatment discontinuation is effectively avoided by contacting the patient using letters , telephone calls or e - mails . mrques contreras et al.6 evaluated the efficacy of telephone and e - mail intervention for therapeutic compliance among 538 patients with mild to moderate hypertension and verified that the group that received phone calls showed higher compliance rates ( 96.2% ) than the group that was only contacted by e - mail ( 91.3% ) or the group that did not receive any intervention ( 69.2% ) . similar data were found in a controlled study recently conducted by bosworth et al.7 in which the self - reported medication adherence was shown to increase by 9% in the group that had received behavioral and educational intervention ( 319 hypertensive subjects ) through telephone calls versus 1% in the group that had not received intervention ( n = 317 ) . friedman et al.23 , through telephone calls associated with usual medical care for six months , compared hypertensive patients who received their usual medical treatment with those who used a monitoring and counseling system . participants with compliance below 80% of the prescribed medication prior to enrollment in the study showed better compliance with the treatment when the telephone monitoring system was used than did patients who did not use the system ( 36% vs. 26% , respectively ) . on the other hand , participants with a compliance level above 80% prior to enrollment in the study did not show any change in compliance , and compliance was comparable in users and non - users of the telephone monitoring system . in our study , regardless of the group to which they were randomized , patients showed compliance rates above 85% at all visits . however , we do not have data regarding medication compliance before enrollment in the study . a study conducted by wu et al.9 randomized 442 non - compliant patients who took five or more medications for chronic illnesses to either receive or not receive telephone counseling . the objective was to investigate the influence of this advice for treatment compliance and patient mortality . the study verified that telephone counseling was associated with a 41% reduction of death risk after two years . these authors used scores to classify the levels of compliance of 1011 patients , and they showed that the lower the level of compliance , the higher the risk of mortality in the long term . the influence of the initial selection of antihypertensive medication in regards to compliance with treatment was evaluated by monane et al.24 the authors verified that the use of newer antihypertensive agents , such as angiotensin converting enzyme inhibitors and calcium channel antagonists , was associated with a compliance 80% when compared with thiazide diuretics in patients with cardiac morbidities and multiple visits to physicians . this may have been the result of better tolerability to newer classes of antihypertensive agents . however , in our study , there was no difference in tolerability between the traditional and current groups , which could explain the similar compliance rates in both groups . on the other hand , just like in monane et al.24 we found that fewer patients in the complicated group had their blood pressure under control in the final visit ( 67% ) , whereas in the next to last study visit , 90% of patients in the uncomplicated group and only 66% in the complicated group had blood pressure levels below 140/90 mmhg . this may be due to comorbidities and the use of other concurrent treatments . the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone callsshowed significant reductions in blood pressure from the randomization visit to the final visit , as a high percentage of patients with good blood pressure control ( bp < 140/90 mmhg ) in the total group ( 74% ) , regardless of an increase in body weight throughout the study . in our study , the high percentage patients with controlled blood pressure at the end of treatment can be explained by the simple drug regimen , the simple drug acquisition provided by the physician at no cost at the end of each visit , a fixed team of physicians and nurses throughout the study period and the easy access to team members in cases of unexpected occurrences by means of unscheduled visits . we observed that in the next - to - last study visit , the uncomplicated , current , traditional , phone calls and no phone calls groups had more patients with controlled blood pressure when compared to the last visit . it is possible that during the last visit the patients were worried about continuing the treatment and receiving the medications . therefore , guidance provided to patients by means of active telephone calls , brochures and group workshops with healthcare professionals is an efficient strategy for reducing treatment discontinuation , the most severe type of noncompliance with treatment . in an attempt to reduce non - adherence to antihypertensive treatment determinants in a developing country , we demonstrated that better blood pressure control was obtained through doctor s visits every two months with the same physician preceded by nursing staff visits with patients receiving all medication necessary for their treatment ; this benefit was independent of comorbidities and the type of treatment used . guidance provided to the patients through active telephone calls , brochures , group workshops with healthcare professionals and donation of all the required medications significantly reduced the treatment discontinuation rates . this study received support from biosintetica assistance and biosinttica laboratory , which donated all of the required medication . in an attempt to reduce non - adherence to antihypertensive treatment determinants in a developing country , we demonstrated that better blood pressure control was obtained through doctor s visits every two months with the same physician preceded by nursing staff visits with patients receiving all medication necessary for their treatment ; this benefit was independent of comorbidities and the type of treatment used . guidance provided to the patients through active telephone calls , brochures , group workshops with healthcare professionals and donation of all the required medications significantly reduced the treatment discontinuation rates . this study received support from biosintetica assistance and biosinttica laboratory , which donated all of the required medication .

objectives : to evaluate the importance of providing guidelines to patients via active telephone calls for blood pressure control and for preventing the discontinuation of treatment among hypertensive patients.introduction:many reasons exist for non - adherence to medical regimens , and one of the strategies employed to improve treatment compliance is the use of active telephone calls.methods:hypertensive patients ( n = 354 ) who could receive telephone calls to remind them of their medical appointments and receive instruction about hypertension were distributed into two groups : a ) uncomplicated hypertensive patients with no other concurrent diseases and b ) complicated - severe hypertensive patients ( mean diastolic 110 mmhg with or without medication ) or patients with comorbidities . all patients , except those excluded ( n = 44 ) , were open - block randomized to follow two treatment regimens ( traditional or current ) and to receive or not receive telephone calls ( phone calls and no phone calls groups , respectively).results : significantly fewer patients in the phone calls group discontinued treatment compared to those in the no phone calls group ( 4 vs. 30 ; p<0.0094 ) . there was no difference in the percentage of patients with controlled blood pressure in the phone calls group and no phone calls group or in the traditional and current groups . the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) was increased at the end of the treatment ( 74% ) , reaching 80% in the uncomplicated group and 67% in the complicated group ( p<0.000001).conclusion : guidance to patients via active telephone calls is an efficient strategy for preventing the discontinuation of antihypertensive treatment .

INTRODUCTION METHODS Blood Pressure Measurement Patient Randomization Statistical Analysis RESULTS a) Control of Blood Pressure b) Treatment Discontinuation c) Tablet Count d) Body Weight e) Adverse Events and Laboratory Tests DISCUSSION Perspectives Sources of Funding

most patients with hypertension do not benefit from treatment.1 data from nhanes / nchs 19992004 showed that 71.8% of individuals were aware of their condition , 61.4% were under current treatment and 35.1% had it under control.2 although the percentage of individuals with controlled blood pressure in brazil is unknown , we estimate it to be low because the percentage of patients with their high blood pressure under control is around 30 - 35% at outpatient clinics that specialize in hypertension , and these clinics do not represent the national reality.3 many reasons exist for non - adherence to medical regimens , including adverse drug effects , poor instructions , poor provider - patient relationship , poor memory , a patient s disagreement with the need for treatment or inability to afford medication.4,5 one of the strategies employed to improve treatment compliance is the use of active telephone calls6 to patients through which they are given guidance and treatment questions are answered . in view of these data , we evaluated the importance of providing guidelines to patients via active telephone calls for blood pressure management and preventing treatment discontinuation in hypertensive patients . the patients had essential hypertension and were able to receive telephone calls to be reminded of their medical appointments and be given guidance about hypertension . patients were excluded on several bases , including blood pressure < 140/90 mmhg without antihypertensive medication , secondary hypertension , white - coat hypertension with systolic pressures 140 mmhg and/or diastolic pressures 90 mmhg at the doctor s office and awake mean systolic pressures < 135 mmhg or awake mean diastolic pressures < 85 mmhg without antihypertensive medication , malignant hypertension and patients with a previous history of hypersensitivity reaction to the study medications . other exclusion criteria were as follows : pregnant women or nursing mothers , the presence of liver dysfunction evidenced by the patient s clinical history or by one of the liver function tests ( levels twice the normal values for alkaline phosphatase , total bilirubin , aspartate aminotransferase ) , patients with clinical conditions that might interfere with the total conformity with the study or those who might have an increased risk for participating in the study , patients with a history of alcoholism , drug abuse or mental disorders that might invalidate the free and informed consent or limit the patient s ability to meet the protocol rules and patients who had participated in any other studies involving investigational drugs or drugs already marketed within the previous month before enrollment in this study or concomitantly with this study . the diagnosis of hypertension was made when the mean values of the last two measurements were as follows : systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg with or without medication at the initial visit ( visit 0 ) . all of the patients underwent ambulatory blood pressure monitoring ( abpm ) , which was performed with a validated oscillometric device ( spacelabs 90207 , spacelabs inc , richmond , wa , usa),18,19 five weeks after the start of the placebo to eliminate cases of patients with white - coat hypertension and , in patients who did not receive placebo , to identify the white - coat effect . according to clinical characteristics , uncomplicated hypertensive patients without complications and without other concurrent diseases and b ) complicated- patients with severe hypertension ( mean diastolic pressure 110 mmhg with or without medication ) or comorbidities such as diabetes mellitus , renal failure ( serum creatinine > 1.4 mg / dl ) , coronary insufficiency , congestive heart failure or prior history of cerebrovascular accident . all patients , from both the complicated and the uncomplicated groups , were open - block randomized to receive active telephone calls ( phone calls group ) or not to receive telephone calls ( no phone calls group ) and to follow two treatment regimens , traditional or thus , after the first visit and randomization , patients from the phone calls group were invited to enroll by telephone in a program called biosinttica assistance , supported by biosinttica laboratory . at the initial stage of treatment ( following eight weeks of treatment with placebo ) , the uncomplicated group received one of the following treatment regimens : a ) traditional treatment with 6.25 mg 2x / day hydrochlorothiazide and 25 mg 2x / day atenolol ; b ) current treatment with 25 mg 2x / day losartan and 2.5 mg 2x / day amlodipine . complicated group did not undergo the treatment period with placebo and was randomized to receive either traditional or current drug regimens similar to the ones administered to the uncomplicated group , though the specifics of each condition were carefully considered . the addition of other antihypertensive agents in the uncomplicated group and the specificities of the patient regimens in the complicated group were performed according to the guidelines from the v brazilian guidelines on arterial hypertension.3 all patients were instructed to take their medication every day at 7:00 am and 7:00 pm , with a variation of up to one hour . the diagnosis of hypertension was made when the mean values of the last two measurements were as follows : systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg with or without medication at the initial visit ( visit 0 ) . all of the patients underwent ambulatory blood pressure monitoring ( abpm ) , which was performed with a validated oscillometric device ( spacelabs 90207 , spacelabs inc , richmond , wa , usa),18,19 five weeks after the start of the placebo to eliminate cases of patients with white - coat hypertension and , in patients who did not receive placebo , to identify the white - coat effect . according to clinical characteristics , complicated- patients with severe hypertension ( mean diastolic pressure 110 mmhg with or without medication ) or comorbidities such as diabetes mellitus , renal failure ( serum creatinine > 1.4 mg / dl ) , coronary insufficiency , congestive heart failure or prior history of cerebrovascular accident . all patients , from both the complicated and the uncomplicated groups , were open - block randomized to receive active telephone calls ( phone calls group ) or not to receive telephone calls ( no phone calls group ) and to follow two treatment regimens , traditional or thus , after the first visit and randomization , patients from the phone calls group were invited to enroll by telephone in a program called biosinttica assistance , supported by biosinttica laboratory . at the initial stage of treatment ( following eight weeks of treatment with placebo ) , the uncomplicated group received one of the following treatment regimens : a ) traditional treatment with 6.25 mg 2x / day hydrochlorothiazide and 25 mg 2x / day atenolol ; b ) current treatment with 25 mg 2x / day losartan and 2.5 mg 2x / day amlodipine . the complicated group did not undergo the treatment period with placebo and was randomized to receive either traditional or current drug regimens similar to the ones administered to the uncomplicated group , though the specifics of each condition were carefully considered . the addition of other antihypertensive agents in the uncomplicated group and the specificities of the patient regimens in the complicated group were performed according to the guidelines from the v brazilian guidelines on arterial hypertension.3 all patients were instructed to take their medication every day at 7:00 am and 7:00 pm , with a variation of up to one hour . 44 were excluded for the following reasons : a ) lack of enrollment in the biosinttica assistance program ( n = 17 ) ; b ) body mass index > 40 kg / m ( n = 6 ) ; c ) secondary hypertension ( n = 4 ) ; d ) white - coat hypertension ( n = 3 ) ; e ) white - coat normotension ( masked hypertension ) ( n = 3 ) ; f ) alcohol use ( n = 2 ) ; g ) classification error ( n = 4 ) ; h ) refusal to do abpm ( n = 2 ) ; i ) loss of blood pressure measurements from the first visit ( n = 1 ) ; j ) pregnancy ( n = 1 ) ; k ) death ( n = 1 ) . the blood pressure was reduced by 1910/2014 mmhg in the uncomplicated group and by 1814/2217 mmhg ( systolic / diastolic ) in the complicated group ( p>0.05 ; n = 354 ) . groups showed significantly lower blood pressures at the end of treatment ( p < 0.00001 ) . traditional and current groups showed significantly lower blood pressures at the end of treatment ( figure 1 ) . the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) was also high at the end of treatment ( 74% ) . on the other hand , the percentage of patients with blood pressure reduced to < 120/80 mmhg was only 29% . there was no difference in the percentage of patients with controlled blood pressure between the phone calls and no phone calls groups or in the reduction of blood pressure and the percentage of patients with controlled blood pressure among the however , patients in the phone calls group ( 80% ) had better blood pressure control than those in the no phone calls group ( 71% ) , though the difference was not statistically significant . uncomplicated and complicated groups had statistically significant differences in the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) ( 80% vs. 67% , respectively ; p<0.000001 ) . at the next to last study visit ( visit 7 ) , 90% and 66% of patients had blood pressure measurements < 140/90 mmhg in the uncomplicated and complicated groups , respectively . however , there was no difference in the percentage of patients with blood pressure < 120/80 mmhg ( 31% on the final visit in both groups , table 3 ) . among patients with a blood pressure < 140/90 mmhg at the final visit , only 3% had received only one type of antihypertensive medication ; most patients ( 56% ) received 2 ( 34% ) or 3 ( 22% ) types of antihypertensive medication ( table 4 ) . a significantly lower number of patients in the group quit the treatment compared to the no phone calls group ( 4 vs. 30 , respectively ; figure 2 ) . there was no difference in the percentage discontinuation in the complicated and uncomplicated groups or in the current and traditional treatment regimens ( p>0.05 ) . patients in the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone calls did not show statistically significant differences in medication intake , as verified by counting the tablets returned at the visits . on the other hand , patients in the current group had a greater number of complaints of dizziness when compared to patients in the traditional treatment group ( 63 vs. 42 , respectively ) . compared to patients in the uncomplicated group , patients in the complicated group presented a larger number the following symptoms : abdominal pain ( 19 vs. 8) , sleepiness ( 32 vs. 10 ) , cough ( 23 vs. 11 ) , weakness ( 23 vs. 11 ) and blurred vision ( 6 vs. 0 ) . conversely , compared to patients in the complicated group , patients in the uncomplicated group presented a larger number of complaints such as pain ( 74 vs. 53 ) and headache ( 80 vs. 59 ) . group reported a 34% rate of symptoms , while a 66% rate of symptoms was reported by patients in the no phone calls group . there was an increase in the 24-hour urinary sodium excretion from the beginning to the end of the study ( 12046 vs. 12945 a marked reduction in blood pressure was seen in patients from both the complicated and uncomplicated groups ( table 2 ) . the blood pressure was reduced by 1910/2014 mmhg in the uncomplicated group and by 1814/2217 mmhg ( systolic / diastolic ) in the complicated group ( p>0.05 ; n = 354 ) . groups showed significantly lower blood pressures at the end of treatment ( p < 0.00001 ) . traditional and current groups showed significantly lower blood pressures at the end of treatment ( figure 1 ) . the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) was also high at the end of treatment ( 74% ) . there was no difference in the percentage of patients with controlled blood pressure between the phone calls and no phone calls groups or in the reduction of blood pressure and the percentage of patients with controlled blood pressure among the however , patients in the phone calls group ( 80% ) had better blood pressure control than those in the no phone calls group ( 71% ) , though the difference was not statistically significant . uncomplicated and complicated groups had statistically significant differences in the percentage of patients with controlled blood pressure ( < 140/90 mmhg ) ( 80% vs. 67% , respectively ; p<0.000001 ) . at the next to last study visit ( visit 7 ) , 90% and 66% of patients had blood pressure measurements < 140/90 mmhg in the uncomplicated and complicated groups , respectively . however , there was no difference in the percentage of patients with blood pressure < 120/80 mmhg ( 31% on the final visit in both groups , table 3 ) . among patients with a blood pressure < 140/90 mmhg at the final visit , only 3% had received only one type of antihypertensive medication ; most patients ( 56% ) received 2 ( 34% ) or 3 ( 22% ) types of antihypertensive medication ( table 4 ) . a significantly lower number of patients in the phone calls group quit the treatment compared to the no phone calls group ( 4 vs. 30 , respectively ; figure 2 ) . there was no difference in the percentage discontinuation in the complicated and uncomplicated groups or in the current and traditional treatment regimens ( p>0.05 ) . patients in the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone calls did not show statistically significant differences in medication intake , as verified by counting the tablets returned at the visits . on the other hand , patients in the current group had a greater number of complaints of dizziness when compared to patients in the traditional treatment group ( 63 vs. 42 , respectively ) . compared to patients in the uncomplicated group , patients in the complicated group presented a larger number the following symptoms : abdominal pain ( 19 vs. 8) , sleepiness ( 32 vs. 10 ) , cough ( 23 vs. 11 ) , weakness ( 23 vs. 11 ) and blurred vision ( 6 vs. 0 ) . conversely , compared to patients in the complicated group , patients in the uncomplicated group presented a larger number of complaints such as pain ( 74 vs. 53 ) and headache ( 80 vs. 59 ) . patients in the phone calls group reported a 34% rate of symptoms , while a 66% rate of symptoms was reported by patients in the no phone calls group . there was an increase in the 24-hour urinary sodium excretion from the beginning to the end of the study ( 12046 vs. 12945 adherence to a medication regimen is generally defined as the extent to which patients take medications as prescribed by their health care providers.20 this can be classified into three aspects of the individual s behavior regarding his / her health : 1 ) take the medication correctly , 2 ) follow the professionals instructions related to diets and lifestyle changes and 3 ) attend medical visits.21 the discontinuation of medication is considered the most serious type of noncompliance with treatment . in our study , the phone calls group showed a significantly lower percentage of patients who discontinued treatment compared with the no phone calls group . a study conducted by wu et al.9 randomized 442 non - compliant patients who took five or more medications for chronic illnesses to either receive or not receive telephone counseling . the influence of the initial selection of antihypertensive medication in regards to compliance with treatment was evaluated by monane et al.24 the authors verified that the use of newer antihypertensive agents , such as angiotensin converting enzyme inhibitors and calcium channel antagonists , was associated with a compliance 80% when compared with thiazide diuretics in patients with cardiac morbidities and multiple visits to physicians . however , in our study , there was no difference in tolerability between the traditional and current groups , which could explain the similar compliance rates in both groups . on the other hand , just like in monane et al.24 we found that fewer patients in the complicated group had their blood pressure under control in the final visit ( 67% ) , whereas in the next to last study visit , 90% of patients in the uncomplicated group and only 66% in the complicated group had blood pressure levels below 140/90 mmhg . the groups : complicated and uncomplicated ; traditional and current ; phone calls and no phone callsshowed significant reductions in blood pressure from the randomization visit to the final visit , as a high percentage of patients with good blood pressure control ( bp < 140/90 mmhg ) in the total group ( 74% ) , regardless of an increase in body weight throughout the study . in our study , the high percentage patients with controlled blood pressure at the end of treatment can be explained by the simple drug regimen , the simple drug acquisition provided by the physician at no cost at the end of each visit , a fixed team of physicians and nurses throughout the study period and the easy access to team members in cases of unexpected occurrences by means of unscheduled visits . we observed that in the next - to - last study visit , the uncomplicated , current , traditional , phone calls and no phone calls groups had more patients with controlled blood pressure when compared to the last visit . therefore , guidance provided to patients by means of active telephone calls , brochures and group workshops with healthcare professionals is an efficient strategy for reducing treatment discontinuation , the most severe type of noncompliance with treatment . in an attempt to reduce non - adherence to antihypertensive treatment determinants in a developing country , we demonstrated that better blood pressure control was obtained through doctor s visits every two months with the same physician preceded by nursing staff visits with patients receiving all medication necessary for their treatment ; this benefit was independent of comorbidities and the type of treatment used . in an attempt to reduce non - adherence to antihypertensive treatment determinants in a developing country , we demonstrated that better blood pressure control was obtained through doctor s visits every two months with the same physician preceded by nursing staff visits with patients receiving all medication necessary for their treatment ; this benefit was independent of comorbidities and the type of treatment used .

flavo - diiron proteins ( fdps ) are widespread in microaerophilic and anaerobic microorganisms , including bacteria , archaea , and a few protozoan parasites . the accumulated genetic and biochemical evidence indicates that fdps are the terminal components of a reductive dioxygen ( o2 ) and/or anaerobic nitric oxide ( no ) scavenging pathways . the fdp - catalyzed four - electron reduction of o2 to water ( o2r activity ) or the two - electron reduction of two no molecules to nitrous oxide ( nor activity ) protects against accumulation of toxic reactive oxygen or nitrogen species . fdps are soluble cytoplasmic enzymes with a head - to - tail each active site consists of a nonheme diiron cluster ( fe1fe2 distance 3.23.6 ) , and a flavin mononucleotide ( fmn ) cofactor located 4 from the diiron site across the subunit interface , as diagrammed in scheme 1 . in almost all structurally characterized fdps , each iron of the diiron site contains two histidine ligands and a terminal monodentate carboxylate ligand from either aspartate or glutamate . a bridging bidentate carboxylate from an aspartate residue and a protonated solvent ligand complete the diiron coordination sphere , resulting in two five - coordinate irons . one fdp structure contains a terminal solvent in place of a his ligand , which is rotated away from fe2 . site - directed mutations show that this substitution has surprisingly minor effects on structure and activities . open or solvent - occupied sixth coordination sites trans to his ligands on each iron are oriented toward a buried pocket , which in some structures contains solvent or other exogenous small molecules . the relatively nonpolar o2 or no diffuse into this pocket and presumably bind to these labile coordination sites during o2r or nor turnover . under aerobic conditions , fefe oxidation state ( fdpox ) , but the diferrous oxidation state is necessary for substrate reduction . at least one function of the fmn is to funnel reducing equivalents from exogenous electron donors to the diiron site . the fully reduced active site , fmnh2fefe ( fdpred ) thus has the capacity to deliver up to four electrons to either one o2 or four nos during o2r or nor turnover . here , we address the nor mechanism of fdps , proposals for which are shown in scheme 2 . all three proposed mechanisms , labeled difeno , sr , and hyp , initiate from a diferrous - mononitrosyl , fe{feno } , which forms as a stable species upon addition of less than 1 equiv of no per diferrous site . difeno and sr both propose that binding of a second no ( step 1difeno , sr ) leads to a diferrous - dinitrosyl ( [ { feno}]2 ) intermediate . steps 2difeno and 2sr represent alternative rate - limiting reactions of the diferrous - dinitrosyl . in step 2difeno , n bond , are protonated , and lose the elements of water , thereby generating n2o and a diferric site . in the alternative rate - limiting step , 2sr , fmnh2 super - reduces the [ { feno}]2 to a highly reactive diferrous - dinitroxyl ( [ { fe(h)no}]2 ) , which decays to diferrous ( fefe ) with release of n2o . the hyp mechanism proposes that the second no reacts directly with the { feno } center of fe{feno } ( step 1hyp ) , leading to a formally diferric - o , n - semibridging hyponitrite intermediate ( fe(n2o2)fe ) , which decays in the rate - limiting step to diferric with release of n2o . of the three proposed mechanisms , only sr requires redox participation of the fmn cofactor prior to or at the rate - limiting step leading to n2o . in order to separate the functions of the fmn and the diiron site , the reaction of no with the diferrous deflavinated ( deflavo ) fdp from thermotoga maritima ( tm ) was examined . the results were consistent with reaction of the fe{feno } with a second no leading to parallel productive ( i.e. , n2o generating ) and unproductive pathways . these results suggested that at least a portion of the diiron sites are capable of catalyzing n2o formation in the absence of fmn , which is consistent with either mechanisms difeno or hyp but not with mechanism sr in scheme 2 . however , more recent studies on a synthetic diferrous - dinitrosyl complex seem to support the feasibility of super - reduction in mechanism sr . the observation of an unusually low n o stretching frequency in the fe{feno } complex of tm fdp led to the proposal that the coordinated no is activated for reaction with a second no , which would seem to support mechanism hyp . in order to clarify these mechanistic ambiguities , we have undertaken rapid kinetics studies to trap and characterize intermediates in the fdp nor reaction . in addition , we have investigated the ultimate oxidation state of the tm fdpred active site upon reaction with excess no . ligands typically show an s = /2 ground spin state exhibiting axial g 4.0 , 2.0 electron paramagnetic resonance ( epr ) spectra and characteristic ligand - to - metal charge transfer uv visible ( uv vis ) absorptions at 650 , 450 , and 330 nm with 450 nm 1000 m cm . the diferrous mononitrosyl , fe{feno } , in fdp shows this characteristic uv vis absorption but exhibits an s = /2 epr spectrum ( g 2.3 , 1.9 ) due to antiferromagnetic coupling between the high spin fe ( s = 2 ) and the s = /2 { feno}. numerous studies of nonheme iron synthetic complexes and proteins have established a unique and characteristic combination of fe mssbauer isomer shift and quadrupole splitting values that unambiguously identify s = /2 { feno } species with n / o - ligand coordination spheres.fe mssbauer spectroscopy can furthermore distinguish between magnetically coupled versus isolated { feno } centers but has not previously been applied to the nitric oxide reactions of fdps . the three flavin redox states , fmnox , fmnh ( semiquinone ) , and fmnh2 , have distinctive and characteristic visible absorption spectra . we have used all of these spectroscopic methods to identify intermediates in reactions of no with tm fdpred , including the first mssbauer spectral characterizations of diiron redox intermediates during catalysis in this class of proteins . our results clarify the roles of the fmn and the diiron site in nor catalysis and can be used to discriminate among the mechanistic possibilities in scheme 2 . all reagents , protein , and media solutions were prepared using water purified with a millipore ultrapurification system to a resistivity of 18 m to minimize trace metal ion contamination . unless otherwise noted , the buffer used for all experiments was 50 mm 3-(n - morpholino)-propanesulfonic acid ( mops ) ph 7.3 , which is hereafter referred to as buffer . protein iron and fmn contents were determined using a standard ferrozine assay and a 461 nm value of 12 000 m cm , respectively , the latter of which was also used to determine protein active - site concentrations . the expression , purification , and flavin enrichment of recombinant tm fdp was performed as previously described . tm nadh / rubredoxin oxidoreductase ( nror),desulfovibrio vulgaris rubredoxin ( rd ) and d. vulgaris fdp were obtained as previously described . for fe enrichment of tm fdp , a 35 ml culture of the tm fdp expression strain was grown in luria a 5 ml portion of this overnight culture was used to inoculate 1 l of m9 medium supplemented with 2 g of n - z amine ( quality biological , inc . ) , 0.05% glucose , and 0.5% glycerol . the culture was incubated with shaking at 37 c for 34 h to an od600 nm of 0.61.0 . protein expression was then induced by addition of 200 mg / l arabinose , followed by 1 mg of fe ( cambridge isotope laboratories , inc . ) , which had been dissolved in 100 l of aqua regia . the induced , fe - treated culture was allowed to grow overnight with shaking at room temperature . isolation , purification , and flavin enrichment of the fe - enriched protein followed the procedures described for the natural isotope abundance tm fdp . tm fdpox solutions ( 602000 m ) in buffer were placed in septum - capped vials and deoxygenated by vacuum / n2 gas purge cycles on a schlenk line before transfer to an anaerobic n2 gas - filled glovebox ( omnilab system , vacuum atmosphere inc . ) . tm fdpox was reduced by addition of 2.2 mol equiv of nadh per fdp active site in the presence of 500 nm nror and 5 m rd . under these conditions , the fmn in tm fdp was immediately reduced to the semiquinone state , as verified by uv vis absorption ( see the results and discussion section ) . overnight reduction under these condition resulted in the four - electron reduced yellow - colored tm fdpred . for the rfq mssbauer samples , the 1 mm tm fdpred solutions were centrifuged at 14 000 g for 10 min to remove a precipitate . the cleared supernatant solutions were then loaded into the rfq apparatus , as described below . purified nitric oxide gas was prepared by bubbling no gas ( praxair , inc . ) through 50 ml of 10 m koh in a septum - sealed 250 ml erlenmeyer flask for 1015 min . twenty milliliter headspace vials ( kimble chase , llc ) were equilibrated in the glovebox overnight , after which the vials were septum - sealed . immediately prior to use , a septum - sealed headspace vial was evacuated using a 50 ml syringe , and approximately 20 ml of no gas from the headspace of the erlenmeyer flask was transferred via syringe into the evacuated vial . for 1.8 mm no stock solutions , 15 ml of anaerobic buffer at room temperature was injected into the no gas - filled headspace vial . the analogous procedure was used to prepare 3 mm no stock solutions , except that the no - filled headspace vial was cooled to 4 c , and 15 ml of buffer chilled to 4 c was injected into the chilled vial . the concentrations of no in the stock solutions were quantified by adding a 25 l aliquot to a buffered solution containing 100 m nadh , 5 m rd , 500 nm nror , and 2 m d. vulgaris fdp . ( the d. vulgaris fdp was used because it consumes no much more rapidly than does tm fdp . ) the concentration of nadh consumed upon the addition of no was determined from a340 nm ( 340 nm = 6200 m cm ) using an ocean optics usb 2000 spectrophotometer . the no concentrations of the stock solutions were calculated from the nadh consumed and the 2no / nadh stoichiometry . stock solutions of disodium 1-[(2-carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate ( proli - nonoate ) ( cayman chemical company ) were prepared by dissolving 10 mg of proli - nonoate in 0.51 ml of deoxygenated 10 mm sodium hydroxide . these stock solutions were stored in the anaerobic glovebox for no longer than 24 h prior to use . over this time period , the proli - nonoate is stable in the hydroxide stock solutions and can be quantified using a 252 nm value of 8400 m cm . at ph 7.4 , proli - nonoate releases 2 mol of no per 1 mol of nonoate with a half - time of 6 s at 22 c . in our hands the no - delivery equivalents in the nonoate stock solutions were therefore quantified by reaction of the released no with feedta . a 50 mm stock of feedta was prepared by mixing a 1:1 ratio ( v / v ) of 1 m edta , ph 7 , with 100 mm fe(nh4)2(so4)26 h2o . aliquots of 520 l of the proli - nonoate stock solution were diluted into 1 ml of buffer , and after 3060 s , a 210-fold molar excess of feedta was added . ( it was found empirically that addition of the stock nonoate directly to feedta solutions apparently inhibited the nonoate decay . ) the concentration of the resulting feedta no complex was determined using a 440 nm value of 900 m cm . all manipulations were carried out in the anaerobic glovebox . a 3200 l volume of 500 m tm fdpred was prepared as described above . a 400 l portion of this tm fdpred solution was pipetted into a mssbauer cup and frozen in a cold well at 77 k. the remaining tm fdpred solution was used for the no titration experiments . for the titration experiments , two stock proli - nonoate solutions were prepared and quantified as described above to contain either 8 or 40 mm no delivery equivalents . to prepare mssbauer samples containing 0.3 , 0.6 , and 0.9 equiv of no , the appropriate volumes ( 7.522.5 l ) of the proli - nonoate stock solution containing 8 mm no delivery equivalents were added to 400 l samples of 500 m fe - enriched tm fdpred . the samples were incubated at room temperature for 5 min , transferred to mssbauer cups , and frozen in a liquid n2-cooled cold well . the mssbauer cups containing the frozen samples were removed from the glovebox and stored at 77 k. the same procedure was followed for titrations with higher no concentrations except that the proli - nonoate stock solution containing 40 mm no delivery equivalents was used . stopped - flow uv vis absorption spectrophotometry was performed using an sx20 stopped - flow spectrometer ( applied photophysics , ltd . ) . the optical path length through the sample cell was either 1 cm or , for more concentrated protein solutions , 0.2 cm . all anaerobic solutions were prepared in the glovebox and pulled into a 5 ml luer - slip syringe and needle ( fisher scientific ) that had been equilibrated in the glovebox overnight . these loaded syringes were then transferred from the glovebox , and the solutions were injected through the loading port into the drive syringe using the protocol described in the sx20 instruction manual . the stopped - flow circuit was made anaerobic by filling the drive syringes and the circuit with an anaerobic solution of 700 m nadh , 500 nm nror , 5 m rd , and 2 m d. vulgaris fdp . the circuit was incubated with this o2-scaveging mixture for at least 1 h and then flushed with deoxygenated water prior to loading the anaerobic reactant solutions . 2.5 ml drive syringes ( hamilton company , usa ) were used for 1:1 ( v / v ) mixing and a 2.5 ml/250 l drive syringe combination was used for 10:1 ( v / v ) mixing . the tm fpdred solutions were always loaded into a 2.5 ml drive syringe , and 1.8 mm no solutions were loaded into the other drive syringe . the solutions in both drive syringes and the optical cell were cooled to 23 c prior to stopped - flow mixing . reactions with substoichiometric no used 1:1 ( v / v ) mixing of a 2 mm anaerobic solution of tm fdpred with a 1.8 mm no solution . reactions with excess no used 1:1 ( v / v ) mixing of 130160 m tm fdpred with a 1.8 mm no solution . spectra of the starting tm fdpred under these conditions were obtained by stopped - flow mixing with anaerobic buffer in place of the no solutions . the no concentrations immediately after stopped - flow mixing were quantified using the same drive syringe - loaded no solution but stopped - flow mixed with 15 mm feedta in place of the protein . the feedta was found to react with no to form feedta no quantitatively within the mixing dead time and to remain stable for at least several minutes . the feedta no was quantified using a 440 nm value of 900 m cm with 440 nm monochromatic ( 2 nm bandwidth ) and photomultiplier tube detector in order to avoid no photodissociation . the concentrations of fdp and no immediately after mixing are listed in the figure captions . a kintek rfq-3 rapid freeze - quench ( rfq ) instrument ( kintek corp . ) was used . the mixing circuit was made anaerobic , and solutions were loaded into the drive syringes as described above for the stopped - flow experiments . two 5 ml drive syringes were used for 1:1 ( v / v ) rapid mixing , and a 5 ml/500 l drive syringe combination was used for 10:1 ( v / v ) mixing . epr samples of tm fdpred reactions with substoichiometric no were prepared by rapid mixing 10:1 ( v / v ) of an anaerobic 200300 m solution of tm fdpred with a 1.8 mm no solution ( prepared at room temperature as described above ) . epr samples of tm fdpred reactions with excess no were prepared similarly , except mixing 1:1 ( v / v ) with 3 mm no solution in buffer at 4 c . epr samples of unreacted tm fdpred were prepared analogously to those for the no reactions but substituting deoxygenated buffer in place of the no solutions . after the desired aging times , 350 l of the reaction mixture was sprayed into liquid isopentane at 150 to 160 c . the resulting snow was collected in a glass funnel immersed in the cold isopentane and packed into the bottom of a 4 mm o.d . the no concentrations delivered to the protein samples were quantified by rapid - mixing the same drive syringe - loaded solution of no with 15 mm feedta in place of the protein and collecting a 350 l , 1 s sprayed and frozen sample as described for the protein samples . the s = /2 epr signal of the resulting feedta no was quantified by comparison to that of an feedta no sample prepared under 1 atm of no and quantified as described above . the packing factor was determined as the ratio of the peak - to - peak intensity of the s = /2 epr signal of a separately sprayed and packed d. vulgaris ferric rd solution of known concentration ( determined using 490 nm = 8800 m cm ) to that of the signal from the same rd sample in which the snow had been thawed and refrozen in liquid n2 . the packing factor was determined to be 0.7 , and the rfq epr spin concentration was corrected by this packing factor . rfq mssbauer samples were prepared using the drive - syringe loading and 1:1 ( v / v ) mixing conditions described above except 1 mm tm fdpred solutions at room temperature were rapid - mixed with either 3 mm no solutions in buffer at 4 c or , for unreacted samples , deoxygenated buffer at 4 c . ( the use of proli - nonoate to achieve higher no concentrations proved to be impractical for our rfq apparatus . ) immediately before loading the cold no solution , its drive syringe and load line were cooled by flushing three times with 4 c deoxygenated buffer . after the desired aging times , 350 l of the reaction mixture was sprayed into a 50 ml falcon tube ( fisher scientific ) containing 40 ml of liquid ethane at 160 to 170 c . ethane was removed from the frozen sample by decanting the liquid followed by evacuation of the headspace with the tube immersed in a dry ice / methanol bath . do not expose liquid ethane to an open flame and perform all manipulations in a well - ventilated area . the resulting snow was transferred and packed with a liquid - n2 cooled spatula to a liquid - n2 cooled rfq mssbauer cup . all of the reported quench times are a sum of the aging time ( minimum 5 ms ) plus an estimated 15 ms freezing time . all rfq epr and mssbauer samples were kept at liquid - n2 temperature in a storage dewar until spectral data collection . epr spectra were collected on a bruker esp 300e x - band spectrometer equipped with a helium continuous flow cryostat model esr 900 or 910 ( oxford instruments , inc . ) . standard parameters used for spectral collection were as follows : modulation frequency , 100 khz ; modulation amplitude , 10.5 g ; conversion time , 81.9 ms ; time constant , 5.1 ms . epr signals were quantified by relative to a 1 mm cuedta standard in 10% glycerol . isomer shifts are reported relative to fe metal at 298 k. the simulations of epr and mssbauer spectra were calculated with least - squares fitting using the program spincount and the standard spin hamiltonian : for the simulations of two interacting metal systems , the spin hamiltonian is expanded to include terms for each site , , , the exchange interaction , , and the standard dipole dipole interaction . throughout this narrative , unless otherwise noted , fdp refers specifically to tm fdp . for reactions with no , fdpox ( fmnox fefe ) was reduced anaerobically with a small molar excess of nadh and catalytic concentrations of the native redox partner proteins , nror and rd . fmnox , the anionic fmn semiquinone ( fmnsq ) , and the fully reduced fmn ( fmnh2 ) are all redox accessible in fdps . these three fmn species dominate the uv vis absorption spectra , as shown in figure 1a . the fefe / fefe and fefe / fefe couples in fdps have higher reduction potentials than those of both the fmnox / fmnsq and fmnsq / fmnh2 redox couples . thus , anaerobic reduction of fdpox with 2.2 mol equiv of nadh per diiron site in the presence of catalytic nror / rd resulted in formation of the fdpsq ( fmnsq fefe ) within the time of manual mixing followed by a slower reduction to fdpred ( fmnred fefe ) . however , as shown in figure 1b , when rd is equimolar with fdp , rather than a catalytic concentration , reduction of fdpox to fdpred occurred within the time of manual mixing with no detectable accumulation of fdpsq . this differing reducing behavior of catalytic versus stoichiometric rd is analogous to that observed for m. thermoacetica fdp and its physiological redox partner protein . the catalytic concentrations used here avoided spectroscopic interference by the redox partner proteins . uv vis absorption spectra of ( a ) before ( fdpox ) , < 1 min after ( fdpsq ) , and several hours after ( fdpred ) addition of 140 m nadh to a solution containing 5 m rd , 500 nm nror , and 60 m fdpox and ( b ) before ( fdpox ) and immediately after ( fdpred ) addition of 140 m nadh to a solution of 50 m rd , 500 nm nror , and 50 m fdpox . the mssbauer spectra in figure 2 confirm the diiron redox states in fdpox and fdpred . the 4.2 k spectrum of fdpox ( figure 2b ) showed a single sharp quadrupole doublet with parameters listed in table 1 . the sharp doublet , as opposed to a broad paramagnetic spectrum , is attributed to antiferromagnetic exchange coupling of the two s = /2 fe ions at the active site to give a s = 0 state lowest in energy . all of the iron in the fdpox converted into a single new quadrupole doublet with parameters given in table 1 . the parameters of the doublet are indicative of high - spin ferrous iron . the mssbauer parameters of both fdpox and fdpred are similar to those of the diiron sites of hemerythrin and ribonucleotide reductase for the corresponding oxidation states . the fdpox mssbauer spectrum is similar to that previously reported for the as - isolated fdp from m. thermoacetica . the single sharp doublets in the spectra of both fdpox and fdpred ( = 0.31 mm / s ) are indicative of equivalent electronic environments for the individual iron centers in the diiron site , which is consistent with the structure showing that each iron has the same type and number of n- and o - donor ligands ( scheme 1 ) . mssbauer spectra ( black bars ) of ( a ) fdpred and ( b ) fdpox recorded at 4.2 k in a magnetic field of 45 mt applied parallel to the -ray direction . the stopped - flow uv vis absorption spectral time course for the reaction of fdpred with substoichiometric no is shown in figure 3 . the spectral absorption changes are essentially complete within 100 ms after mixing ( figure 3a ) . vis absorption difference spectra were obtained by subtracting the fdpred spectrum from the spectra following mixing with no . the 100 ms difference spectrum ( figure 3a , inset ) has absorption features at 418 ( 418 nm = 1500 m cm per no ) , 456 ( 456 nm = 1450 m cm ) and 633 nm ( 633 nm = 400 m cm ) . the extinction coefficients on the basis of the substoichiometric no concentration are consistent with complete formation of one { feno } per diiron site within the mixing dead time . this absorption difference spectrum is very similar to the absolute spectrum obtained when less than or equal to 1 equiv of no was manually mixed with the deflavinated diferrous fdp . only very minor absorption increases centered at 390 and 500 nm occurred between 47 ms and 100 s ( figure 3b ) . difference spectra ( figure s1 , supporting information ) indicate that these slower absorption increases are due to accumulation of a very minor amount of anionic fmnsq . no spectral features of fmnox developed on the 100 s time scale with substoichiometric no . ( a ) 100 ms and ( b ) 100 s stopped - flow uv vis absorption spectral time courses for reactions of fdpred with 0.8 equiv of no per diiron site at 3 c . the spectra indicated as fdpred were obtained upon stopped - flow mixing fdpred solution with deoxygenated buffer in place of the no solution . inset in panel a shows the difference absorption spectrum obtained by subtracting the fdpred spectrum from the 100 ms spectrum . concentrations after mixing : 880 m fdpred and 680 m no . the difference extinction axis in the inset is per molar of added no . an rfq epr sample of fdpred obtained 200 ms after mixing with substoichiometric no is shown in figure 4 . a minor s = /2 signal ( not shown ) accounted for less than 2% of the diiron sites . the epr spectrum was dominated by an s = /2 signal ( g = 2.39 , 1.97 , 1.70 ) that was essentially identical to that previously assigned to fe{feno } resulting from manual mixing of fdpred with less than or equal to 1 equiv of no . the sharp signal at g = 2 was attributed to a small amount of fmnsq , and the signal at g = 1.97 was attributed to a small amount of free no . the g = 2.10 signal showed variable intensities relative to the fe{feno } signal among several samples and has also been reported previously in manually mixed samples of the same reaction . this signal was attributed to a magnetic dipolar interaction between a small portion of s = /2 fe{feno } ( using the g values listed above ) and s = /2 fmnsq ( g = 2.0 ) at a distance of 4.5 ( a dipolar term was added to the standard spin hamiltonian ) . the simulated concentration of this interacting system was never more than 10% of that of the noninteracting fe{feno } component . the s = /2 concentration in the rfq sample of figure 4 is in reasonable agreement with the added no concentration ( 110 m s = /2 vs 130 m added no ) . vis absorption spectral time course in figure 3 and the rfq epr spectrum in figure 4 shows that reaction of fdpred with substoichiometric no results in rapid and nearly quantitative formation of fe{feno } , which is stable for at least several minutes . since stopped - flow time courses with substoichiometric no showed no significant fmnh2 oxidation , the substoichiometric no - reacted fdp species can be confidently formulated as fmnh2fe{feno}. rfq epr spectra of fdpred ( blue spectrum ) and 200 ms after mixing fdpred with 0.5 equiv of no at 4 c ( green spectrum ) . the black trace overlaying the green spectrum is the sum of simulated fe{feno } and simulated dipole - coupled between a minor portion ( < 3% ) of fe{feno } and fmnsq at a distance of 4.5 . a radical species ( < 1% ) at g = 2.0 attributed to fmnsq has been clipped in both experimental spectra . conditions immediately after mixing : 250 m fdpred and 130 m no in 50 mm mops , ph 7.3 . instrumental parameters : microwaves , 9.645 ghz , 0.26 mw ; temperature , 4 k. mssbauer spectra were obtained for fdpred manually titrated with nonoate stock solutions . to ensure complete reaction , the nonoate - titrated samples were incubated anaerobically for 5 min at room temperature before freezing . the mssbauer spectra for these samples were recorded at temperatures of 80 k ( figure 5a , no magnetic field ) and 4 k ( figure 5b , magnetic field of 45 mt ) . table 1 lists the parameters for each species , and table 2 lists the corresponding percent compositions of iron species . prior to addition of no , the spectra at both temperatures showed a single doublet from the diferrous state of fdpred . at 80 k , titration with no up to 1.5 equiv showed that the area of the doublet from fdpred diminished concomitant with the development of two new doublets with equal areas ( = 1.12 mm / s , eq = 1.98 mm / s ; = 0.67 mm / s , eq = 1.53 mm / s ) . the new doublets are assigned to the fe and { feno } centers , respectively , of the fe{feno } complex . the values of = 0.67 mm / s and eq = 1.53 mm / s unambiguously identify an [ feno ] complex . the relative contributions of each doublet are shown in figure s2 ( supporting information ) for a selection of three spectra from figure 5a . the sums of all spectral species at the amounts listed in table 2 are the theoretical lines overlaid on the spectra . at 4.2 k ( figure 5b ) , the fe{feno } complex appeared as a broad paramagnetic pattern as expected for a s = /2 species . this broad spectrum is dependent on the magnetic properties of both iron ions and is not straightforward to simulate . on the basis of the high - temperature spectra , this complex was 90% of the iron in the 1.5 equiv sample . a minor amount ( 5% ) of the fdpred doublet spectrum and the doublets of the other species at the same percentages as that of the high - temperature data produced the fits shown in figure 5b . the isomer shifts , quadrupole splittings , and paramagnetic broadening of the s = /2 species are reminiscent of that for the s = /2 fe{feno } complex of hemerythrin . from 1.5 to 2.5 equiv of no , the proportions of diferrous ( 8% ) and fe{feno } ( 87% ) remained relatively stable . the addition of 5 equiv of no resulted in complete conversion to an antiferromagnetically coupled diferric spectrum that is indistinguishable from that of fdpox . fe mssbauer spectra ( vertical bars ) of 500 m fdpred titrated with no . spectra were recorded at ( a ) 80 k in zero field or ( b ) 4 k in a magnetic field of 45 mt applied parallel to the -ray direction . the blue traces overlaying the experimental spectra are the resultant simulation sums of the components using the species of table 1 in the amounts of table 2 . epr samples of fdpred plus 0.3 , 0.6 , and 0.9 equiv of no were prepared at the same time and from the same protein material as the mssbauer samples of figure 5 . the epr spectra of these three samples ( figure s3 , supporting information ) showed that the amount of the s = /2 fe{feno } complex quantitatively agreed with the proportion of the paramagnetically broadened component in the mssbauer spectra of figure 5 , indicating that this broadened feature in the mssbauer spectra originated from the s = /2 fe{feno } complex . these spectral titrations confirm that the fefe site in fdpred reacts with substoichiometric no to form fe{feno}. the mssbauer data also show that the fefe site can be oxidized quantitatively to the diferric ( fefe ) state by no only slightly in excess ( 5 equiv ) of the hypothetical minimum needed to remove four electrons from the fdpred active site . iron species other than those listed in table 2 were not detected in significant amounts during this titration . the minor species l , listed in table 2 , is discussed below in the context of the rfq experiments . these rfq results also show that , with 3 equiv of no per diferrous site ( i.e. , between the 2.5 and 5 equiv listed in table 2 ) , at 2 min reaction time , a minor portion of the diferric species characteristic of fdpox appears . stopped - flow mixing of fdpred with excess no resulted in the uv vis absorption spectral time courses shown in figure 6 . a rapid phase that was complete within 130 ms ( figure 6a ) preceded a much slower phase that occurred over the course of 120 s ( panel b ) . the inset to figure 6a shows the difference spectra obtained by subtracting the fdpred absorption spectrum from the 130 ms time course spectra . these difference spectra clearly show the development of the { feno } species . on the basis of comparison with the substoichiometric no difference spectrum in figure 3a , the shoulder at 418 nm in the 1.3 ms difference spectrum in figure 6a indicates formation of fe{feno } within the mixing dead time . over the course of 130 ms , the 418 nm shoulder became less prominent , and a spectrum with absorptions centered at 340 , 453 , and 630 nm developed . the absorption features and extinction coefficient of the 130 ms difference spectrum in figure 6a ( 453nm/2fe 2000 m cm ) indicates substantial formation of additional { feno } beyond fe{feno } and as will become clear from the mssbauer results described below can be assigned to formation of a diferrous - dinitrosyl ( [ { feno}]2 ) complex . the 130 ms time course with excess no can thus be confidently ascribed to successive formation of fe{feno } and [ { feno}]2 from the fefe site in fdpred . single value decomposition and global analysis of the 130 ms spectral time course ( supporting information , figure s4 ) reinforce this interpretation . ( a ) 130 ms and ( b ) 130 s stopped - flow uv vis absorption spectral time courses for reactions of fdpred ( 70 m ) with excess no ( 900 m ) at 3 c . the spectrum indicated as fdpred was obtained upon stopped - flow mixing the fdpred solution with deoxygenated buffer in place of the no solution . panel a spectra were collected in 13 ms intervals after the 1.3 ms spectrum . inset of panel a shows difference absorption spectra obtained by subtracting the fdpred spectrum from the time course spectra ( color - coded as in the main panel ) . the slower portion of the stopped - flow spectral time course shown in figure 6b was dominated by the intense absorption features of fmnox , which accumulated quantitatively over 120 s. spectral features attributable to fmnsq were not apparent . comparison of the time course in figure 6a with that in figure 6b shows that the majority of the diiron sites formed [ { feno}]2 prior to any significant oxidation of fmnh2 . the quantitative oxidation of fmnh2 to fmnox in the presence of excess no is in stark contrast to the lack of any detectable fmnox formation in the stopped - flow spectral time course with substoichiometric no ( figure 3 ) . the stopped - flow spectral time courses in figures 3 and 6 thus indicate that the successive formation of fe{feno } and [ { feno}]2 precedes fmnh2 oxidation . an rfq epr time course for fdpred reacted with a similarly large excess of no as for the stopped - flow experiments ( figure s5 , supporting information ) showed a low intensity s = /2 fe{feno } signal at 130 ms ( 0.2 spins/2fe ) , which decreased at longer quench times . accounting for 10% of the total iron did not appreciably vary with quench time and is attributed to a small portion of mononuclear iron sites . the majority of the diiron sites were thus epr silent under rfq conditions analogous to those used for the stopped - flow spectra in figure 6 . figure s6 ( supporting information ) shows an analogous stopped - flow absorption spectral time course for the tm diferrous deflavo - fdp reaction with excess no . the time scales and spectral features of diferrous dinitrosyl formation and decay in figure s6 ( supporting information ) are very similar to those of the formation of diferrous dinitrosyl and fmnh2 oxidation , respectively , in the fdpred shown in figure 6 . notably the deflavo - fdp spectral time courses did not require subtraction of any fmn spectral changes . as note previously , only a portion of the diferrous sites turn over in the deflavo - fdp , which is why the diferrous dinitrosyl absorption features in figure s6 ( supporting information ) do not decay completely . however , as noted previously , the portion of the diferrous deflavo - fdp sites that did turn over generated n2o . the rfq fe mssbauer spectral time course for reaction of fdpred with a 3 equiv of no per diiron site is shown in figure 7 , for spectra recorded at 80 k ( a ) and 4 k ( b ) . these spectra provided definitive evidence for the formation of a transient s = 0 [ { feno}]2 complex . the spectra labeled fdpred in figure 7 were obtained from a sample prepared by rfq mixing fdpred with no - free buffer . these spectra showed that 20% of the diiron sites were in the diferric state ( fdpox ) with the remainder present as fdpred . the diferric species was not present in any of the subsequent samples mixed with no until the 120 s time point and does not appear in the fdpred spectrum of figure 2 . therefore , the diferric species in the fdpred spectrum of figure 7 was presumed to arise from o2 contamination . fe mssbauer spectra ( vertical bars ) for rfq of fdpred mixed with 3 equiv of no per active site at 15 c and quenched at the times listed near each spectrum . the spectra were recorded at ( a ) 80 k in zero magnetic field or ( b ) 4 k in a magnetic field of 45 mt applied parallel to the -ray direction . the colored traces are simulations of species assigned to fefe ( blue ) , fe ( green ) { feno } ( red ) of fe{feno } , l ( light blue ) , h ( pink ) , and diferric ( purple ) , respectively , using the parameters listed in table 1 and percent compositions listed in table 3 ; black traces are the sums of the simulated species spectra . concentrations immediately after mixing : 500 m fdpred and 1500 m no . the quench times subsequent to mixing with no are indicated in figure 7 . the spectra showed clear evidence four distinct species from the enzymatic active site : fdpred , fdpox , s = /2 fe{feno } , and a transient species h. in addition , two minority species are present in variable amount in the samples of the time course : isolated s = /2 { feno } , and species l. the two minority species in total account for less than 10% of the iron in any of the time points . the parameters for each complex are listed in table 1 , and their corresponding percentages of the total iron at the various quench times are listed in table 3 . all of the 80 k spectra showed a species having two doublets of equal intensity ( red and green lorentzian simulations of figure 7a ) . these two doublets have parameters identical to those assigned to the fe{feno } complex obtained in the no titration ( figure 4 and table 1 ) . similarly , all the 4 k spectra ( figure 7b ) showed the same broad paramagnetic pattern attributable to the s = /2 fe{feno } complex , at an area equal to that of the corresponding red and green simulated doublets in the 80 k spectra . the fe{feno } complex was present at the earliest time point ( 20 ms ) and constituted the majority of the iron throughout the time course . the fits ( black lines ) in figure 7b are the sum of the fe{feno } spectrum and the other doublets discussed below . the spectral area of each species comprising the summation in each of the 4.2 k spectra were constrained to be equal to the areas of the corresponding species of the 80 k spectra . from mssbauer the parameters for two other doublets , labeled h ( for high reactivity ) and l ( for low reactivity ) , are listed in table 1 . species h was observed only for reaction times ranging from 0.2 to 2 s , whereas l was observed as a minority species ( 5% of total iron ) over the entire time course . by 200 ms , no diferrous remained , and h was present at 41% of the total iron . by 2 s , h had decreased to 28% total iron and was accompanied by a proportional increase in fe{feno}. at and after 20 s , the majority of the iron was fe{feno}. at 2 min , the amount of fe{feno } decreased slightly , owing to the oxidation of iron , as demonstrated by the presence of a diferric species attributable to fdpox . the presence of a relatively small proportion of fdpox compared to that of the no titration ( figure 4 ) can be attributed to the modest excess of no in these rfq samples . as indicated by the simulations of figure 7 , species h showed a single quadrupole doublet . the isomer shift and quadrupole splitting of species h ( table 1 ) clearly identify all the iron of this species as high - spin s = /2 { feno}. the absence of a paramagnetic pattern could be attributed to either an exchange interaction between the iron ions or fast electronic relaxation . mssbauer spectra were recorded in high magnetic field ( 7.5 t ) to resolve this ambiguity and further characterize the molecular species . the red - colored spectrum in figure 8 is of the same 200 ms sample used to obtain the low - field spectrum in figure 7 . the blue - colored spectrum in figure 8 is of the fdpred and 2 equiv of no sample from the titration series of figure 5 and is displayed at 55% of the area of the 200 ms spectrum . the 2 equiv of no titration sample was chosen for its relatively high proportion of the fe{feno } species . the other species identified by simulation in figure 8a ( black line ) is from an isolated s = /2 { feno } species , which accounts for 6% of the total iron in the sample . the parameters ( see figure caption ) for this minority species are typical of a mononuclear s = /2 { feno } center but can not be accurately determined due to the low amount and is virtually imperceptible in the low - field spectra . this minor mononuclear species could be due to no reactions with either iron - deficient active sites or adventitious iron . the green - colored spectrum of figure 8b is the difference of the three spectra of figure 8a ( red minus blue minus black ) . the simulation on the difference spectrum in figure 8b ( black line ) is for a diamagnetic species ( s = 0 ) using the low - field parameters of h , which accounted for 39% of the total iron in the sample , and in agreement with the amount of species h determined at low field . the mssbauer parameters and magnetic behavior unambiguously identify species h as an antiferromagnetic exchange - coupled pair of equivalent s = /2 { feno } units , [ { feno}]2 . the variation of the exchange interaction energy , j , in simulations of two identical exchange - coupled s = /2 { feno } centers ( assuming d = 14 cm indicated j 40 cm ( ) . simulations using values of j less than 40 cm resulted in a shift of the outer lines perceptibly inward of the data . species l was a minor component in both the rfq and titration samples and persisted to significantly longer reaction times ( 30 min ) . the similarity of the mssbauer parameters of l and h suggests that l is also a s = 0 [ { feno}]2 species but with low or no corresponding catalytic activity . as was the case for the fdpox and fdpred mssbauer spectra , the sharp line width of species h ( = 0.30 mm / s ) is consistent with the individual { feno } centers having the same type and number of n- and o - donor ligands , including a single no . ( a ) fe mssbauer spectra ( temperature , 4 k ; magnetic field , 7.5 t ) of ( red bars ) of rfq mixed fdpred with 3 equiv of no per active site at 15 c and quenched at 200 ms ( the same 200 ms sample used for figure 7 ) , and ( blue bars ) fdpred + 2 equiv of no from the titration series of figure 5 , displayed at 59% of the area of the red spectrum . the black line in ( a ) is a simulation for s = /2 , d = 15 cm , e / d = 0.02 , aiso = 25 t , = 0.65 mm / s , eq = 1.9 mm / s , = 0.4 , 6% of area of red spectrum . ( b ) the green line is the difference of the three spectra in a ( red minus blue minus black ) . the black line is a simulation for s = 0 , = 0.77 mm / s , eq = 1.85 mm / s , = 1 , 37% of area of red spectrum . previous attempts to obtain michaelis menten parameters for the tm fdp nor activity were hampered by its unusually high km and the difficulty of achieving milllimolar no concentrations in the assay solutions . this barrier was overcome by using proli - nonoate stock solutions for no delivery and the activity assay conditions described in the supporting information . menten fit to the steady - state activity data ( figure s7 , supporting information ) gave km = 1000 m and kcat = 0.6 s. the mssbauer titration data in table 2 , the rfq epr spin quantification from figure 4 , and previously reported epr spin quantifications all showed nearly quantitative fe{feno } formation upon reaction of fdpred with less than or equal to 1 equiv of no per diiron site . as described above , an rfq epr time course ( figure s5 , supporting information ) showed transient minor amounts of fe{feno } even at 1500 m no ( > 10 equiv per diiron site ) . these quantifications indicate that the first no binds to the diiron site with much higher affinity than does the second . the stopped - flow time courses with 0.8 equiv of no per diiron site in figure 3 show formation of a stable fe{feno } without significant fmnh2 oxidation , the latter of which occurred only with excess no ( figure 6 ) . therefore , the steady - state km likely reflects the much lower affinity for the second no . the results described here together with those from previously reported studies lead us to the mechanistic conclusions summarized in scheme 3 . starting from fdpred ( fmnh2fefe ) , the first no reacts relatively rapidly ( submillisecond ) and with high affinity , resulting in fmnh2fe{feno } ( s = /2 diiron site ) . this form is stable at less than or equal to 1 equiv of no . with excess no , the fmnh2fe{feno } reacts with a second no over the course of 100 ms to form an fmnh2[{feno}]2 intermediate ( s = 0 , diiron site , mssbauer species h ) . in the rate - limiting step for the first turnover , the [ { feno}]2 converts to diferric over the course of 2 min , leading to a species we formulate as fmnh2fefe . this species does not accumulate due to rapid internal electron transfer , leading to fmnox fefe , as manifested by fmnh2 oxidation in the uv vis absorption spectral time course with excess no . fefe then reacts with either one or two more no analogously to that for the first turnover , ultimately leading to fdpox at a sufficiently large excess ( > 4 equiv ) of no . the reaccumulation of fe{feno } in the rfq mssbauer spectral time course ( table 3 ) with 3 equiv of no and its accumulation in the manual no titration with less than 4 eq no ( table 2 ) are consistent with this scenario . scheme 3 together with the high km for binding the second no explains the less than quantitative accumulation of the [ { feno}]2 species h and diferric in the mssbauer rfq time course with 3 equiv of no ( table 3 ) . the results reflected in scheme 3 can be used to distinguish among the proposed mechanisms in scheme 2 . therefore , in the absence of exogenous reducing equivalents , the sr mechanism would result in formation of a maximum of one n2o per four - electron reduced active site and could not attain the four - electron oxidized state , fdpox , even with excess no . however , our results show that , at 5 equiv of no , the active site of fdpred undergoes quantitative four - electron oxidation to fmnox production of two n2o per fdpred active site from this reaction is also supported by previous fourier transform ir data . inherent ability of the [ { feno}]2 ( s = 0 ) site to turn over 2no n2o in fdp is also supported by previously reported results on a deflavo - fdp . a comparison of the stopped - flow uv vis absorption spectral time courses in figure 6 and figure s6 ( supporting information ) show that the first turnover of the diferrous - dinitrosyl in fdp occurs on the same time scale as that in the deflavo - fdp . the hyp mechanism in scheme 2 does not include [ { feno}]2 and is therefore also inconsistent with our results . however , the cumulative results are fully consistent with the difeno mechanism , in which [ { feno}]2 converts spontaneously to fefe and n2o in the rate - limiting step for both turnovers ( r.d.s . in scheme 3 ) . n2o formation from two no requires n n bond formation and an n o bond cleavage . while the timing and sequence of these bond formation and cleavage events in fdp remain mysterious , our results suggest some restrictions on these processes in fdp . first and foremost is the apparent requirement for an antiferromagnetically coupled [ { feno}]2 species . the lower limit determined for the antiferromagnetic coupling in this [ { feno}]2 species , j 40 cm , is consistent with retention of the 1,3-bridging carboxylate together with either a hydroxo or oxo bridge , as shown in scheme 1 . this bridging motif would place the two { feno } centers in close proximity , thereby facilitating n n bond formation from [ { feno}]2 involves a diferric - hyponitrite intermediate . however , at least under our conditions , such a species would have to be a nonaccumulating transient species between [ { feno}]2 and fefe and n2o . formation of n2o from [ { feno}]2 presumably requires cleavage of only one of the two substrate n o bonds . therefore , it might seem that some asymmetry must arise in the two { feno } centers despite the very similar coordination spheres around fe1 and fe2 ( scheme 1 ) and the indistinguishable mossbauer parameters of the two { feno } centers . on the other hand , reduction of a symmetrical diruthenium dinitrosyl complex has been reported to form a diruthenium complex with a symmetrically n n - bridging formally hyponitrito ligand , which upon addition of a proton source , yielded n2o . we are unaware of any verified synthetic nonheme iron hyponitrito complexes . while the diferrous mononitrosyl ( fe{feno } ) must be inherently asymmetrical , it is currently unclear whether the no selectively binds to the iron proximal or distal to the fmn . o stretching frequency is abnormally low in the diferrous mononitrosyl complex of this fdp , implying a potentially activated fe no character , which was attributed to a semibridging interaction with the other iron , as depicted in scheme 3 . the n o stretching frequencies are not yet known for the magnetically coupled diferrous dinitrosyl ( species h ) identified in the present study . protonation of a substrate oxygen by either a proximal amino acid side chain or solvent ( possibly the bridging solvent ligand ) is presumably an additional requirement for n o bond cleavage leading to n2o formation . however , we have recently determined by stopped - flow spectroscopy that the rates of neither formation of the diferrous dinitrosyl nor fmnh2 oxidation with excess no show an isotope effect after equilibration of the fdpred in d2o ( frederick , r. e. ; kurtz , d. m. , jr . , this negative result indicates that any proton transfer must occur after the rate - limiting step leading to n2o formation . therefore , it is tempting to speculate that the rate - limiting process involves n n bond formation followed by rapid protonation of a transient hyponitrito ligand . in any case , n bond formation and n o bond cleavage originate from what appears to be a structurally and electronically symmetrical antiferromagnetically coupled [ { feno}]2 site in fdp . our preliminary results on fdps with significantly higher nor activities and higher second no binding affinities support this conclusion .

the unique active site of flavo - diiron proteins ( fdps ) consists of a nonheme diiron - carboxylate site proximal to a flavin mononucleotide ( fmn ) cofactor . fdps serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria , archaea , and some protozoan parasites . nitric oxide is reduced to nitrous oxide by the four - electron reduced ( fmnh2feiifeii ) active site . in order to clarify the nitric oxide reductase mechanism , we undertook a multispectroscopic presteady - state investigation , including the first mssbauer spectroscopic characterization of diiron redox intermediates in fdps . a new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous - dinitrosyl ( s = 0 , [ { feno}7]2 ) species . this species has an exchange energy , j 40 cm1 ( js1 s2 ) , which is consistent with a hydroxo or oxo bridge between the two irons . the results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous - mononitrosyl ( s = 1/2 , feii{feno}7 ) and the s = 0 diferrous - dinitrosyl species . in the rate - determining process , the diferrous - dinitrosyl converts to diferric ( feiiifeiii ) and by inference n2o . the proximal fmnh2 then rapidly rereduces the diferric site to diferrous ( feiifeii ) , which can undergo a second 2no n2o turnover . this pathway is consistent with previous results on the same deflavinated and flavinated fdp , which detected n2o as a product ( hayashibiochemistry2010 , 49 , 704020669924 ) . our results do not support other proposed mechanisms , which proceed either via super - reduction of [ { feno}7]2 by fmnh2 or through feii{feno}7 directly to a diferric - hyponitrite intermediate . the results indicate that an s = 0 [ { feno}7}]2 complex is a proximal precursor to n n bond formation and n o bond cleavage to give n2o and that this conversion can occur without redox participation of the fmn cofactor .

Introduction Experimental Procedures Results and Discussion

flavo - diiron proteins ( fdps ) are widespread in microaerophilic and anaerobic microorganisms , including bacteria , archaea , and a few protozoan parasites . the fdp - catalyzed four - electron reduction of o2 to water ( o2r activity ) or the two - electron reduction of two no molecules to nitrous oxide ( nor activity ) protects against accumulation of toxic reactive oxygen or nitrogen species . fdps are soluble cytoplasmic enzymes with a head - to - tail each active site consists of a nonheme diiron cluster ( fe1fe2 distance 3.23.6 ) , and a flavin mononucleotide ( fmn ) cofactor located 4 from the diiron site across the subunit interface , as diagrammed in scheme 1 . all three proposed mechanisms , labeled difeno , sr , and hyp , initiate from a diferrous - mononitrosyl , fe{feno } , which forms as a stable species upon addition of less than 1 equiv of no per diferrous site . difeno and sr both propose that binding of a second no ( step 1difeno , sr ) leads to a diferrous - dinitrosyl ( [ { feno}]2 ) intermediate . steps 2difeno and 2sr represent alternative rate - limiting reactions of the diferrous - dinitrosyl . in step 2difeno , n bond , are protonated , and lose the elements of water , thereby generating n2o and a diferric site . in the alternative rate - limiting step , 2sr , fmnh2 super - reduces the [ { feno}]2 to a highly reactive diferrous - dinitroxyl ( [ { fe(h)no}]2 ) , which decays to diferrous ( fefe ) with release of n2o . the hyp mechanism proposes that the second no reacts directly with the { feno } center of fe{feno } ( step 1hyp ) , leading to a formally diferric - o , n - semibridging hyponitrite intermediate ( fe(n2o2)fe ) , which decays in the rate - limiting step to diferric with release of n2o . of the three proposed mechanisms , only sr requires redox participation of the fmn cofactor prior to or at the rate - limiting step leading to n2o . in order to separate the functions of the fmn and the diiron site , the reaction of no with the diferrous deflavinated ( deflavo ) fdp from thermotoga maritima ( tm ) was examined . the results were consistent with reaction of the fe{feno } with a second no leading to parallel productive ( i.e. these results suggested that at least a portion of the diiron sites are capable of catalyzing n2o formation in the absence of fmn , which is consistent with either mechanisms difeno or hyp but not with mechanism sr in scheme 2 . however , more recent studies on a synthetic diferrous - dinitrosyl complex seem to support the feasibility of super - reduction in mechanism sr . the observation of an unusually low n o stretching frequency in the fe{feno } complex of tm fdp led to the proposal that the coordinated no is activated for reaction with a second no , which would seem to support mechanism hyp . in order to clarify these mechanistic ambiguities , we have undertaken rapid kinetics studies to trap and characterize intermediates in the fdp nor reaction . ligands typically show an s = /2 ground spin state exhibiting axial g 4.0 , 2.0 electron paramagnetic resonance ( epr ) spectra and characteristic ligand - to - metal charge transfer uv visible ( uv vis ) absorptions at 650 , 450 , and 330 nm with 450 nm 1000 m cm . the diferrous mononitrosyl , fe{feno } , in fdp shows this characteristic uv vis absorption but exhibits an s = /2 epr spectrum ( g 2.3 , 1.9 ) due to antiferromagnetic coupling between the high spin fe ( s = 2 ) and the s = /2 { feno}. we have used all of these spectroscopic methods to identify intermediates in reactions of no with tm fdpred , including the first mssbauer spectral characterizations of diiron redox intermediates during catalysis in this class of proteins . our results clarify the roles of the fmn and the diiron site in nor catalysis and can be used to discriminate among the mechanistic possibilities in scheme 2 . overnight reduction under these condition resulted in the four - electron reduced yellow - colored tm fdpred . the packing factor was determined as the ratio of the peak - to - peak intensity of the s = /2 epr signal of a separately sprayed and packed d. vulgaris ferric rd solution of known concentration ( determined using 490 nm = 8800 m cm ) to that of the signal from the same rd sample in which the snow had been thawed and refrozen in liquid n2 . the packing factor was determined to be 0.7 , and the rfq epr spin concentration was corrected by this packing factor . fmnox , the anionic fmn semiquinone ( fmnsq ) , and the fully reduced fmn ( fmnh2 ) are all redox accessible in fdps . thus , anaerobic reduction of fdpox with 2.2 mol equiv of nadh per diiron site in the presence of catalytic nror / rd resulted in formation of the fdpsq ( fmnsq fefe ) within the time of manual mixing followed by a slower reduction to fdpred ( fmnred fefe ) . uv vis absorption spectra of ( a ) before ( fdpox ) , < 1 min after ( fdpsq ) , and several hours after ( fdpred ) addition of 140 m nadh to a solution containing 5 m rd , 500 nm nror , and 60 m fdpox and ( b ) before ( fdpox ) and immediately after ( fdpred ) addition of 140 m nadh to a solution of 50 m rd , 500 nm nror , and 50 m fdpox . the sharp doublet , as opposed to a broad paramagnetic spectrum , is attributed to antiferromagnetic exchange coupling of the two s = /2 fe ions at the active site to give a s = 0 state lowest in energy . the single sharp doublets in the spectra of both fdpox and fdpred ( = 0.31 mm / s ) are indicative of equivalent electronic environments for the individual iron centers in the diiron site , which is consistent with the structure showing that each iron has the same type and number of n- and o - donor ligands ( scheme 1 ) . at 4.2 k ( figure 5b ) , the fe{feno } complex appeared as a broad paramagnetic pattern as expected for a s = /2 species . the isomer shifts , quadrupole splittings , and paramagnetic broadening of the s = /2 species are reminiscent of that for the s = /2 fe{feno } complex of hemerythrin . from 1.5 to 2.5 equiv of no , the proportions of diferrous ( 8% ) and fe{feno } ( 87% ) remained relatively stable . the epr spectra of these three samples ( figure s3 , supporting information ) showed that the amount of the s = /2 fe{feno } complex quantitatively agreed with the proportion of the paramagnetically broadened component in the mssbauer spectra of figure 5 , indicating that this broadened feature in the mssbauer spectra originated from the s = /2 fe{feno } complex . the mssbauer data also show that the fefe site can be oxidized quantitatively to the diferric ( fefe ) state by no only slightly in excess ( 5 equiv ) of the hypothetical minimum needed to remove four electrons from the fdpred active site . on the basis of comparison with the substoichiometric no difference spectrum in figure 3a , the shoulder at 418 nm in the 1.3 ms difference spectrum in figure 6a indicates formation of fe{feno } within the mixing dead time . the absorption features and extinction coefficient of the 130 ms difference spectrum in figure 6a ( 453nm/2fe 2000 m cm ) indicates substantial formation of additional { feno } beyond fe{feno } and as will become clear from the mssbauer results described below can be assigned to formation of a diferrous - dinitrosyl ( [ { feno}]2 ) complex . the stopped - flow spectral time courses in figures 3 and 6 thus indicate that the successive formation of fe{feno } and [ { feno}]2 precedes fmnh2 oxidation . the time scales and spectral features of diferrous dinitrosyl formation and decay in figure s6 ( supporting information ) are very similar to those of the formation of diferrous dinitrosyl and fmnh2 oxidation , respectively , in the fdpred shown in figure 6 . as note previously , only a portion of the diferrous sites turn over in the deflavo - fdp , which is why the diferrous dinitrosyl absorption features in figure s6 ( supporting information ) do not decay completely . these spectra provided definitive evidence for the formation of a transient s = 0 [ { feno}]2 complex . therefore , the diferric species in the fdpred spectrum of figure 7 was presumed to arise from o2 contamination . the colored traces are simulations of species assigned to fefe ( blue ) , fe ( green ) { feno } ( red ) of fe{feno } , l ( light blue ) , h ( pink ) , and diferric ( purple ) , respectively , using the parameters listed in table 1 and percent compositions listed in table 3 ; black traces are the sums of the simulated species spectra . the spectra showed clear evidence four distinct species from the enzymatic active site : fdpred , fdpox , s = /2 fe{feno } , and a transient species h. in addition , two minority species are present in variable amount in the samples of the time course : isolated s = /2 { feno } , and species l. the two minority species in total account for less than 10% of the iron in any of the time points . similarly , all the 4 k spectra ( figure 7b ) showed the same broad paramagnetic pattern attributable to the s = /2 fe{feno } complex , at an area equal to that of the corresponding red and green simulated doublets in the 80 k spectra . the other species identified by simulation in figure 8a ( black line ) is from an isolated s = /2 { feno } species , which accounts for 6% of the total iron in the sample . the simulation on the difference spectrum in figure 8b ( black line ) is for a diamagnetic species ( s = 0 ) using the low - field parameters of h , which accounted for 39% of the total iron in the sample , and in agreement with the amount of species h determined at low field . the variation of the exchange interaction energy , j , in simulations of two identical exchange - coupled s = /2 { feno } centers ( assuming d = 14 cm indicated j 40 cm ( ) . the similarity of the mssbauer parameters of l and h suggests that l is also a s = 0 [ { feno}]2 species but with low or no corresponding catalytic activity . as was the case for the fdpox and fdpred mssbauer spectra , the sharp line width of species h ( = 0.30 mm / s ) is consistent with the individual { feno } centers having the same type and number of n- and o - donor ligands , including a single no . ( a ) fe mssbauer spectra ( temperature , 4 k ; magnetic field , 7.5 t ) of ( red bars ) of rfq mixed fdpred with 3 equiv of no per active site at 15 c and quenched at 200 ms ( the same 200 ms sample used for figure 7 ) , and ( blue bars ) fdpred + 2 equiv of no from the titration series of figure 5 , displayed at 59% of the area of the red spectrum . the black line is a simulation for s = 0 , = 0.77 mm / s , eq = 1.85 mm / s , = 1 , 37% of area of red spectrum . the stopped - flow time courses with 0.8 equiv of no per diiron site in figure 3 show formation of a stable fe{feno } without significant fmnh2 oxidation , the latter of which occurred only with excess no ( figure 6 ) . starting from fdpred ( fmnh2fefe ) , the first no reacts relatively rapidly ( submillisecond ) and with high affinity , resulting in fmnh2fe{feno } ( s = /2 diiron site ) . with excess no , the fmnh2fe{feno } reacts with a second no over the course of 100 ms to form an fmnh2[{feno}]2 intermediate ( s = 0 , diiron site , mssbauer species h ) . in the rate - limiting step for the first turnover , the [ { feno}]2 converts to diferric over the course of 2 min , leading to a species we formulate as fmnh2fefe . therefore , in the absence of exogenous reducing equivalents , the sr mechanism would result in formation of a maximum of one n2o per four - electron reduced active site and could not attain the four - electron oxidized state , fdpox , even with excess no . however , our results show that , at 5 equiv of no , the active site of fdpred undergoes quantitative four - electron oxidation to fmnox production of two n2o per fdpred active site from this reaction is also supported by previous fourier transform ir data . inherent ability of the [ { feno}]2 ( s = 0 ) site to turn over 2no n2o in fdp is also supported by previously reported results on a deflavo - fdp . a comparison of the stopped - flow uv vis absorption spectral time courses in figure 6 and figure s6 ( supporting information ) show that the first turnover of the diferrous - dinitrosyl in fdp occurs on the same time scale as that in the deflavo - fdp . however , the cumulative results are fully consistent with the difeno mechanism , in which [ { feno}]2 converts spontaneously to fefe and n2o in the rate - limiting step for both turnovers ( r.d.s . n2o formation from two no requires n n bond formation and an n o bond cleavage . the lower limit determined for the antiferromagnetic coupling in this [ { feno}]2 species , j 40 cm , is consistent with retention of the 1,3-bridging carboxylate together with either a hydroxo or oxo bridge , as shown in scheme 1 . this bridging motif would place the two { feno } centers in close proximity , thereby facilitating n n bond formation from [ { feno}]2 involves a diferric - hyponitrite intermediate . formation of n2o from [ { feno}]2 presumably requires cleavage of only one of the two substrate n o bonds . therefore , it might seem that some asymmetry must arise in the two { feno } centers despite the very similar coordination spheres around fe1 and fe2 ( scheme 1 ) and the indistinguishable mossbauer parameters of the two { feno } centers . on the other hand , reduction of a symmetrical diruthenium dinitrosyl complex has been reported to form a diruthenium complex with a symmetrically n n - bridging formally hyponitrito ligand , which upon addition of a proton source , yielded n2o . o stretching frequency is abnormally low in the diferrous mononitrosyl complex of this fdp , implying a potentially activated fe no character , which was attributed to a semibridging interaction with the other iron , as depicted in scheme 3 . the n o stretching frequencies are not yet known for the magnetically coupled diferrous dinitrosyl ( species h ) identified in the present study . protonation of a substrate oxygen by either a proximal amino acid side chain or solvent ( possibly the bridging solvent ligand ) is presumably an additional requirement for n o bond cleavage leading to n2o formation . however , we have recently determined by stopped - flow spectroscopy that the rates of neither formation of the diferrous dinitrosyl nor fmnh2 oxidation with excess no show an isotope effect after equilibration of the fdpred in d2o ( frederick , r. e. ; kurtz , d. m. , jr . therefore , it is tempting to speculate that the rate - limiting process involves n n bond formation followed by rapid protonation of a transient hyponitrito ligand . in any case , n bond formation and n o bond cleavage originate from what appears to be a structurally and electronically symmetrical antiferromagnetically coupled [ { feno}]2 site in fdp .

fungal endophytes of the genus neotyphodium and epichlo are widespread in temperate grasses of the poaceae subfamily pooideae [ 1 , 2 ] . in agronomically important pasture grasses such as perennial ryegrass ( lolium perenne l. ) and tall fescue ( festuca arundinacea schreb . l. arundinaceum ) , the respective symbionts ( n. lolii [ latch , christensen , and samuels ] glenn , bacon , and hanlin and n. coenophialum [ morgan - jones and gams ] glenn , bacon , and hanlin ) confer both beneficial and detrimental agronomic traits [ 37 ] . molecular genetic marker - based studies have contributed to knowledge of endophyte genetics , taxonomy , and phylogeny . neotyphodium species were originally placed in the form ( asexual ) genus acremonium , but were reclassified into the new form genus neotyphodium when sequence analysis of ribosomal rna - encoding ( rdna ) genes indicated a monophyletic group with the sexual epichlo species [ 1 , 9 ] . phylogenetic analysis of genes for conserved proteins such as the -tubulin gene ( tub2 ) , translation elongation factor 1- ( tefa ) , and actin ( actg ) also provided evidence for close relationships between neotyphodium and epichlo species [ 1017 ] . although taxa such as n. lolii are haploid in nature , other neotyphodium species were shown to contain multiple gene copies and conform to heteroploid genomic constitutions . the single or multiple gene copies of asexual neotyphodium species appear to correspond to those of specific haploid epichlo species . this observation has been interpreted to support a hybrid origin for heteroploid taxa : for instance , n. coenophialum has been proposed to have arisen through hybridisation and subsequent nuclear fusion events involving the extant taxa e. typhina , e. baconii , and e. festucae . the relative genome sizes of haploid and heteroploid endophytes ( c. 30 mb for n. lolii ; c. 60 mb for n. coenophialum ) lend some support to this hypothesis , subject to the possibility of selective gene loss subsequent to hybridisation events . phylogenetic relationships between endophyte taxa are hence complex and reticulated . sequence analysis of individual gene loci may be used to infer such relationships based on affinities between shared genomes . however , performance differences between individual genes have been observed . the resolution capacity provided by rdna and acta genes was low in comparison to other genes [ 1215 ] , possibly due to homoplasy effects . heteroploid - like neotyphodium species also display aneuploidy for some loci , such as the rdna gene , limiting resolution of complete phylogenies . a broader survey of gene classes is hence desirable to further clarify affinities between endophyte taxa . simple sequence repeats ( ssrs ) or microsatellites have been widely used for analysis of genetic variation within and between closely related species . a high rate of mutation renders ssr array length polymorphism particularly useful for intraspecific genetic studies . however , sequence analysis has revealed complex mechanisms controlling allele size variation , limiting the efficiency of interspecific phylogenetic analysis . repeat number variation is thought to arise from polymerase slippage during replication , but constraints on threshold size for allele expansion and on allele size range are evident . in addition , interruptions of the repeat structure tend to stabilise ssr loci . constraints on allele size may consequently lead to inaccurate assessment of phylogenetic divergence between taxa . size homoplasy of distinct alleles arising from insertions , deletions , and base substitutions in the ssr flanking regions are also common [ 2730 ] . changes in flanking regions appear to occur independently of changes in the ssr repeat array [ 28 , 30 ] . due to these factors , allelic variation of ssr loci , as assessed by amplicon size variation , is appropriate only for phenetic analysis and not suitable for phylogenetic reconstruction . in contrast , several studies have performed phylogenetic interpretation through analysis of ssr - flanking sequence regions . the resolving power of evolutionary studies using individual structural genes may be constrained by limited divergence , and studies of a small number of gene loci may not be representative of whole - genome variation . however , the abundant genomic distribution of ssrs [ 3335 ] permits phylogenetic assessment across the transcriptional units of multiple gene classes . ssr - flanking regions have been used for phylogenetic analysis of multiple organisms [ 31 , 32 , 36 , 37 ] , resolving relationships to otherwise inaccessible levels [ 31 , 32 , 37 ] . consistent with these previous studies , gene - associated ssr loci have previously been shown to discriminate endophyte taxa based on size polymorphism , but did not permit phylogenetic analysis . the present study describes the comparison of sequences that flank the ssr array in 5 independently selected gene loci , across 23 distinct fungal endophyte isolates . the derived data have determined the extent of molecular variation underlying ssr size polymorphism , confirmed current models for genome affinities , inferred phylogenetic relationships and models of genome evolution ( including a role for selective gene loss ) , and elucidated the genomic origin of several previously unclassified neotyphodium taxa . phylogenetic analysis was performed on 20 endophyte isolates representing three neotyphodium and five epichlo taxa , as well as three neotyphodium isolates which could not be assigned to known taxa based on their morphological characters ( a. leuchtmann , pers . comm . ) , and a tall fescue endophyte taxon ( fatg-2 ) which has yet to be allocated a linnean name ( table 1 ) . genomic amplicons obtained with primer pairs designed to five est - ssr loci ( ncesta1ab04 , ncesta1fh03 , ncesta1ag07 , nlesta1gf09 , and nlesta1nf04 ) were analysed . amplicons from haploid taxa were analysed by direct sequencing , with the exception of locus nlesta1nf04 for which sequencing was performed on purified plasmids containing the cloned amplicon . sequencing reactions were performed in 10 l reaction volumes containing 4 l sequencing reagent premix from the dyenamic et terminator cycle sequencing kit ( amersham biosciences , little chalfont , uk ) , 0.5 m of forward or reverse primer for the locus of interest and 5 l of amplicon in a thermocycler ( geneamp ; pe applied biosystems , forster city , california , usa . ) programmed for 20 seconds at 92c followed by 30 cycles of 20 s at 95c , 15 s at 50c , 2 minutes at 60c , then 10 min at 60c . sequencing products were purified using autoseq 96 plates ( amersham biosciences ) , dried at 80c for 30 min and resuspended in 5 l of sterile milli - q water before analysis on a abi prism 3700 automated sequencer ( pe applied biosystems ) . for multiple products amplified in a single reaction from nonhaploid taxa , the purified products were cloned into pgem - t easy vector ( promega , madison , wisconsin , u.s.a . ) and transformed into competent cells . consensus sequences were derived through analysis of several independently isolated clones or direct sequencing of both strands . sequences were compared using sequencher ( version 4.0 ) ( gene codes corporation , ann arbor , michigan , u.s.a . ) . blastx ( version 2.2.1 and 2.2.6 ) was used to search for similarities between the est sequence of ssr loci and protein sequences in the protein databases available from the national centre for biological information ( nr , pdb and swissprot ; http://www.ncbi.nlm.nih.gov/blast/ ) . the dna sequences of unique amplicons were prepared for phylogenetic analysis by compilation in fasta format into a single file for sequence alignment in clustalx ( version 1.8 ) . manual realignment of sequences removed primer termini and polymorphic ssr arrays and converted insertion - deletion ( indel ) regions into single multistate characters . sequence alignments were analysed by clustering or tree searching methods available in phylip ( version 3.6a3 ) ( j. felsenstein , university of washington , seattle , washington , u.s.a . , available from http://evolution.gs.washington.edu/phylip.html ) . for parsimony analysis , sequences were analysed with indels either removed , or coded as single multistate characters . where multiple trees were resolved , the kishino - hasegawa - templeton ( kht ) test [ 41 , 42 ] or shimodaira - hasegawa ( sh ) test was used to test for significant differences . the robustness of the trees was measured by the bootstrap method with 1000 replicates . a bootstrap value of 70% or greater was considered to be well supported . for maximum likelihood ( ml ) and distance - based analysis distance matrices were obtained using the f84 model [ 42 , 45 ] and clustered using the fitch - margoliash ( fm ) method or neighbor - joining ( nj ) method . the transition / transversion ratio was estimated using the tree - puzzle program ( version 5.0 ) or by the ml method . to estimate the transition / transversion ratio by the ml method , different possible values for the transition / transversion ratio were evaluated in multiple runs to find the value with the maximum likelihood estimate . the same approach was used to estimate the among - site rate heterogeneity by the ml method . to estimate the among - site rate heterogeneity by the minimum evolution ( me ) method , distance matrices generated for each site were analysed and the total branch length was taken as the estimated value of the rate of change for each of the sites . genomic amplicons from five est - ssr loci , ranging in length from 181385 bp , were characterised from 12 neotyphodium and 10 epichlo isolates , as well as fatg-2 ( table 2 ) . the sequences of two loci ( ncesta1ab04 and nlesta1gf09 ) shared amino acid sequence similarity with hypothetical or predicted proteins of neurospora crassa and magnoporthe grisea and were mainly composed of coding sequence ( cds ) as well as 5-untranslated region ( 5-utr ) and intron sequences , respectively . the sequences of the remaining three loci did not show similarity with any proteins in public databases . amplification products from locus ncesta1ga07 were predominantly composed of intron sequence , based on comparison of est and genomic dna sequences . size polymorphisms between taxa for the selected loci resulted from variation at a number of indel sites ( table 2 ) . differences also occurred in the repeat unit number of the ssr array for three loci ( ncesta1fh03 , ncesta1ga07 and nlesta1nf04 ; data not shown ) , accounting for the majority of the observed size polymorphisms . a number of sequence haplotypes ( defined here as amplicons with multiple sequence variant content , but clearly related to a single common reference ) for each locus were identified across the sample set , with identity observed between those of several neotyphodium and epichlo isolates . single haplotypes for individual genes were observed for n. lolii , unclassified neotyphodium isolate 9727 , and the different epichlo species , while n. coenophialum , fatg-2 , n. uncinatum , and unclassified neotyphodium isolates 9303/2 and 9728 generated multiple haplotypes . the number of haplotypes present in these species varied between loci , but with a maximum of three for n. coenophialum and neotyphodium isolates 9303/2 and 9728 , and two for fatg-2 and n. uncinatum . variation was observed in cloning efficiency of different pcr products for those species possessing multiple haplotypes . aberrant haplotypes , which were likely to be chimeras generated by pcr - mediated recombination , were also obtained . pairwise comparisons identified between 2351 informative characters for the different loci ( table 2 ) . the proportion of informative characters ranged from 14% ( locus nlesta1gf09 ) to 31% ( locus nlesta1nf04 ) , but was c. 20% for the other loci . a similar proportion of informative characters occurred in both the coding and noncoding sequences of the eligible loci . loci were analysed through sequence alignment ( supplementary material : appendices 15 available at doi:10.4067/2011/921312 ) individually , rather than as a combined dataset , due to variation of both inferred ploidy level and number of observed haplotypes between different ssr loci from heteroploid isolates . between one and four trees were resolved for the different loci using the parsimony method ( figures 15 ) . the multiple trees obtained for loci ncesta1ab04 ( figure 1 ) , nlesta1gf09 ( figure 4 ) and nlesta1nf04 ( figure 5 ) only differed in the placement of one or two species . more variation was evident in the branching of the multiple trees identified for the locus ncesta1ga07 ( figure 3 ) . the trees , however , were not found to be significantly different in the kht or sh tests . similar trees were resolved for the different loci using the ml , fm , and nj methods ( data not shown ) . in tests performed using the ml and me methods , no significant differences were detected in the rate of change between coding and non - coding sequences or between exon and intron sequences for eligible loci ( data not shown ) . phylogenetic analysis of the different loci revealed similar genomic relationships among endophyte species , as summarised in a network format ( figure 6 ) . close relationships between haplotypes from different taxa were deduced to indicate partial or complete commonality of genome content . some locus - dependent differences in tree topology ( figures 15 ) were observed , but specific taxa consistently grouped together . in most instances , the epichlo species were separated into two groups , separation being supported by bootstrap analysis . the first contained e. festucae , e. baconii , and e. bromicola , and the second contained e. typhina , e. clarkii , and e. sylvatica , neotyphodium species being included within both groups . group 1 epichlo species were further divided into distinct branches according to their taxonomic classification , while group 2 endophytes showed higher levels of genetic similarity . these relationships were observed in most of the trees and were also supported by bootstrap analysis . the single haplotypes derived from both n. lolii and e. festucae were grouped together in all trees and were identical in structure for two of the loci ( ncesta1fh03 and nlesta1gf09 ) . multiple haplotypes from n. coenophialum , fatg-2 , and n. uncinatum were consistently associated with counterparts from specific neotyphodium and epichlo species . n. coenophialum and n. uncinatum shared identical or very closely related haplotypes for all five loci . the remaining haplotypes common to n. coenophialum and fatg-2 grouped with the corresponding single haplotypes from e. festucae and n. lolii . for a subset of the target loci , n. uncinatum - derived sequences grouped with the corresponding haplotypes from e. bromicola . neotyphodium isolate 9727 produced single haplotypes from each locus that were either identical or very similar to the haplotypes common between n. coenophialum and n. uncinatum , and grouped most closely with those derived from e. sylvatica . neotyphodium isolates 9303/2 and 9728 were closely related , all locus - specific haplotypes showing a high degree of sequence similarity . one of three subclasses of derived haplotypes grouped to form a distinct well - supported group with those from e. festucae , n. lolii , n. coenophialum , and fatg-2 . isolates 9303/2 and 9728 exhibited a second haplotype sub - class that grouped with counterparts from e. bromicola and n. uncinatum , and the same class was present in isolate over two loci . the remaining haplotype sub - class ( observed in isolate 9303/2 for four loci , and in isolate 9728 for one locus ) grouped with the equivalent haplotypes from e. typhina and e. clarkii . the application of ssr markers for phylogenetic analysis is limited by two main factors : complex molecular evolution of ssr loci and the occurrence of size homoplasy between distinct ssr alleles . sequence analysis of selected ssr loci in the current study has demonstrated that these factors influence the generic inability of ssr markers to resolve phylogenetic relationships among endophyte species . changes in the ssr array repeat number appeared to be independent of flanking region changes : some of the locus nlesta1nf04-derived haplotypes from multiple e. festucae isolate differed for ssr array number , but exhibited identity for flanking sequence , while others showed the converse relationship . ssr allele size homoplasy occurred between different endophyte taxa of distinct origins as a result of insertions , deletions , and base substitutions in both the ssr motif and flanking sequences , as observed for the locus ncesta1fh03-specific e. festucae and e. baconii - related n. coenophialum haplotypes . endophyte ssr locus arrays were highly variable , and differences in repeat unit number generally accounted for allele size variation between closely related endophyte species , while indel and base substitution incidence increased when comparisons were made between more distantly related taxa . the results of this and other studies [ 30 , 31 ] suggest that size variation may provide a relatively accurate measure of genetic variation between closely related species . although homoplasy was not taken into account , ssrs have previously proven useful for genetic discrimination within and between endophyte species . presumably , the inherently variable nature of ssrs and the large number of loci analysed reduced the potential biasing effects of individual loci . the complex nature of ssr loci , however , demonstrates the critical value of sequence level analysis for phylogenetic inference . the flanking regions of gene - associated ssrs were highly conserved within and between endophyte taxa ( 80%100% sequence identity across coding and non - coding regions ) , supporting a common origin for these species [ 1 , 9 ] . despite this level of sequence conservation , the different individual loci obtained similar genetic relationships , consistent with previous studies of other genes . differences in the power of the individual loci to resolve relationships were identified due to variation in number of informative characters and composition ( exon , intron , coding , or non - coding ) of amplicons . in other studies , ssr - flanking sequences from different loci have been aggregated to increase the number of informative characters and improve resolution of phylogenetic relationships [ 31 , 32 , 36 , 37 ] . however , variation of inferred ploidy level and of number of haplotypes derived from different loci in heteroploids would potentially bias such aggregation studies . the close relationships between taxa were in accordance with those predicted from genes more commonly used for phylogenetic analysis such as rdna , tubb , tefa , and actg . single locus - specific haplotypes were obtained from all epichlo species and from n. lolii , the latter being closely related to e. festucae . other neotyphodium species contained multiple haplotypes that were similar to those from different epichlo species . occurrence of different haplotype subclasses in n. coenophialum , fatg-2 , and n. uncinatum is consistent with the heteroploid or nonhaploid nature of these species [ 11 , 14 ] and indicates the presence of multiple genomes ( figure 6 ) . neotyphodium isolates that could not be assigned to known morphological classes also appear to differ from characterised taxa at the molecular level . isolate 9303/2 and isolate 9728 have been assigned to taxonomic groupings heutg-2 and lptg-2 , respectively , based on phylogenetic analysis of the tefa and tubb genes ( a. leuchtmann , pers . analysis of ssr - flanking regions in this study , however , suggests that the isolates show closer affinities than formerly predicted . reported the detection of two haplotype classes for each isolate , closely related to those from e. bromicola and e. typhina ( heutg ) and from e. festucae and e. typhina ( lptg-2 ) . however , both flanking sequence analysis , as well as phenetic studies based on a larger number of ssr loci , detected a third haplotype sub - class for both isolates and suggested common affinities with e. festucae , e. bromicola , and e. typhina , respectively . accurate inference of phylogenetic relationships among neotyphodium and epichlo species consequently requires characterisation of a number of different genomic loci . although isolates 9303/2 and 9728 share common affinities , dna - based phylogenetic and phenetic analyses suggest mutual genetic divergence and placement in taxonomic groups with different relative gene content . although similar - sized haplotypes were detected , ssr polymorphism between these isolates was greater than that detected within n. coenophialum , n. lolii , and e. festucae . in addition , 9303/2 and 9728 failed to cluster together in an aflp - derived phenogram , which represents a genome - wide assessment of genetic polymorphism . ssr polymorphism analysis also detected substantial differences in the number of locus - specific haplotypes : isolate 9728 produced a higher proportion of single haplotype classes . e. festucae - related haplotypes were detected in both isolates for all five loci , while e. bromicola - like and e. typhina - like sequence variants were observed more frequently in isolate 9303/2 than isolate 9728 and were not represented between all loci . neotyphodium species with common phylogenetic affinities are known to occur in several different grass species . the lptg-2 , n. tembladerae , and n. australiense endophytes , which are resident in l. perenne , poa huecu , and echinopogon ovatus , respectively , all appear to be phylogenetically related to e. festucae and e. typhina [ 10 , 16 ] . however , these endophytes appear to be related to different e. typhina strains and also differ in their genome structure [ 10 , 13 , 16 ] . differences in transcript levels associated with different gene - specific sequence variants , as observed for the 60s ribosomal protein - encoding gene in this study , may also contribute to phenotypic trait variation between different heteroploid endophyte taxa . two asexual endophyte species , n. huerfanum and n. tembladerae , are known to occur in festuca arizonica . phylogenetic analysis of the third unclassified neotyphodium isolate ( 9727 ) , which was also derived from f. arizonica , suggests that it may belong to the former taxon . isolate 9727 produced single haplotypes and these sequences , like those of the n. huerfanum tefa and tubb loci , are closely related to the inferred e. typhina - related haplotypes from n. coenophialum and n. uncinatum ( section 4.2 ) . these results were also supported by ssr polymorphism - based phenetic analysis , in which 9727 clustered with e. typhina , e. clarkii , and e. sylvatica , while in aflp analysis the isolate clustered with n. uncinatum . due to their close phylogenetic relationships with specific epichlo species , neotyphodium species have been proposed to have originated from these sexual endophyte taxa either directly through the loss of the sexual state , or through interspecific hybridisation of distinct epichlo and neotyphodium species . the first process is proposed to have given rise to haploid neotyphodium species such as n. lolii , while the heteroploid neotyphodium species such as n. coenophialum , fatg-2 , and n. uncinatum may have arisen through the second evolutionary process . because epichlo species form unique mating populations [ 4951 ] and neotyphodium species are not known to sporulate in vivo , this second mode of evolution is thought to have been a parasexual process involving somatic fusion of endophyte hyphae . this hypothesis does , however , require physical colocation between endophyte taxa that generally occur in distinct host species . in addition , mechanisms of gene loss following nuclear fusion are necessary to account for the observed genomic composition of contemporary heteroploid taxa , as a range of studies [ 2 , 10 , 11 , 13 , 14 , 16 , 17 ] have shown that extant neotyphodium species do not appear to have the full complement of genes present in phylogenetically related epichlo species . loss of genes involved in sexual reproduction and pathogenicity would be a prerequisite for such genomic rearrangement events , as well as genes vulnerable to dosage - dependent effects . it is also formally possible that sexual epichlo species may have arisen from asexual neotyphodium species in response to selective environmental pressures , a mechanism requiring both gene loss and gene gain , possibly through horizontal gene transfer . mechanisms for both processes have been inferred through comparisons of different fungal taxa at the whole genome levels and may have been facilitated by structural features such as presence of conserved repetitive elements . in conclusion , this study demonstrates the application of ssr - flanking sequences to studies of genome affinities between pasture grass fungal endophyte species for clarification of novel modes of genome evolution . the inferred affinities were consistent with those obtained from gene loci that are more commonly used in molecular phylogenetics , but provided a more extensive survey of genomic loci , that may be ultimately extended to whole genome comparisons based.on second - generation sequencing technologies .

fungal species of the neotyphodium and epichlo genera are endophytes of pasture grasses showing complex differences of life - cycle and genetic architecture . simple sequence repeat ( ssr ) markers have been developed from endophyte - derived expressed sequence tag ( est ) collections . although ssr array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa , the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects . in contrast , nonrepetitive sequence regions that flank ssrs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes . consistent patterns of relationships between specific taxa were apparent across multiple target loci , confirming previous studies of genome evolution based on variation of individual genes . evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content . results were compatible with the current view that some asexual neotyphodium species arose following interspecific hybridisation between sexual epichlo ancestors . phylogenetic analysis of ssr - flanking regions , in combination with the results of previous studies with other est - derived ssr markers , further permitted characterisation of neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics .

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

fungal endophytes of the genus neotyphodium and epichlo are widespread in temperate grasses of the poaceae subfamily pooideae [ 1 , 2 ] . in agronomically important pasture grasses such as perennial ryegrass ( lolium perenne l. ) and tall fescue ( festuca arundinacea schreb . l. arundinaceum ) , the respective symbionts ( n. lolii [ latch , christensen , and samuels ] glenn , bacon , and hanlin and n. coenophialum [ morgan - jones and gams ] glenn , bacon , and hanlin ) confer both beneficial and detrimental agronomic traits [ 37 ] . molecular genetic marker - based studies have contributed to knowledge of endophyte genetics , taxonomy , and phylogeny . neotyphodium species were originally placed in the form ( asexual ) genus acremonium , but were reclassified into the new form genus neotyphodium when sequence analysis of ribosomal rna - encoding ( rdna ) genes indicated a monophyletic group with the sexual epichlo species [ 1 , 9 ] . phylogenetic analysis of genes for conserved proteins such as the -tubulin gene ( tub2 ) , translation elongation factor 1- ( tefa ) , and actin ( actg ) also provided evidence for close relationships between neotyphodium and epichlo species [ 1017 ] . although taxa such as n. lolii are haploid in nature , other neotyphodium species were shown to contain multiple gene copies and conform to heteroploid genomic constitutions . the single or multiple gene copies of asexual neotyphodium species appear to correspond to those of specific haploid epichlo species . phylogenetic relationships between endophyte taxa are hence complex and reticulated . sequence analysis of individual gene loci may be used to infer such relationships based on affinities between shared genomes . however , performance differences between individual genes have been observed . heteroploid - like neotyphodium species also display aneuploidy for some loci , such as the rdna gene , limiting resolution of complete phylogenies . a broader survey of gene classes is hence desirable to further clarify affinities between endophyte taxa . simple sequence repeats ( ssrs ) or microsatellites have been widely used for analysis of genetic variation within and between closely related species . a high rate of mutation renders ssr array length polymorphism particularly useful for intraspecific genetic studies . in addition , interruptions of the repeat structure tend to stabilise ssr loci . constraints on allele size may consequently lead to inaccurate assessment of phylogenetic divergence between taxa . size homoplasy of distinct alleles arising from insertions , deletions , and base substitutions in the ssr flanking regions are also common [ 2730 ] . changes in flanking regions appear to occur independently of changes in the ssr repeat array [ 28 , 30 ] . due to these factors , allelic variation of ssr loci , as assessed by amplicon size variation , is appropriate only for phenetic analysis and not suitable for phylogenetic reconstruction . in contrast , several studies have performed phylogenetic interpretation through analysis of ssr - flanking sequence regions . the resolving power of evolutionary studies using individual structural genes may be constrained by limited divergence , and studies of a small number of gene loci may not be representative of whole - genome variation . however , the abundant genomic distribution of ssrs [ 3335 ] permits phylogenetic assessment across the transcriptional units of multiple gene classes . ssr - flanking regions have been used for phylogenetic analysis of multiple organisms [ 31 , 32 , 36 , 37 ] , resolving relationships to otherwise inaccessible levels [ 31 , 32 , 37 ] . consistent with these previous studies , gene - associated ssr loci have previously been shown to discriminate endophyte taxa based on size polymorphism , but did not permit phylogenetic analysis . the present study describes the comparison of sequences that flank the ssr array in 5 independently selected gene loci , across 23 distinct fungal endophyte isolates . the derived data have determined the extent of molecular variation underlying ssr size polymorphism , confirmed current models for genome affinities , inferred phylogenetic relationships and models of genome evolution ( including a role for selective gene loss ) , and elucidated the genomic origin of several previously unclassified neotyphodium taxa . phylogenetic analysis was performed on 20 endophyte isolates representing three neotyphodium and five epichlo taxa , as well as three neotyphodium isolates which could not be assigned to known taxa based on their morphological characters ( a. leuchtmann , pers . amplicons from haploid taxa were analysed by direct sequencing , with the exception of locus nlesta1nf04 for which sequencing was performed on purified plasmids containing the cloned amplicon . sequencing reactions were performed in 10 l reaction volumes containing 4 l sequencing reagent premix from the dyenamic et terminator cycle sequencing kit ( amersham biosciences , little chalfont , uk ) , 0.5 m of forward or reverse primer for the locus of interest and 5 l of amplicon in a thermocycler ( geneamp ; pe applied biosystems , forster city , california , usa . ) for multiple products amplified in a single reaction from nonhaploid taxa , the purified products were cloned into pgem - t easy vector ( promega , madison , wisconsin , u.s.a . ) consensus sequences were derived through analysis of several independently isolated clones or direct sequencing of both strands . blastx ( version 2.2.1 and 2.2.6 ) was used to search for similarities between the est sequence of ssr loci and protein sequences in the protein databases available from the national centre for biological information ( nr , pdb and swissprot ; http://www.ncbi.nlm.nih.gov/blast/ ) . the dna sequences of unique amplicons were prepared for phylogenetic analysis by compilation in fasta format into a single file for sequence alignment in clustalx ( version 1.8 ) . where multiple trees were resolved , the kishino - hasegawa - templeton ( kht ) test [ 41 , 42 ] or shimodaira - hasegawa ( sh ) test was used to test for significant differences . the robustness of the trees was measured by the bootstrap method with 1000 replicates . to estimate the transition / transversion ratio by the ml method , different possible values for the transition / transversion ratio were evaluated in multiple runs to find the value with the maximum likelihood estimate . to estimate the among - site rate heterogeneity by the minimum evolution ( me ) method , distance matrices generated for each site were analysed and the total branch length was taken as the estimated value of the rate of change for each of the sites . genomic amplicons from five est - ssr loci , ranging in length from 181385 bp , were characterised from 12 neotyphodium and 10 epichlo isolates , as well as fatg-2 ( table 2 ) . amplification products from locus ncesta1ga07 were predominantly composed of intron sequence , based on comparison of est and genomic dna sequences . size polymorphisms between taxa for the selected loci resulted from variation at a number of indel sites ( table 2 ) . differences also occurred in the repeat unit number of the ssr array for three loci ( ncesta1fh03 , ncesta1ga07 and nlesta1nf04 ; data not shown ) , accounting for the majority of the observed size polymorphisms . a number of sequence haplotypes ( defined here as amplicons with multiple sequence variant content , but clearly related to a single common reference ) for each locus were identified across the sample set , with identity observed between those of several neotyphodium and epichlo isolates . single haplotypes for individual genes were observed for n. lolii , unclassified neotyphodium isolate 9727 , and the different epichlo species , while n. coenophialum , fatg-2 , n. uncinatum , and unclassified neotyphodium isolates 9303/2 and 9728 generated multiple haplotypes . the number of haplotypes present in these species varied between loci , but with a maximum of three for n. coenophialum and neotyphodium isolates 9303/2 and 9728 , and two for fatg-2 and n. uncinatum . pairwise comparisons identified between 2351 informative characters for the different loci ( table 2 ) . the proportion of informative characters ranged from 14% ( locus nlesta1gf09 ) to 31% ( locus nlesta1nf04 ) , but was c. 20% for the other loci . a similar proportion of informative characters occurred in both the coding and noncoding sequences of the eligible loci . loci were analysed through sequence alignment ( supplementary material : appendices 15 available at doi:10.4067/2011/921312 ) individually , rather than as a combined dataset , due to variation of both inferred ploidy level and number of observed haplotypes between different ssr loci from heteroploid isolates . the multiple trees obtained for loci ncesta1ab04 ( figure 1 ) , nlesta1gf09 ( figure 4 ) and nlesta1nf04 ( figure 5 ) only differed in the placement of one or two species . more variation was evident in the branching of the multiple trees identified for the locus ncesta1ga07 ( figure 3 ) . similar trees were resolved for the different loci using the ml , fm , and nj methods ( data not shown ) . phylogenetic analysis of the different loci revealed similar genomic relationships among endophyte species , as summarised in a network format ( figure 6 ) . close relationships between haplotypes from different taxa were deduced to indicate partial or complete commonality of genome content . some locus - dependent differences in tree topology ( figures 15 ) were observed , but specific taxa consistently grouped together . in most instances , the epichlo species were separated into two groups , separation being supported by bootstrap analysis . the single haplotypes derived from both n. lolii and e. festucae were grouped together in all trees and were identical in structure for two of the loci ( ncesta1fh03 and nlesta1gf09 ) . multiple haplotypes from n. coenophialum , fatg-2 , and n. uncinatum were consistently associated with counterparts from specific neotyphodium and epichlo species . for a subset of the target loci , n. uncinatum - derived sequences grouped with the corresponding haplotypes from e. bromicola . neotyphodium isolates 9303/2 and 9728 were closely related , all locus - specific haplotypes showing a high degree of sequence similarity . the remaining haplotype sub - class ( observed in isolate 9303/2 for four loci , and in isolate 9728 for one locus ) grouped with the equivalent haplotypes from e. typhina and e. clarkii . the application of ssr markers for phylogenetic analysis is limited by two main factors : complex molecular evolution of ssr loci and the occurrence of size homoplasy between distinct ssr alleles . sequence analysis of selected ssr loci in the current study has demonstrated that these factors influence the generic inability of ssr markers to resolve phylogenetic relationships among endophyte species . changes in the ssr array repeat number appeared to be independent of flanking region changes : some of the locus nlesta1nf04-derived haplotypes from multiple e. festucae isolate differed for ssr array number , but exhibited identity for flanking sequence , while others showed the converse relationship . ssr allele size homoplasy occurred between different endophyte taxa of distinct origins as a result of insertions , deletions , and base substitutions in both the ssr motif and flanking sequences , as observed for the locus ncesta1fh03-specific e. festucae and e. baconii - related n. coenophialum haplotypes . the results of this and other studies [ 30 , 31 ] suggest that size variation may provide a relatively accurate measure of genetic variation between closely related species . although homoplasy was not taken into account , ssrs have previously proven useful for genetic discrimination within and between endophyte species . presumably , the inherently variable nature of ssrs and the large number of loci analysed reduced the potential biasing effects of individual loci . the complex nature of ssr loci , however , demonstrates the critical value of sequence level analysis for phylogenetic inference . the flanking regions of gene - associated ssrs were highly conserved within and between endophyte taxa ( 80%100% sequence identity across coding and non - coding regions ) , supporting a common origin for these species [ 1 , 9 ] . despite this level of sequence conservation , the different individual loci obtained similar genetic relationships , consistent with previous studies of other genes . differences in the power of the individual loci to resolve relationships were identified due to variation in number of informative characters and composition ( exon , intron , coding , or non - coding ) of amplicons . in other studies , ssr - flanking sequences from different loci have been aggregated to increase the number of informative characters and improve resolution of phylogenetic relationships [ 31 , 32 , 36 , 37 ] . however , variation of inferred ploidy level and of number of haplotypes derived from different loci in heteroploids would potentially bias such aggregation studies . the close relationships between taxa were in accordance with those predicted from genes more commonly used for phylogenetic analysis such as rdna , tubb , tefa , and actg . single locus - specific haplotypes were obtained from all epichlo species and from n. lolii , the latter being closely related to e. festucae . other neotyphodium species contained multiple haplotypes that were similar to those from different epichlo species . neotyphodium isolates that could not be assigned to known morphological classes also appear to differ from characterised taxa at the molecular level . isolate 9303/2 and isolate 9728 have been assigned to taxonomic groupings heutg-2 and lptg-2 , respectively , based on phylogenetic analysis of the tefa and tubb genes ( a. leuchtmann , pers . analysis of ssr - flanking regions in this study , however , suggests that the isolates show closer affinities than formerly predicted . however , both flanking sequence analysis , as well as phenetic studies based on a larger number of ssr loci , detected a third haplotype sub - class for both isolates and suggested common affinities with e. festucae , e. bromicola , and e. typhina , respectively . accurate inference of phylogenetic relationships among neotyphodium and epichlo species consequently requires characterisation of a number of different genomic loci . although isolates 9303/2 and 9728 share common affinities , dna - based phylogenetic and phenetic analyses suggest mutual genetic divergence and placement in taxonomic groups with different relative gene content . in addition , 9303/2 and 9728 failed to cluster together in an aflp - derived phenogram , which represents a genome - wide assessment of genetic polymorphism . e. festucae - related haplotypes were detected in both isolates for all five loci , while e. bromicola - like and e. typhina - like sequence variants were observed more frequently in isolate 9303/2 than isolate 9728 and were not represented between all loci . neotyphodium species with common phylogenetic affinities are known to occur in several different grass species . differences in transcript levels associated with different gene - specific sequence variants , as observed for the 60s ribosomal protein - encoding gene in this study , may also contribute to phenotypic trait variation between different heteroploid endophyte taxa . phylogenetic analysis of the third unclassified neotyphodium isolate ( 9727 ) , which was also derived from f. arizonica , suggests that it may belong to the former taxon . isolate 9727 produced single haplotypes and these sequences , like those of the n. huerfanum tefa and tubb loci , are closely related to the inferred e. typhina - related haplotypes from n. coenophialum and n. uncinatum ( section 4.2 ) . these results were also supported by ssr polymorphism - based phenetic analysis , in which 9727 clustered with e. typhina , e. clarkii , and e. sylvatica , while in aflp analysis the isolate clustered with n. uncinatum . due to their close phylogenetic relationships with specific epichlo species , neotyphodium species have been proposed to have originated from these sexual endophyte taxa either directly through the loss of the sexual state , or through interspecific hybridisation of distinct epichlo and neotyphodium species . the first process is proposed to have given rise to haploid neotyphodium species such as n. lolii , while the heteroploid neotyphodium species such as n. coenophialum , fatg-2 , and n. uncinatum may have arisen through the second evolutionary process . because epichlo species form unique mating populations [ 4951 ] and neotyphodium species are not known to sporulate in vivo , this second mode of evolution is thought to have been a parasexual process involving somatic fusion of endophyte hyphae . this hypothesis does , however , require physical colocation between endophyte taxa that generally occur in distinct host species . in addition , mechanisms of gene loss following nuclear fusion are necessary to account for the observed genomic composition of contemporary heteroploid taxa , as a range of studies [ 2 , 10 , 11 , 13 , 14 , 16 , 17 ] have shown that extant neotyphodium species do not appear to have the full complement of genes present in phylogenetically related epichlo species . it is also formally possible that sexual epichlo species may have arisen from asexual neotyphodium species in response to selective environmental pressures , a mechanism requiring both gene loss and gene gain , possibly through horizontal gene transfer . mechanisms for both processes have been inferred through comparisons of different fungal taxa at the whole genome levels and may have been facilitated by structural features such as presence of conserved repetitive elements . in conclusion , this study demonstrates the application of ssr - flanking sequences to studies of genome affinities between pasture grass fungal endophyte species for clarification of novel modes of genome evolution . the inferred affinities were consistent with those obtained from gene loci that are more commonly used in molecular phylogenetics , but provided a more extensive survey of genomic loci , that may be ultimately extended to whole genome comparisons based.on second - generation sequencing technologies .

systemic lupus erythematosus ( sle ) is an autoimmune disease of unknown origin characterized by inflammation of multiple organs ( kidneys , brain , heart , liver , lungs , joints , muscles , skin , etc.).1 the kidney has usually been considered the most frequently affected organ in systemic lupus erythematosus , the heart and the pulmonary vessels , however , may also be seriously involved.2 pulmonary involvement is relatively frequent in adult patients rather than children.3 pulmonary hypertension is the most severe forms of lupus associated pulmonary involvement , although they occur infrequently in children with sle.4 although several mechanisms are involved in pathogenesis of pulmonary hypertension in sle , the real causes are yet unknown . the hypothesis of pulmonary vasculitis , with deposits of immunocomplexes and complements on the pulmonary artery walls , thromboembolic blockage in pulmonary vessels , possibly related to antibodies ( anticardiolipin antibody and lupus anticoagulant ) , and vasospasms , are suggested by a greater frequency of raynaud s phenomenon in these patients.5 the prevalence of pulmonary arterial hypertension ( pah ) in patients with lupus is largely unknown , but has been reported to approximate 6%15% in adult patients , in whom it is most commonly associated with raynaud s phenomenon.6 there have been very few studies addressing the prevalence of pah in childhood - onset sle , which is reported to approximate 4%8% using transthoracic echocardiography.7 the determination of the real prevalence and magnitude of pulmonary hypertension in sle has been difficult over the years due to the fact that the only method with sufficient sensitivity for determining pulmonary pressures and confirming the existence of pulmonary hypertension has been the catheterization of the right chambers with direct measurement of pulmonary pressures . the advent of two - dimensional doppler echocardiography , a method thought to be of similar sensitivity as right chamber catheterization for measuring pulmonary pressure , allowed the study of a greater number of patients , with or without symptoms.5 pulmonary hypertension is often silent , with the development of clinical symptoms often signaling the presence of advanced disease , associated with poor survival . this has prompted the need for methods of detecting pah , including annual echocardiographic screening of asymptomatic individuals with systemic autoimmunity.8 the sensitivity and specificity of doppler echocardiography ( de ) for estimating pulmonary artery pressure are 0.79 to 1.0 and 0.68 to 0.98 , respectively.9 de can evaluate the degree of ph , prognosticating variables ( ra and rv enlargement , pericardial effusion , rv ejection fraction ) , and other potential causes of ph ( left ventricular systolic / diastolic dysfunction , valvular heart disease , and intracardiac shunting).10 the aim of this study was to screen for asymptomatic pulmonary hypertension in systemic lupus erythematosus using doppler echocardiography , and to correlate it with inflammatory parameters of the disease . this is a cross - sectional study conducted on sle patients attending outpatient clinic of rheumatology and rehabilitation department at minia university hospital , egypt , over one year . all patients were females and met the 1982 american college of rheumatology revised criteria for sle11 with no overlap syndrome . seventy four female sle patients were recruited , 66 adults ( aged from 19 to 45 years ) , and 8 children ( aged from 11 to 15 years ) . all the patients were subjected to a thorough history taking , general examination , and rheumatological evaluation which included the assessment of sle disease activity index ( sledai).12 esr was measured using westergren method and rheumatoid factor was performed using latex fixation test . antinuclear antibody ( ana ) , anti - double stranded dna antibody ( anti - dsdna ) , and igg anti - cardiolipin antibodies ( acl antibodies ) were assayed by immuno - fluorescence technique and elisa . other routine laboratory tests as complete blood picture , renal function test , urinalysis ( for pyuria , hematuria , casts ) , and 24-hour urine for protein were done for all patients . a - p and lateral views were done for all patients to exclude evidence of parenchymal lung disease . two - dimensional ( 2d ) , m - mode , color flow mapping and spectral doppler echocardiography were done using a vivid 3 expert ge echocardiography machine ( ge vingmed ultrasound , horten , norway , model vivid 3 expert ) , with a 5 mhz transducer for children patients . patients were examined in the parasternal long and short axis , and four chamber projections . to estimate pulmonary artery systolic pressure ( pasp ) , the maximum transtricuspid pressure gradient was calculated using the simplified bernoulli equation.13,14 the estimate of right atrial pressure , 10 mmhg , was added to the pressure gradient to calculate the right ventricular systolic pressure , which was considered equal to the pasp in the absence of right ventricular outflow obstruction . the doppler recordings were reviewed by at least one echocardiographer who had no previous knowledge of the patient . right ventricular dilatation was defined as the ventricular area greater than the left ventricular area obtained by planimetry from the apical four - chamber view . right ventricular dilatation with flattening of the ventricular septum was considered indicative of right ventricular overload . pulmonary hypertension was diagnosed if the peak systolic pressure gradient at the tricuspid valve was more than 30 mmhg . the severity of ph is classified as : mild ( pasp < 45 mmhg ) , moderate ( pasp from 45 to 59 mmhg ) or severe ( pasp > 60 mmhg).13 accordingly , patients enrolled in our study were divided into two groups : group i : patients with pah.group ii : patients without pah . qualitative variables , such as symptoms , age , sex were represented by numbers and percentages . student s t - test ( t ) was used when comparing the means of quantitative variables . chi - squared test ( x ) was used to compare frequencies ( numbers and percentages ) for qualitative variables . odds ratios ( or ) and 95% confidence intervals ( 95% cis ) were calculated . this is a cross - sectional study conducted on sle patients attending outpatient clinic of rheumatology and rehabilitation department at minia university hospital , egypt , over one year . all patients were females and met the 1982 american college of rheumatology revised criteria for sle11 with no overlap syndrome . seventy four female sle patients were recruited , 66 adults ( aged from 19 to 45 years ) , and 8 children ( aged from 11 to 15 years ) . all the patients were subjected to a thorough history taking , general examination , and rheumatological evaluation which included the assessment of sle disease activity index ( sledai).12 esr was measured using westergren method and rheumatoid factor was performed using latex fixation test . antinuclear antibody ( ana ) , anti - double stranded dna antibody ( anti - dsdna ) , and igg anti - cardiolipin antibodies ( acl antibodies ) were assayed by immuno - fluorescence technique and elisa . other routine laboratory tests as complete blood picture , renal function test , urinalysis ( for pyuria , hematuria , casts ) , and 24-hour urine for protein were done for all patients . a - p and lateral views were done for all patients to exclude evidence of parenchymal lung disease . two - dimensional ( 2d ) , m - mode , color flow mapping and spectral doppler echocardiography were done using a vivid 3 expert ge echocardiography machine ( ge vingmed ultrasound , horten , norway , model vivid 3 expert ) , with a 5 mhz transducer for children patients . patients were examined in the parasternal long and short axis , and four chamber projections . special attention was given to the detection of tricuspid regurgitation . to estimate pulmonary artery systolic pressure ( pasp ) , the maximum transtricuspid pressure gradient was calculated using the simplified bernoulli equation.13,14 the estimate of right atrial pressure , 10 mmhg , was added to the pressure gradient to calculate the right ventricular systolic pressure , which was considered equal to the pasp in the absence of right ventricular outflow obstruction . the doppler recordings were reviewed by at least one echocardiographer who had no previous knowledge of the patient . right ventricular dilatation was defined as the ventricular area greater than the left ventricular area obtained by planimetry from the apical four - chamber view . right ventricular dilatation with flattening of the ventricular septum was considered indicative of right ventricular overload . pulmonary hypertension was diagnosed if the peak systolic pressure gradient at the tricuspid valve was more than 30 mmhg . the severity of ph is classified as : mild ( pasp < 45 mmhg ) , moderate ( pasp from 45 to 59 mmhg ) or severe ( pasp > 60 mmhg).13 accordingly , patients enrolled in our study were divided into two groups : group i : patients with pah.group ii : patients without pah . qualitative variables , such as symptoms , age , sex were represented by numbers and percentages . student s t - test ( t ) was used when comparing the means of quantitative variables . chi - squared test ( x ) was used to compare frequencies ( numbers and percentages ) for qualitative variables . odds ratios ( or ) and 95% confidence intervals ( 95% cis ) were calculated . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . linear regression ( stepwise method ) was performed to evaluate all clinical and laboratory parameters . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . linear regression ( stepwise method ) was performed to evaluate all clinical and laboratory parameters . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . linear regression ( stepwise method ) was performed to evaluate all clinical and laboratory parameters . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . part of these patients represents variable degrees of pulmonary hypertension.15 pulmonary hypertension in lupus patients has been described since 1973.1618 the prevalence rate of pah in sle patients ranges from 0.5% to 14%.19,20 in a serial study of 28 patients with sle , the prevalence of ph measured by echocardiogram increased from 14% to 43% with 5 years of follow - up.21 prabu et al22 provides important data on the prevalence of pah in a large cohort of sle patients . the prevalence of pah was found to be 4.2% , which was based on echocardiographic screening . moreover , only 3 of the 12 patients had severe disease [ systolic pulmonary arterial pressure ( pap ) > 40 mmhg ] . these results are of particular interest , because the cohort , in contrast to other reports , has a community , non - tertiary background and seems not biased by the patient selection , and therefore might very well be considered as representative of the general sle population . yeh et al7 reported the prevalence of pah in juvenile sle patients which range from 4% to 8% using transthoracic echocardiography . yet , there are no studies using doppler echocardiography as a screening tool for detection of asymptomatic pulmonary hypertension in juvenile sle patients . echocardiography provides both estimates of pulmonary artery pressure and an assessment of cardiac structure and function . these features justify its application as the most commonly used screening tool in patients with suspected pah.9 in the present study , doppler echocardiography was used as a screening tool for detection of asymptomatic pulmonary hypertension in sle patients . pah was found in 8 of 74 sle female patients ( 10.8% ) , they were 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . asherson et al23 have reported that , patients with sle - ph are predominantly women of child - bearing potential aged from 18 to 40 years with a 10 to 1 ratio of female over male . fos et al24 reported that systolic pap > 35 mmhg in 12 ( 13% ) out of 93 sle patients , 10 women and 2 men . in the present study , raynaud s phenomenon was found in 5 adult - onset sle patients with pulmonary hypertension ( 62.5% ) . in agreement with our results haas20 reported that up to 58% of 105 patients ( most of whom were female ) with both ph and sle had raynaud s phenomenon . asherson et al25 found raynaud s phenomenon in 50% of adult sle raising the possibility of vasospasm as an aetiological factor . however asherson et al23 reported that 75% of patients with sle - ph have raynaud s phenomenon , which is higher than the expected rate of 25% among all patients with sle . the striking correlation between the occurrence of raynaud s phenomenon and sle - ph suggests that pulmonary arterial vasospasm may also be involved in the pathogenesis of sle - ph . raynaud s phenomenon is part of a systemic vascular response that includes a decrease in size of the pulmonary capillary bed , which may in turn result in muscular necrosis and secondary inflammation.26 no signs of vasculitis were found in any case in the present study . however , asherson et al23 have reported that one - third of patients with pulmonary hypertension in their study had evidence of peripheral cutaneous vasculitis , livedo reticularis , and digital gangrene . in the present study , sle patients with pah showed statistically significant higher number of cases with positive rheumatoid factor ( rf ) ( 50% ) and positive anticardiolipin antibodies ( 87.5% ) in comparison to sle patients without pah ( p = 0.02 , p = 0.008 respectively ) . in agreement with our results asherson et al and wilson et al reported that patients with sle - ph are universally positive for ana . antibodies to ribonuclear protein ( rnp ) and rheumatoid factor ( rf ) are often present in sle - ph , although no pathogenic role has been postulated . the prevalence of rf has been reported to be as high as 50% to 80% in sle - ph . the frequency of ph in patients with sle and positive antiphospholipid antibodies is considerably higher than in patients with sle and negative antiphospholipid antibodies ( 83% versus 25%).23,27 lian et al28 have found positive rheumatoid factor in 46.3% and positive anticardiolipin antibodies in 53.7% of patients with sle - ph . however , fos et al24 have reported a very high frequency of anticardiolipin antibodies ( 75% ) in their patients and they suggested that these autoantibodies might facilitate the formation of microthrombi in the pulmonary vasculature and thereby contribute to spap elevation , but the relationship between them and ph in sle remains controversial . in our sle patients with pah , doppler echocardiography detected elevated pulmonary artery systolic pressure in 8 patients ( 10.8% ) , being mild in 5 , moderate in 2 , and severe in one patient , right ventricular dilatation in 1 patient , ( 12.5% ) , valvular thickening in 3 patients , ( 37.5% ) , tricuspid regurge ( mild in 1 , moderate in 5 , and severe in 2 patients ) , and even mild pericardial effusion in 2 patients , ( 25% ) before symptom onset . lian et al28 reported that , raynaud s phenomenon , anticardiolipin antibodies , and anti - u1rnp were associated with increased odds ratio ( or = 3.204 , or = 3.753 , or = 5.393 respectively ) , and were considered as significant independent predictors of pah in sle ( p = 0.01 , p = 0.004 , p = 0.001 respectively ) . but in the current study , raynaud s phenomenon , rheumatoid factor , and anti - cardiolipin antibodies were associated with increased risk for pah ( or = 4.44 , or = 7.25 , or = 12.25 respectively ) . while only positive rheumatoid factor and positive acl were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) in the linear regression analysis . study findings by robbins et al29 indicate that the duration of sle does not correlate with the development of pah , although many patients with sle develop pah within the first 5 years . these findings coincided with our results , as the duration of sle was not correlated with pah and sle patients with pah had shorter disease duration ( 0.36 yr ) in comparison to sle patients without pah ( 0.116 yr ) , but the difference not statistically significant . sle disease related risk factors , the disease activity score ( sledai ) , esr , anti - ds dna and renal affection in sle patients enrolled in the present study did not differ significantly between patients with pah and those without , and they were nt correlated with pah . in agreement with our results , asherson et al23 and robbins et al29 reported that ph was unrelated to the severity or activity of sle such as high anti ds - dna and/or grossly elevated esr , and can occur when non - pulmonary disease activity is quiescent . in our sle patients , doppler echocardiography could detect elevated pulmonary artery systolic pressure in 10.8% , degree of pulmonary hypertension , right ventricular dilatation , valvular thickening , tricuspid regurge , and even mild pericardial effusion before symptom onset . sle patients with pah showed significant higher number of cases with positive rheumatoid factor ( 50% ) and positive anticardiolipin antibodies ( 87.5% ) in comparison to sle patients without pah . echocardiography should be performed routinely once a year ( as a screening tool ) and consideration should be given to screening sle patients with positive anti - cardiolipin antibodies , and rheumatoid factor ; as they were significant predictors of pulmonary hypertension in sle . further study is required to determine if patients with asymptomatic pulmonary hypertension will benefit from therapy in terms of morbidity and mortality .

introduction : pulmonary arterial hypertension ( pah ) is a serious and often fatal complication of systemic lupus erythematosus ( sle ) . because the diagnosis of pah often is made years after symptom onset , early diagnostic strategies are essential . doppler echocardiography currently is considered the noninvasive screening test of choice for evaluating pulmonary hypertension.aim:screening for asymptomatic pulmonary hypertension in systemic lupus erythematosus patients using doppler echocardiography , and correlating it with inflammatory parameters of the disease.patients and methods : doppler echocardiography was performed in 74 patients with systemic lupus erythematosus over one year ( 66 adult and 8 juvenile ) , adult sle included 57 patients with adult - onset and 9 patients with childhood - onset . pulmonary hypertension was diagnosed if the peak systolic pressure gradient at the tricuspid valve was more than 30 mmhg . all patients were subjected to full history taking , rheumatological examination , laboratory studies and chest x-ray.results:in seventy four sle patients , the pulmonary hypertension was detected in 8 patients ( 10.8% ) , 7 adult - onset sle patients ( aged from 19 to 30 years ) and 1 juvenile sle ( aged 12 years ) . the range of pulmonary artery systolic pressure was 3461.2 mmhg ( 43.19 9.28 ) . no significant differences between patients with and those without pulmonary hypertension as regard clinical features . significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies were found in patients with pulmonary hypertension versus those without ( p = 0.02 , p = 0.008 respectively ) . positive rheumatoid factor and acl were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle disease activity index ( sledai ) , esr , and anti - ds dna.conclusion:patients with sle have an increased risk of pulmonary arterial hypertension . echocardiography should be used as a screening tool in patients at high risk for development of pulmonary hypertension . positive anti - cardiolipin antibodies and rheumatoid factor were significant predictors of pulmonary hypertension in our study .

Introduction Patients and Methods Study population Clinical evaluation Laboratory studies Chest x-ray Echocardiographic evaluation Statistical analysis Results Demographic data of all SLE patients are shown in ( Comparison of patients with and without pulmonary arterial hypertension Discussion Conclusion

systemic lupus erythematosus ( sle ) is an autoimmune disease of unknown origin characterized by inflammation of multiple organs ( kidneys , brain , heart , liver , lungs , joints , muscles , skin , etc. ).1 the kidney has usually been considered the most frequently affected organ in systemic lupus erythematosus , the heart and the pulmonary vessels , however , may also be seriously involved.2 pulmonary involvement is relatively frequent in adult patients rather than children.3 pulmonary hypertension is the most severe forms of lupus associated pulmonary involvement , although they occur infrequently in children with sle.4 although several mechanisms are involved in pathogenesis of pulmonary hypertension in sle , the real causes are yet unknown . the hypothesis of pulmonary vasculitis , with deposits of immunocomplexes and complements on the pulmonary artery walls , thromboembolic blockage in pulmonary vessels , possibly related to antibodies ( anticardiolipin antibody and lupus anticoagulant ) , and vasospasms , are suggested by a greater frequency of raynaud s phenomenon in these patients.5 the prevalence of pulmonary arterial hypertension ( pah ) in patients with lupus is largely unknown , but has been reported to approximate 6%15% in adult patients , in whom it is most commonly associated with raynaud s phenomenon.6 there have been very few studies addressing the prevalence of pah in childhood - onset sle , which is reported to approximate 4%8% using transthoracic echocardiography.7 the determination of the real prevalence and magnitude of pulmonary hypertension in sle has been difficult over the years due to the fact that the only method with sufficient sensitivity for determining pulmonary pressures and confirming the existence of pulmonary hypertension has been the catheterization of the right chambers with direct measurement of pulmonary pressures . the advent of two - dimensional doppler echocardiography , a method thought to be of similar sensitivity as right chamber catheterization for measuring pulmonary pressure , allowed the study of a greater number of patients , with or without symptoms.5 pulmonary hypertension is often silent , with the development of clinical symptoms often signaling the presence of advanced disease , associated with poor survival . this has prompted the need for methods of detecting pah , including annual echocardiographic screening of asymptomatic individuals with systemic autoimmunity.8 the sensitivity and specificity of doppler echocardiography ( de ) for estimating pulmonary artery pressure are 0.79 to 1.0 and 0.68 to 0.98 , respectively.9 de can evaluate the degree of ph , prognosticating variables ( ra and rv enlargement , pericardial effusion , rv ejection fraction ) , and other potential causes of ph ( left ventricular systolic / diastolic dysfunction , valvular heart disease , and intracardiac shunting).10 the aim of this study was to screen for asymptomatic pulmonary hypertension in systemic lupus erythematosus using doppler echocardiography , and to correlate it with inflammatory parameters of the disease . this is a cross - sectional study conducted on sle patients attending outpatient clinic of rheumatology and rehabilitation department at minia university hospital , egypt , over one year . seventy four female sle patients were recruited , 66 adults ( aged from 19 to 45 years ) , and 8 children ( aged from 11 to 15 years ) . all the patients were subjected to a thorough history taking , general examination , and rheumatological evaluation which included the assessment of sle disease activity index ( sledai).12 esr was measured using westergren method and rheumatoid factor was performed using latex fixation test . antinuclear antibody ( ana ) , anti - double stranded dna antibody ( anti - dsdna ) , and igg anti - cardiolipin antibodies ( acl antibodies ) were assayed by immuno - fluorescence technique and elisa . other routine laboratory tests as complete blood picture , renal function test , urinalysis ( for pyuria , hematuria , casts ) , and 24-hour urine for protein were done for all patients . to estimate pulmonary artery systolic pressure ( pasp ) , the maximum transtricuspid pressure gradient was calculated using the simplified bernoulli equation.13,14 the estimate of right atrial pressure , 10 mmhg , was added to the pressure gradient to calculate the right ventricular systolic pressure , which was considered equal to the pasp in the absence of right ventricular outflow obstruction . pulmonary hypertension was diagnosed if the peak systolic pressure gradient at the tricuspid valve was more than 30 mmhg . the severity of ph is classified as : mild ( pasp < 45 mmhg ) , moderate ( pasp from 45 to 59 mmhg ) or severe ( pasp > 60 mmhg).13 accordingly , patients enrolled in our study were divided into two groups : group i : patients with pah.group ii : patients without pah . this is a cross - sectional study conducted on sle patients attending outpatient clinic of rheumatology and rehabilitation department at minia university hospital , egypt , over one year . seventy four female sle patients were recruited , 66 adults ( aged from 19 to 45 years ) , and 8 children ( aged from 11 to 15 years ) . all the patients were subjected to a thorough history taking , general examination , and rheumatological evaluation which included the assessment of sle disease activity index ( sledai).12 esr was measured using westergren method and rheumatoid factor was performed using latex fixation test . antinuclear antibody ( ana ) , anti - double stranded dna antibody ( anti - dsdna ) , and igg anti - cardiolipin antibodies ( acl antibodies ) were assayed by immuno - fluorescence technique and elisa . other routine laboratory tests as complete blood picture , renal function test , urinalysis ( for pyuria , hematuria , casts ) , and 24-hour urine for protein were done for all patients . to estimate pulmonary artery systolic pressure ( pasp ) , the maximum transtricuspid pressure gradient was calculated using the simplified bernoulli equation.13,14 the estimate of right atrial pressure , 10 mmhg , was added to the pressure gradient to calculate the right ventricular systolic pressure , which was considered equal to the pasp in the absence of right ventricular outflow obstruction . pulmonary hypertension was diagnosed if the peak systolic pressure gradient at the tricuspid valve was more than 30 mmhg . the severity of ph is classified as : mild ( pasp < 45 mmhg ) , moderate ( pasp from 45 to 59 mmhg ) or severe ( pasp > 60 mmhg).13 accordingly , patients enrolled in our study were divided into two groups : group i : patients with pah.group ii : patients without pah . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . according to doppler echocardiographic findings , pah was detected in 8 patients ( 10.8% ) out of seventy four sle patients . sle patients were classified into two groups : group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years).group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . group i : included 8 patients who had pah : 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . group ii : included : 66 patients who had no pah ( 50 adult - onset sle , 9 childhood - onset sle and 7 juvenile sle ) . comparison of patients with and without pah to find if there s any association of pah with inflammatory , sle - related or other risk factors for pulmonary arterial hypertension had showed that , no significant differences between the two groups regarding , demographic and clinical features ( table 2 ) , while laboratory parameters had significantly higher frequencies of rheumatoid factor and anti - cardiolipin antibodies in patients with pah ( p = 0.02 , p = 0.008 respectively ) as in ( table 3 ) . no signs of vasculitis , anti - phospholipid syndrome and/or lung affection were found in patients with pah . pulmonary artery pressure was differed significantly between the two groups subset ( p < 0.001 ) . the degree of pulmonary arterial hypertension was mild in 5 , moderate in 2 , and severe in one patient as shown in ( table 4 ) . different degrees of pulmonary hypertension as found in our patients are represented in ( fig . odds ratio of possible risk factors for development of pah in sle patients are shown in ( table 5 ) . among all factors , both rheumatoid factor and acl positivity were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) ( table 5 ) . no significant correlations were found between pulmonary artery pressure , disease duration , sle - dai , esr , and anti - ds dna . table 6 summarizes clinical data , laboratory features and pasp in sle patients with pah . part of these patients represents variable degrees of pulmonary hypertension.15 pulmonary hypertension in lupus patients has been described since 1973.1618 the prevalence rate of pah in sle patients ranges from 0.5% to 14%.19,20 in a serial study of 28 patients with sle , the prevalence of ph measured by echocardiogram increased from 14% to 43% with 5 years of follow - up.21 prabu et al22 provides important data on the prevalence of pah in a large cohort of sle patients . yeh et al7 reported the prevalence of pah in juvenile sle patients which range from 4% to 8% using transthoracic echocardiography . yet , there are no studies using doppler echocardiography as a screening tool for detection of asymptomatic pulmonary hypertension in juvenile sle patients . these features justify its application as the most commonly used screening tool in patients with suspected pah.9 in the present study , doppler echocardiography was used as a screening tool for detection of asymptomatic pulmonary hypertension in sle patients . pah was found in 8 of 74 sle female patients ( 10.8% ) , they were 7 adult - onset sle ( aged from 19 to 30 yr ) and 1 juvenile sle ( aged 12 years ) . in the present study , raynaud s phenomenon was found in 5 adult - onset sle patients with pulmonary hypertension ( 62.5% ) . raynaud s phenomenon is part of a systemic vascular response that includes a decrease in size of the pulmonary capillary bed , which may in turn result in muscular necrosis and secondary inflammation.26 no signs of vasculitis were found in any case in the present study . however , asherson et al23 have reported that one - third of patients with pulmonary hypertension in their study had evidence of peripheral cutaneous vasculitis , livedo reticularis , and digital gangrene . in the present study , sle patients with pah showed statistically significant higher number of cases with positive rheumatoid factor ( rf ) ( 50% ) and positive anticardiolipin antibodies ( 87.5% ) in comparison to sle patients without pah ( p = 0.02 , p = 0.008 respectively ) . antibodies to ribonuclear protein ( rnp ) and rheumatoid factor ( rf ) are often present in sle - ph , although no pathogenic role has been postulated . the frequency of ph in patients with sle and positive antiphospholipid antibodies is considerably higher than in patients with sle and negative antiphospholipid antibodies ( 83% versus 25%).23,27 lian et al28 have found positive rheumatoid factor in 46.3% and positive anticardiolipin antibodies in 53.7% of patients with sle - ph . in our sle patients with pah , doppler echocardiography detected elevated pulmonary artery systolic pressure in 8 patients ( 10.8% ) , being mild in 5 , moderate in 2 , and severe in one patient , right ventricular dilatation in 1 patient , ( 12.5% ) , valvular thickening in 3 patients , ( 37.5% ) , tricuspid regurge ( mild in 1 , moderate in 5 , and severe in 2 patients ) , and even mild pericardial effusion in 2 patients , ( 25% ) before symptom onset . lian et al28 reported that , raynaud s phenomenon , anticardiolipin antibodies , and anti - u1rnp were associated with increased odds ratio ( or = 3.204 , or = 3.753 , or = 5.393 respectively ) , and were considered as significant independent predictors of pah in sle ( p = 0.01 , p = 0.004 , p = 0.001 respectively ) . but in the current study , raynaud s phenomenon , rheumatoid factor , and anti - cardiolipin antibodies were associated with increased risk for pah ( or = 4.44 , or = 7.25 , or = 12.25 respectively ) . while only positive rheumatoid factor and positive acl were significantly associated with occurrence of pah in sle ( p = 0.007 , p = 0.006 respectively ) in the linear regression analysis . study findings by robbins et al29 indicate that the duration of sle does not correlate with the development of pah , although many patients with sle develop pah within the first 5 years . these findings coincided with our results , as the duration of sle was not correlated with pah and sle patients with pah had shorter disease duration ( 0.36 yr ) in comparison to sle patients without pah ( 0.116 yr ) , but the difference not statistically significant . sle disease related risk factors , the disease activity score ( sledai ) , esr , anti - ds dna and renal affection in sle patients enrolled in the present study did not differ significantly between patients with pah and those without , and they were nt correlated with pah . in our sle patients , doppler echocardiography could detect elevated pulmonary artery systolic pressure in 10.8% , degree of pulmonary hypertension , right ventricular dilatation , valvular thickening , tricuspid regurge , and even mild pericardial effusion before symptom onset . sle patients with pah showed significant higher number of cases with positive rheumatoid factor ( 50% ) and positive anticardiolipin antibodies ( 87.5% ) in comparison to sle patients without pah . echocardiography should be performed routinely once a year ( as a screening tool ) and consideration should be given to screening sle patients with positive anti - cardiolipin antibodies , and rheumatoid factor ; as they were significant predictors of pulmonary hypertension in sle .

the design and the rationale for the addition - europe study have previously been reported ( 17,18 ) . in brief , the danish arm of the addition - europe study ( addition - denmark ) consisted of two phases : 1 ) a screening phase and 2 ) a pragmatic cluster - randomized parallel - group trial . in five danish regions , 744 general practices were invited to participate . of these , 190 agreed to participate and were allocated to screening and randomized to provide either routine care or intensive multifactorial treatment of patients with diabetes detected by screening . the screening phase took place in 20012006 and consisted of a population - based stepwise screening program to identify individuals with screen - detected diabetes . it was conducted among the participating general practitioners patients aged 4069 years without known diabetes . the recruitment and selection methods have previously been described in detail ( 19,20 ) . in total , 1,533 individuals were diagnosed with diabetes according to the 1999 world health organization criteria ( 21 ) and were enrolled in the trial . in 2009 , a follow - up examination was performed . in two of five study centers ( holstebro hospital and steno diabetes center ) , measurements of central hemodynamics were carried out and successfully obtained in 456 of 486 attending patients . these patients constitute our study sample . of the 456 patients , 420 had measurements of both apwv and central blood pressure , 9 had measurements of apwv only , and 27 had measurements of central blood pressure only ( supplementary fig . the reasons for missing measurements were as follows : station closure owing to insufficient staff ( n = 27 ) , irregular pulse ( apwv , n = 4 ; central blood pressure and augmentation index , n = 12 ) , and for apwv , that the carotid or femoral artery pulse could not be found ( n = 26 ) . the study was approved by the local ethics committee ( region midt , denmark ) and was conducted in accordance with the 1996 helsinki declaration . written informed consent was obtained from each patient at baseline and at follow - up . the specific characteristics of the interventions to promote intensive treatment have previously been described in detail ( 17 ) . we aimed to educate and support general practitioners and practice nurses in target - driven management ( using medication and promotion of healthy lifestyle ) of hyperglycemia , hypertension , and hypercholesterolemia based on the stepwise regimen used in the steno-2 study ( 22,23 ) . practice personnel were provided with educational material for patients , and patients were sent reminders if annual check - up appointments were overdue . practices received additional funding to support the delivery of extra care added to the usual care and consultations . although targets for treatment were specified and classes of medication recommended , decisions on prescriptions , including choice of individual drugs , were made by general practitioners and patients in cooperation . the general practitioners in the routine care group were provided with diagnostic test results only ; their patients with screen - detected diabetes received standard care of diabetes according to the danish national guidelines ( 24 ) . in this secondary analysis , the outcomes were central and peripheral hemodynamics : apwv , central systolic and diastolic blood pressure , pulse pressure , augmented pressure , augmentation index , brachial systolic and diastolic blood pressure , and brachial pulse pressure . the staff obtaining the outcome measures was blinded for randomization group . with the patient in a supine position , brachial blood pressure was measured after 10 min of rest ( omron m6 comfort ; omron healthcare , milton keynes , u.k . ) . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . the tonometer was used to capture the wave forms at the carotid and subsequently at the femoral artery simultaneously with an electrocardiogram recording using the intersecting tangent ( 27 ) . the distance from the suprasternal notch to the carotid artery was measured with a tape measure and from the suprasternal notch to the femoral artery with an anthropometer ( seca ; medical scales and measuring systems , hamburg , germany ) to avoid overestimation of the distance and subsequently the velocity in obese individuals . the path length was determined by subtracting the carotid - sternal notch distance from the femoral - sternal notch distance . in each patient , apwv was measured twice . if the difference of apwv between the two measurements was larger than 0.5 m / s , a third measurement was taken . in the statistical analysis , the average of the two closest measurements in each patient was used . with the patient in a supine position , peripheral pressure waveforms were recorded at the radial artery using applanation tonometry ( version 8 , sphygmocor system ; atcor medical ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central pulse pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial systolic and diastolic blood pressures were measured three times after a 10-min rest with an automated oscillometric blood pressure recorder ( omron m6 comfort ) with the patient in a sitting position . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . health assessments at baseline and at follow - up were performed at the danish addition study centers by trained staff blinded for randomization group following standard operating procedures ( 6 ) . briefly , height , weight , waist circumference , and brachial blood pressure were measured . in venous blood samples , hba1c , serum total urinary albumin and urinary creatinine were measured on spot urine . at baseline and follow - up , self - report questionnaires were used to collect information on ethnicity , smoking status , alcohol consumption , medication , and cvd ( previous myocardial infarction and stroke ) . furthermore , at follow - up we collected information on self - reported episodes with angina pectoris or arrhythmia and self - reported cardiovascular revascularization . patient characteristics at baseline and at follow - up are presented as means ( sd ) or , in cases of skewed distributions , as median ( interquartile range [ iqr ] ) . the effect of intensive multifactorial treatment on central hemodynamics was analyzed by mixed - effects models with adjustment for clustering ( randomization by general practice ) and heart rate at time of measurement , taking its direct functional effect on the hemodynamic markers into account . the analysis of apwv was also adjusted for mean blood pressure at time of measurement . in addition to regression coefficients , standardized regression coefficients ( sd change in the outcome ) are presented . aortic stiffness is associated with the intrasubject variation of the central hemodynamic measurements ( the higher the level of stiffness , the larger the intrasubject variation ) . to assure a full spectrum of aortic stiffness , , we subsequently performed a subanalysis , excluding measurements with large intrasubject variation according to the manufacturer s guidelines ( 30,31 ) . in the subanalysis of apwv , measurements with a ratio > 0.2 between apwv sem and apwv were excluded . for the measurements of central blood pressure and augmentation index , the software provides an operator index based on the height of the pulse waves , variations in the height of the pulse waves , diastolic variation , and deviation in the shape of the pulse waves . in the subanalysis of central blood pressure and augmentation index , measurements with an operator index < 75 were excluded . analyses were performed using sas version 9.2 ( sas institute , cary , nc ) . the screening phase took place in 20012006 and consisted of a population - based stepwise screening program to identify individuals with screen - detected diabetes . it was conducted among the participating general practitioners patients aged 4069 years without known diabetes . the recruitment and selection methods have previously been described in detail ( 19,20 ) . in total , 1,533 individuals were diagnosed with diabetes according to the 1999 world health organization criteria ( 21 ) and were enrolled in the trial . in 2009 , a follow - up examination was performed . in two of five study centers ( holstebro hospital and steno diabetes center ) , measurements of central hemodynamics were carried out and successfully obtained in 456 of 486 attending patients . 420 had measurements of both apwv and central blood pressure , 9 had measurements of apwv only , and 27 had measurements of central blood pressure only ( supplementary fig . 1 ) . the reasons for missing measurements were as follows : station closure owing to insufficient staff ( n = 27 ) , irregular pulse ( apwv , n = 4 ; central blood pressure and augmentation index , n = 12 ) , and for apwv , that the carotid or femoral artery pulse could not be found ( n = 26 ) . the study was approved by the local ethics committee ( region midt , denmark ) and was conducted in accordance with the 1996 helsinki declaration . written informed consent was obtained from each patient at baseline and at follow - up . the specific characteristics of the interventions to promote intensive treatment have previously been described in detail ( 17 ) . we aimed to educate and support general practitioners and practice nurses in target - driven management ( using medication and promotion of healthy lifestyle ) of hyperglycemia , hypertension , and hypercholesterolemia based on the stepwise regimen used in the steno-2 study ( 22,23 ) . practice personnel were provided with educational material for patients , and patients were sent reminders if annual check - up appointments were overdue . practices received additional funding to support the delivery of extra care added to the usual care and consultations . although targets for treatment were specified and classes of medication recommended , decisions on prescriptions , including choice of individual drugs , were made by general practitioners and patients in cooperation . the general practitioners in the routine care group were provided with diagnostic test results only ; their patients with screen - detected diabetes received standard care of diabetes according to the danish national guidelines ( 24 ) . in this secondary analysis , the outcomes were central and peripheral hemodynamics : apwv , central systolic and diastolic blood pressure , pulse pressure , augmented pressure , augmentation index , brachial systolic and diastolic blood pressure , and brachial pulse pressure . the staff obtaining the outcome measures was blinded for randomization group . with the patient in a supine position , brachial blood pressure was measured after 10 min of rest ( omron m6 comfort ; omron healthcare , milton keynes , u.k . ) . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . the tonometer was used to capture the wave forms at the carotid and subsequently at the femoral artery simultaneously with an electrocardiogram recording using the intersecting tangent ( 27 ) . the distance from the suprasternal notch to the carotid artery was measured with a tape measure and from the suprasternal notch to the femoral artery with an anthropometer ( seca ; medical scales and measuring systems , hamburg , germany ) to avoid overestimation of the distance and subsequently the velocity in obese individuals . the path length was determined by subtracting the carotid - sternal notch distance from the femoral - sternal notch distance . in each patient , apwv was measured twice . if the difference of apwv between the two measurements was larger than 0.5 m / s , a third measurement was taken . in the statistical analysis , the average of the two closest measurements in each patient was used . with the patient in a supine position , peripheral pressure waveforms were recorded at the radial artery using applanation tonometry ( version 8 , sphygmocor system ; atcor medical ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial systolic and diastolic blood pressures were measured three times after a 10-min rest with an automated oscillometric blood pressure recorder ( omron m6 comfort ) with the patient in a sitting position . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . with the patient in a supine position , brachial blood pressure was measured after 10 min of rest ( omron m6 comfort ; omron healthcare , milton keynes , u.k . ) . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . the tonometer was used to capture the wave forms at the carotid and subsequently at the femoral artery simultaneously with an electrocardiogram recording using the intersecting tangent ( 27 ) . the distance from the suprasternal notch to the carotid artery was measured with a tape measure and from the suprasternal notch to the femoral artery with an anthropometer ( seca ; medical scales and measuring systems , hamburg , germany ) to avoid overestimation of the distance and subsequently the velocity in obese individuals . the path length was determined by subtracting the carotid - sternal notch distance from the femoral - sternal notch distance . in each patient , apwv was measured twice . if the difference of apwv between the two measurements was larger than 0.5 m / s , a third measurement was taken . in the statistical analysis , with the patient in a supine position , peripheral pressure waveforms were recorded at the radial artery using applanation tonometry ( version 8 , sphygmocor system ; atcor medical ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial systolic and diastolic blood pressures were measured three times after a 10-min rest with an automated oscillometric blood pressure recorder ( omron m6 comfort ) with the patient in a sitting position . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . health assessments at baseline and at follow - up were performed at the danish addition study centers by trained staff blinded for randomization group following standard operating procedures ( 6 ) . briefly , height , weight , waist circumference , and brachial blood pressure were measured . in venous blood samples , hba1c , serum total urinary albumin and urinary creatinine were measured on spot urine . at baseline and follow - up , self - report questionnaires were used to collect information on ethnicity , smoking status , alcohol consumption , medication , and cvd ( previous myocardial infarction and stroke ) . furthermore , at follow - up we collected information on self - reported episodes with angina pectoris or arrhythmia and self - reported cardiovascular revascularization . patient characteristics at baseline and at follow - up are presented as means ( sd ) or , in cases of skewed distributions , as median ( interquartile range [ iqr ] ) . for proportions , the effect of intensive multifactorial treatment on central hemodynamics was analyzed by mixed - effects models with adjustment for clustering ( randomization by general practice ) and heart rate at time of measurement , taking its direct functional effect on the hemodynamic markers into account . the analysis of apwv was also adjusted for mean blood pressure at time of measurement . in addition to regression coefficients , standardized regression coefficients ( sd change in the outcome ) are presented . aortic stiffness is associated with the intrasubject variation of the central hemodynamic measurements ( the higher the level of stiffness , the larger the intrasubject variation ) . to assure a full spectrum of aortic stiffness , no measurements were excluded in the primary analysis . for comparison , we subsequently performed a subanalysis , excluding measurements with large intrasubject variation according to the manufacturer s guidelines ( 30,31 ) . in the subanalysis of apwv , measurements with a ratio > 0.2 between apwv sem and apwv were excluded . for the measurements of central blood pressure and augmentation index , the software provides an operator index based on the height of the pulse waves , variations in the height of the pulse waves , diastolic variation , and deviation in the shape of the pulse waves . in the subanalysis of central blood pressure and augmentation index , measurements with an operator index < 75 were excluded . analyses were performed using sas version 9.2 ( sas institute , cary , nc ) . in our study sample , 61% of the patients were men , mean age was 59 years at screening , and mean follow - up time was 6.2 years . at screening , modifiable cardiovascular risk factors were elevated ( table 1 ) . furthermore , 38% of the patients were treated with antihypertensive drugs , and lipid - lowering treatment was used by only 10% in the routine care group and by 12% in the intensive treatment group . during follow - up , hba1c , total cholesterol , ldl cholesterol , triglycerides , creatinine , and the proportion of smokers decreased in both treatment arms , whereas waist circumference , bmi , and hdl cholesterol did not change ( table 2 ) . at follow - up , the medication use was more or less similar between treatment groups . only ace inhibitor or angiotensin receptor blocker ( arb ) , aspirin , overall glucose - lowering drugs , and other glucose - lowering drugs , which mainly accounted for repaglinide , were more frequently used by the intensive treatment group . baseline characteristics of the study sample follow - up characteristics of the study sample at follow - up , mean apwv was 9.8 ( sd 2.2 ) m / s in the routine care group and 9.4 ( 2.1 ) m / s in the intensive treatment group ( table 2 ) . with the cluster randomization and heart rate at time of measurement taken into account , apwv was 0.51 m / s lower ( 95% ci 0.96 to 0.05 ; p = 0.03 ) in the intensive treatment group compared with routine care ( table 3 ) . we found no statistically significant differences between treatment arms in brachial systolic blood pressure , diastolic blood pressure , or pulse pressure or central systolic blood pressure , diastolic blood pressure , pulse pressure , augmented pressure , or augmentation index . when we excluded apwv measurements with large intrasubject variation ( 72 out of a total of 1,140 measurements ) , 15 patients were excluded from the analysis ( 2 from routine care and 13 from intensive treatment group ) , and the difference in apwv between treatment arms was 0.39 m / s ( 0.82 to 0.05 ) . for the analysis of central blood pressure and augmentation index , excluding measurements with an operator index < 75 ( 178 measurements of 1,046 ; 27 patients , 6 from routine care and 21 from intensive treatment ) did not change the results substantially . effect of intensive treatment compared with routine care on peripheral and central hemodynamics comparison of the intervention effect on peripheral and central hemodynamics in standardized analyses showed that the largest intervention effect was on apwv ( fig . 1 ) and , subsequently , central diastolic and systolic blood pressure . the effect on the peripheral hemodynamics was further down the line or in the opposite direction . gray , model 1 : adjusted for clustering ( general practice ) and heart rate at time of measurement ; black , model 2 : model 1 plus adjustment for age and sex . in this secondary analysis of a subset of 456 individuals with screen - detected type 2 diabetes who participated in the addition - denmark trial , we found that individuals attending practices randomized to provide intensive treatment had lower aortic stiffness based on apwv after 6 years of follow - up compared with those attending practices randomized to provide routine care . furthermore , we found a statistically nonsignificant indication of lower central systolic and diastolic blood pressure , augmented pressure , and augmentation index in the intensive treatment group . our results should be seen in the light of the findings from the addition - europe trial , which found a statistically significant difference in the cardiovascular risk factors between routine care and intensive treatment : hba1c , brachial blood pressure , total cholesterol , ldl cholesterol , and triglycerides and a statistically nonsignificant 17% risk reduction of a composite cardiovascular end point after 5.3 years ( 6 ) . in the addition - europe trial , there was an indication that the rate of cardiovascular events started to separate after 4 years of treatment . similar findings have been observed in other trials of intensive treatment in patients with established type 2 diabetes ( 5,22,32 ) , indicating that in the presence of a relatively low absolute event rate , the effect of cardiovascular risk reduction takes some time to translate into a lower rate of hard events . our findings lend support to the notion that during the early phases of cardiovascular risk lowering , subclinical functional and/or structural cardiovascular changes occur that underlie the later effect of treatment on hard cardiovascular events . however , the occurrence of hard cardiovascular events in this early time window may still depend on the state of subclinical cardiovascular function and structure before the start of treatment . based on this effect - lag concept and our results , it is possible that the incidence curves in the addition - europe trial will continue to separate as cardiovascular events continue to accrue in the posttrial follow - up . the level of cardiovascular risk factors at follow - up was similar between treatment arms . nonetheless , we found a difference in aortic stiffness . similarly , the steno-2 study also found a small or nonexistent difference in several of the cardiovascular risk factors after a mean follow - up of 7.8 years , and the cardiovascular events were still reduced by 53% in the intensive treatment group ( 22 ) . addressing several cardiovascular risk factors simultaneously , which is usually the approach in general practice , therefore seems to lower cardiovascular risk . because of the pragmatic study design and the treatment strategy , which covered several treatment components and features to provide intensive treatment , we can not conclude on the impact of each single treatment component ; we can speculate on the mechanisms involved only . in our study , brachial systolic blood pressure , central systolic blood pressure , central pulse pressure , central augmented pressure , and augmentation index were not significantly lower in the intensive treatment group compared with the routine care group at follow - up . this is in contrast to what we had expected , given that the brachial blood pressure decreased significantly in the intensive treatment group in the addition - europe trial . further , central aortic pressures and augmentation index do not necessarily reflect the same arterial wall properties as measured by apwv . the velocity of the pulse wave assessed at the carotid and femoral artery is a direct measure of the elasticity of the aorta , whereas augmented pressure , augmentation index , central systolic blood pressure , and pulse pressure are determined by the reflective properties of the entire arterial tree , distance to the reflection sites , the velocity of the forward and reflected wave , and stroke volume ( 33 ) . the reflective properties of the arterial wall can be modulated independently of aortic stiffening ( 34,35 ) and vice versa ( 11,36 ) . in line with our study , karalliedde et al . ( 36 ) found a reduction only in apwv in diabetic patients treated with an arb over 24 weeks compared with a calcium channel blocker , despite similar reductions in augmentation index and brachial and central pulse pressure . ( 11 ) found a reduced apwv in individuals with stable coronary artery disease treated with an ace inhibitor for 4.5 years compared with placebo and no difference in augmentation index and brachial and central pulse pressure between the two groups . in our study , the first antihypertensive drug of choice in the intensive treatment group was ace inhibitor , and at follow - up , 72% of the intensive treatment arm patients were treated with either an ace inhibitor or arb compared with 62% in the routine care arm . this could be the main reason for lower apwv in the intensive treatment group , as previous studies have shown that blockade of the renin - angiotensin system has a beneficial effect on the elastic properties of the arterial wall ( 37 ) . in the analysis of apwv , we also adjusted for mean blood pressure at time of measurement to see whether the effect on apwv was driven by the effect on mean blood pressure , and the effect was attenuated only modestly . hence , the effect of intensive multifactorial treatment is independent of the direct effect on blood pressure , suggesting that the intervention had a direct effect on the vessel wall . the treatment algorithm in the intensive treatment group included statins to all patients and aspirin to patients receiving antihypertensive medication . at follow - up , 80% of patients in the intensive treatment group were treated with statins compared with 72% in the routine care group , and aspirin was more frequently used in the intensive treatment group . the difference in statin treatment could also explain part of the difference in aortic stiffness at follow - up , as a few studies have shown that statin treatment lowers aortic stiffness ( 9,12 ) , whereas the destiffening effect of aspirin remains speculative . the most frequently used glucose - lowering drug in our study was metformin , which has a beneficial effect on cardiovascular risk ( 4 ) . nevertheless , a meta - analysis has shown that metformin does not reduce brachial systolic and diastolic blood pressure ( 38 ) . few studies have examined the effect of glucose - lowering treatment on aortic stiffness ( 15,39 ) , and studies conducted among individuals with diabetes are sparse ( 15 ) . there was no difference in bmi or waist circumference at follow - up between the treatment arms , and the proportion of smokers decreased equally in the treatment arms during follow - up . it is therefore unlikely that the lifestyle component of the intensive treatment intervention led to the observed aortic stiffness effects . this study is the first examining the effect of intensive multifactorial treatment on central hemodynamics among individuals with screen - detected diabetes in general practice . all patients were screened and treated by their general practitioner , showing that the screening strategy and intervention are feasible in general practice . during follow - up , the evidence - based guidelines of diabetes care in denmark ( 24,40 ) changed toward the treatment targets in the intensive treatment arm , which could have evened out the effect of intensive treatment . nevertheless , we found a lower aortic stiffness in patients treated intensively . this suggests either that the cardiovascular risk factor levels differed more markedly in the earliest years of the trial and had an effect on aortic stiffness that carried forward or that even relatively small differences in cardiovascular risk factor levels can impact aortic stiffness if consistent over 6 years . the main limitation of this secondary trial analysis is that our central hemodynamic outcomes were not prespecified before initiation of the study . statistically , the addition of nine outcomes increases the possibility of getting statistically significant results simply by chance . however , the nine outcomes should not be regarded as independent , as they are all expressions of the functional and structural processes underlying the prespecified outcome : cvd . the number of patients in the intensive treatment group was larger than in the routine care group , even though there were no differences in the number and types of practices in the two groups . the practices were randomized before screening and inclusion of patients , but it seems that the intervention allocation enhanced the focus on screening and thereby inclusion of patients in the intensive treatment practices . still , the patients did not differ between groups regarding cardiovascular risk factors at baseline . another limitation is the lack of measurements of central hemodynamics at baseline , but based on the study design and similar levels of cardiovascular risk factors , we believe it is reasonable to assume that the level of aortic stiffness and central blood pressure were similar in both groups at baseline and , consequently , that the observed differences occurred during the study . because of the study design , the general practices were not blinded to treatment allocation , but the research personnel conducting the measurements at follow - up were unaware of the treatment allocation . several prospective observational studies have shown that a lower level of aortic stiffness is associated with a lower cardiovascular risk , as reported in a recent meta - analysis ( 7 ) . with our results extrapolated based on estimates from this meta - analysis , which included studies with various measures of aortic stiffness , the difference of 0.51 m / s would correspond with a relative cvd risk reduction of 7% by intensive multifactorial treatment compared with routine care . however , we can not conclude that reducing aortic stiffness also reduces the incidence of cardiovascular events , even though this hypothesis might be plausible . to confirm this theory , we need to follow our population for future cardiovascular events . in this secondary analysis of 456 patients with screen - detected diabetes in a cluster randomized trial , we found that patients treated intensively by general practitioners have a lower level of apwv than individuals receiving routine care after 6 years of treatment . this means that screening for type 2 diabetes followed by intensive multifactorial treatment in general practice leads to lower aortic stiffness , a key intermediate cardiovascular outcome , during the time window when treatment effects on hard cardiovascular outcomes are not yet observable .

objectivediabetes is associated with increased brachial and central blood pressure and aortic stiffness . we examined the effect of intensive multifactorial treatment in general practice on indices of peripheral and central hemodynamics among patients with screen - detected diabetes.research design and methodsas part of a population - based screening and intervention study in general practice , 1,533 danes aged 4069 years were clinically diagnosed with screen - detected diabetes . general practitioners were randomized to provide intensive multifactorial treatment or routine care . after a mean follow - up of 6.2 years , an unselected subsample of 456 patients underwent central hemodynamic assessments by applanation tonometry . central pressure was derived from the radial pulse wave . aortic stiffness was assessed as carotid - femoral pulse wave velocity ( apwv ) . the intervention effect on each index of central hemodynamics was analyzed by mixed - effects models adjusted for heart rate , cluster randomization , age , and sex.resultsat screening , median age was 59.2 years ( interquartile range 55.264.6 ) ; 289 patients ( 63% ) were in the intensive treatment group , and 278 patients ( 61% ) were men . patients in the intensive treatment group had a 0.51 m / s ( 95% ci 0.96 to 0.05 , p = 0.03 ) lower apwv compared with routine care . respective differences for central augmentation index ( 0.84% [ 2.54 to 0.86 ] ) , pulse pressure ( 0.28 mmhg [ 1.75 to 2.32 ] ) , and systolic ( 1.42 mmhg [ 4.47 to 1.64 ] ) and diastolic ( 1.79 mmhg [ 3.72 to 0.14 ] ) blood pressure were not statistically significant.conclusionsintensive multifactorial treatment of screen - detected diabetes during 6 years in general practice has a significant impact on aortic stiffness , whereas the effects on other hemodynamic measures are smaller and not statistically significant .

RESEARCH DESIGN AND METHODS Population Intervention Outcomes Aortic pulse wave velocity. Central blood pressure and augmentation index. Peripheral blood pressure. Measurements at baseline and at follow-up Statistical analysis RESULTS CONCLUSIONS

of these , 190 agreed to participate and were allocated to screening and randomized to provide either routine care or intensive multifactorial treatment of patients with diabetes detected by screening . the screening phase took place in 20012006 and consisted of a population - based stepwise screening program to identify individuals with screen - detected diabetes . in total , 1,533 individuals were diagnosed with diabetes according to the 1999 world health organization criteria ( 21 ) and were enrolled in the trial . in two of five study centers ( holstebro hospital and steno diabetes center ) , measurements of central hemodynamics were carried out and successfully obtained in 456 of 486 attending patients . of the 456 patients , 420 had measurements of both apwv and central blood pressure , 9 had measurements of apwv only , and 27 had measurements of central blood pressure only ( supplementary fig . the reasons for missing measurements were as follows : station closure owing to insufficient staff ( n = 27 ) , irregular pulse ( apwv , n = 4 ; central blood pressure and augmentation index , n = 12 ) , and for apwv , that the carotid or femoral artery pulse could not be found ( n = 26 ) . we aimed to educate and support general practitioners and practice nurses in target - driven management ( using medication and promotion of healthy lifestyle ) of hyperglycemia , hypertension , and hypercholesterolemia based on the stepwise regimen used in the steno-2 study ( 22,23 ) . the general practitioners in the routine care group were provided with diagnostic test results only ; their patients with screen - detected diabetes received standard care of diabetes according to the danish national guidelines ( 24 ) . in this secondary analysis , the outcomes were central and peripheral hemodynamics : apwv , central systolic and diastolic blood pressure , pulse pressure , augmented pressure , augmentation index , brachial systolic and diastolic blood pressure , and brachial pulse pressure . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central pulse pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . briefly , height , weight , waist circumference , and brachial blood pressure were measured . patient characteristics at baseline and at follow - up are presented as means ( sd ) or , in cases of skewed distributions , as median ( interquartile range [ iqr ] ) . the effect of intensive multifactorial treatment on central hemodynamics was analyzed by mixed - effects models with adjustment for clustering ( randomization by general practice ) and heart rate at time of measurement , taking its direct functional effect on the hemodynamic markers into account . aortic stiffness is associated with the intrasubject variation of the central hemodynamic measurements ( the higher the level of stiffness , the larger the intrasubject variation ) . for the measurements of central blood pressure and augmentation index , the software provides an operator index based on the height of the pulse waves , variations in the height of the pulse waves , diastolic variation , and deviation in the shape of the pulse waves . in the subanalysis of central blood pressure and augmentation index , measurements with an operator index < 75 were excluded . the screening phase took place in 20012006 and consisted of a population - based stepwise screening program to identify individuals with screen - detected diabetes . it was conducted among the participating general practitioners patients aged 4069 years without known diabetes . in total , 1,533 individuals were diagnosed with diabetes according to the 1999 world health organization criteria ( 21 ) and were enrolled in the trial . in two of five study centers ( holstebro hospital and steno diabetes center ) , measurements of central hemodynamics were carried out and successfully obtained in 456 of 486 attending patients . 420 had measurements of both apwv and central blood pressure , 9 had measurements of apwv only , and 27 had measurements of central blood pressure only ( supplementary fig . the reasons for missing measurements were as follows : station closure owing to insufficient staff ( n = 27 ) , irregular pulse ( apwv , n = 4 ; central blood pressure and augmentation index , n = 12 ) , and for apwv , that the carotid or femoral artery pulse could not be found ( n = 26 ) . we aimed to educate and support general practitioners and practice nurses in target - driven management ( using medication and promotion of healthy lifestyle ) of hyperglycemia , hypertension , and hypercholesterolemia based on the stepwise regimen used in the steno-2 study ( 22,23 ) . the general practitioners in the routine care group were provided with diagnostic test results only ; their patients with screen - detected diabetes received standard care of diabetes according to the danish national guidelines ( 24 ) . in this secondary analysis , the outcomes were central and peripheral hemodynamics : apwv , central systolic and diastolic blood pressure , pulse pressure , augmented pressure , augmentation index , brachial systolic and diastolic blood pressure , and brachial pulse pressure . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial systolic and diastolic blood pressures were measured three times after a 10-min rest with an automated oscillometric blood pressure recorder ( omron m6 comfort ) with the patient in a sitting position . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . from the supine systolic and diastolic blood pressure , mean blood pressure was calculated ( diastolic blood pressure + 0.4 pulse pressure ) ( 25 ) . the velocity of the pulse wave was then assessed between the right carotid and femoral sites by applanation tonometry using the sphygmocor device ( version 8 ; atcor medical ) and a high - fidelity tonometer , which is a validated method of measuring apwv ( 26 ) . based on a built - in generalized transfer function using the supine brachial systolic and diastolic blood pressure for calibration , central waveforms were calculated from the radial arterial waveforms . from the central waveforms , central systolic and diastolic blood pressure , central augmented pressure was calculated as the contribution of the pulse wave reflection to central systolic pressure , and central augmentation index was calculated as the ratio between central augmented pressure and central pulse pressure . brachial systolic and diastolic blood pressures were measured three times after a 10-min rest with an automated oscillometric blood pressure recorder ( omron m6 comfort ) with the patient in a sitting position . brachial pulse pressure was calculated as the difference between brachial systolic and diastolic blood pressure . patient characteristics at baseline and at follow - up are presented as means ( sd ) or , in cases of skewed distributions , as median ( interquartile range [ iqr ] ) . for proportions , the effect of intensive multifactorial treatment on central hemodynamics was analyzed by mixed - effects models with adjustment for clustering ( randomization by general practice ) and heart rate at time of measurement , taking its direct functional effect on the hemodynamic markers into account . aortic stiffness is associated with the intrasubject variation of the central hemodynamic measurements ( the higher the level of stiffness , the larger the intrasubject variation ) . for the measurements of central blood pressure and augmentation index , the software provides an operator index based on the height of the pulse waves , variations in the height of the pulse waves , diastolic variation , and deviation in the shape of the pulse waves . in the subanalysis of central blood pressure and augmentation index , measurements with an operator index < 75 were excluded . in our study sample , 61% of the patients were men , mean age was 59 years at screening , and mean follow - up time was 6.2 years . furthermore , 38% of the patients were treated with antihypertensive drugs , and lipid - lowering treatment was used by only 10% in the routine care group and by 12% in the intensive treatment group . during follow - up , hba1c , total cholesterol , ldl cholesterol , triglycerides , creatinine , and the proportion of smokers decreased in both treatment arms , whereas waist circumference , bmi , and hdl cholesterol did not change ( table 2 ) . only ace inhibitor or angiotensin receptor blocker ( arb ) , aspirin , overall glucose - lowering drugs , and other glucose - lowering drugs , which mainly accounted for repaglinide , were more frequently used by the intensive treatment group . baseline characteristics of the study sample follow - up characteristics of the study sample at follow - up , mean apwv was 9.8 ( sd 2.2 ) m / s in the routine care group and 9.4 ( 2.1 ) m / s in the intensive treatment group ( table 2 ) . with the cluster randomization and heart rate at time of measurement taken into account , apwv was 0.51 m / s lower ( 95% ci 0.96 to 0.05 ; p = 0.03 ) in the intensive treatment group compared with routine care ( table 3 ) . we found no statistically significant differences between treatment arms in brachial systolic blood pressure , diastolic blood pressure , or pulse pressure or central systolic blood pressure , diastolic blood pressure , pulse pressure , augmented pressure , or augmentation index . when we excluded apwv measurements with large intrasubject variation ( 72 out of a total of 1,140 measurements ) , 15 patients were excluded from the analysis ( 2 from routine care and 13 from intensive treatment group ) , and the difference in apwv between treatment arms was 0.39 m / s ( 0.82 to 0.05 ) . for the analysis of central blood pressure and augmentation index , excluding measurements with an operator index < 75 ( 178 measurements of 1,046 ; 27 patients , 6 from routine care and 21 from intensive treatment ) did not change the results substantially . effect of intensive treatment compared with routine care on peripheral and central hemodynamics comparison of the intervention effect on peripheral and central hemodynamics in standardized analyses showed that the largest intervention effect was on apwv ( fig . the effect on the peripheral hemodynamics was further down the line or in the opposite direction . gray , model 1 : adjusted for clustering ( general practice ) and heart rate at time of measurement ; black , model 2 : model 1 plus adjustment for age and sex . in this secondary analysis of a subset of 456 individuals with screen - detected type 2 diabetes who participated in the addition - denmark trial , we found that individuals attending practices randomized to provide intensive treatment had lower aortic stiffness based on apwv after 6 years of follow - up compared with those attending practices randomized to provide routine care . furthermore , we found a statistically nonsignificant indication of lower central systolic and diastolic blood pressure , augmented pressure , and augmentation index in the intensive treatment group . our results should be seen in the light of the findings from the addition - europe trial , which found a statistically significant difference in the cardiovascular risk factors between routine care and intensive treatment : hba1c , brachial blood pressure , total cholesterol , ldl cholesterol , and triglycerides and a statistically nonsignificant 17% risk reduction of a composite cardiovascular end point after 5.3 years ( 6 ) . similar findings have been observed in other trials of intensive treatment in patients with established type 2 diabetes ( 5,22,32 ) , indicating that in the presence of a relatively low absolute event rate , the effect of cardiovascular risk reduction takes some time to translate into a lower rate of hard events . based on this effect - lag concept and our results , it is possible that the incidence curves in the addition - europe trial will continue to separate as cardiovascular events continue to accrue in the posttrial follow - up . similarly , the steno-2 study also found a small or nonexistent difference in several of the cardiovascular risk factors after a mean follow - up of 7.8 years , and the cardiovascular events were still reduced by 53% in the intensive treatment group ( 22 ) . addressing several cardiovascular risk factors simultaneously , which is usually the approach in general practice , therefore seems to lower cardiovascular risk . because of the pragmatic study design and the treatment strategy , which covered several treatment components and features to provide intensive treatment , we can not conclude on the impact of each single treatment component ; we can speculate on the mechanisms involved only . in our study , brachial systolic blood pressure , central systolic blood pressure , central pulse pressure , central augmented pressure , and augmentation index were not significantly lower in the intensive treatment group compared with the routine care group at follow - up . this is in contrast to what we had expected , given that the brachial blood pressure decreased significantly in the intensive treatment group in the addition - europe trial . the velocity of the pulse wave assessed at the carotid and femoral artery is a direct measure of the elasticity of the aorta , whereas augmented pressure , augmentation index , central systolic blood pressure , and pulse pressure are determined by the reflective properties of the entire arterial tree , distance to the reflection sites , the velocity of the forward and reflected wave , and stroke volume ( 33 ) . ( 36 ) found a reduction only in apwv in diabetic patients treated with an arb over 24 weeks compared with a calcium channel blocker , despite similar reductions in augmentation index and brachial and central pulse pressure . ( 11 ) found a reduced apwv in individuals with stable coronary artery disease treated with an ace inhibitor for 4.5 years compared with placebo and no difference in augmentation index and brachial and central pulse pressure between the two groups . in our study , the first antihypertensive drug of choice in the intensive treatment group was ace inhibitor , and at follow - up , 72% of the intensive treatment arm patients were treated with either an ace inhibitor or arb compared with 62% in the routine care arm . this could be the main reason for lower apwv in the intensive treatment group , as previous studies have shown that blockade of the renin - angiotensin system has a beneficial effect on the elastic properties of the arterial wall ( 37 ) . in the analysis of apwv , we also adjusted for mean blood pressure at time of measurement to see whether the effect on apwv was driven by the effect on mean blood pressure , and the effect was attenuated only modestly . hence , the effect of intensive multifactorial treatment is independent of the direct effect on blood pressure , suggesting that the intervention had a direct effect on the vessel wall . the treatment algorithm in the intensive treatment group included statins to all patients and aspirin to patients receiving antihypertensive medication . at follow - up , 80% of patients in the intensive treatment group were treated with statins compared with 72% in the routine care group , and aspirin was more frequently used in the intensive treatment group . the difference in statin treatment could also explain part of the difference in aortic stiffness at follow - up , as a few studies have shown that statin treatment lowers aortic stiffness ( 9,12 ) , whereas the destiffening effect of aspirin remains speculative . nevertheless , a meta - analysis has shown that metformin does not reduce brachial systolic and diastolic blood pressure ( 38 ) . few studies have examined the effect of glucose - lowering treatment on aortic stiffness ( 15,39 ) , and studies conducted among individuals with diabetes are sparse ( 15 ) . there was no difference in bmi or waist circumference at follow - up between the treatment arms , and the proportion of smokers decreased equally in the treatment arms during follow - up . it is therefore unlikely that the lifestyle component of the intensive treatment intervention led to the observed aortic stiffness effects . this study is the first examining the effect of intensive multifactorial treatment on central hemodynamics among individuals with screen - detected diabetes in general practice . all patients were screened and treated by their general practitioner , showing that the screening strategy and intervention are feasible in general practice . during follow - up , the evidence - based guidelines of diabetes care in denmark ( 24,40 ) changed toward the treatment targets in the intensive treatment arm , which could have evened out the effect of intensive treatment . this suggests either that the cardiovascular risk factor levels differed more markedly in the earliest years of the trial and had an effect on aortic stiffness that carried forward or that even relatively small differences in cardiovascular risk factor levels can impact aortic stiffness if consistent over 6 years . the number of patients in the intensive treatment group was larger than in the routine care group , even though there were no differences in the number and types of practices in the two groups . the practices were randomized before screening and inclusion of patients , but it seems that the intervention allocation enhanced the focus on screening and thereby inclusion of patients in the intensive treatment practices . another limitation is the lack of measurements of central hemodynamics at baseline , but based on the study design and similar levels of cardiovascular risk factors , we believe it is reasonable to assume that the level of aortic stiffness and central blood pressure were similar in both groups at baseline and , consequently , that the observed differences occurred during the study . several prospective observational studies have shown that a lower level of aortic stiffness is associated with a lower cardiovascular risk , as reported in a recent meta - analysis ( 7 ) . with our results extrapolated based on estimates from this meta - analysis , which included studies with various measures of aortic stiffness , the difference of 0.51 m / s would correspond with a relative cvd risk reduction of 7% by intensive multifactorial treatment compared with routine care . in this secondary analysis of 456 patients with screen - detected diabetes in a cluster randomized trial , we found that patients treated intensively by general practitioners have a lower level of apwv than individuals receiving routine care after 6 years of treatment . this means that screening for type 2 diabetes followed by intensive multifactorial treatment in general practice leads to lower aortic stiffness , a key intermediate cardiovascular outcome , during the time window when treatment effects on hard cardiovascular outcomes are not yet observable .

in resource - limited countries , the prevalence of chronic hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) infection is often high , and populations with a high prevalence of hbv and hcv usually overlap with those seriously affected by hiv . in a study from northern india , the reported prevalence of hbv and hcv co - infection in hiv - infected patients was 53% and 24% , respectively . a study in tanzania reported 173% and 181% of hiv - infected patients were co - infected with hbv and hcv , respectively , and an earlier report from thailand showed that the prevalence of hbv infection was 87% and hcv infection 78% in hiv - infected patients . accumulating evidence suggests that hiv co - infection adversely affects the clinical course of hepatitis . increased hbv carriage rates , greater levels of hbv viraemia , more rapid decline in hbv surface antibody , increased reactivation episodes , and faster progression to liver cirrhosis are all characteristic of hiv / hbv co - infected patients [ 5 , 6 ] . in hiv / hcv co - infected patients , faster progression of fibrosis resulting in decompensated cirrhosis have been shown in previous studies [ 7 , 8 ] . as patients on highly active antiretroviral therapy ( haart ) survive much longer , liver failure is becoming the major cause of death in patients with hepatitis co - infection . current international aids society guidelines for the management of hiv recommend that hiv / viral hepatitis co - infected patients should start haart , the same as hiv mono - infected patients . moreover , initiation of haart is recommended regardless of cd4 cell count when treatment for hbv is considered . however , there is a big gap between the recommendation of the guidelines and the real - life situation in resource - limited countries . while access to haart has markedly increased , even in resource - limited countries , overall haart coverage remains as low as 36% ( 95% ci 3339% ) based on 2010 who guidelines ( treatment initiation at cd4 cell count < 350 cells/l . moreover , monitoring ( viral load testing and genotyping ) and treatment for hepatitis are not available due to the cost in most resource - limited countries . thus , we assume that there are still large groups of patients with hiv / chronic hepatitis co - infection who are not receiving haart in resource - limited countries . for better management of these patients , it is important to know the association between hepatitis co - infection and the natural history of hiv infection . the effect of viral hepatitis co - infection on hiv progression and all - cause mortality before initiating haart remains uncertain . most studies conducted in the late 1990s and early 2000s did not include hiv viral load in their analysis . some studies presented a more rapid progression to aids and reduced survival in patients who have chronic hbv or hcv infection [ 1215 ] while others have shown conflicting results [ 1619 ] . in the majority of previous studies examining hbv and hcv co - infection , the main transmission mode of hiv in participants was homosexual intercourse or injecting drug use ( idu ) and none were conducted with a substantial sample size in asian or african countries where the majority of hiv - infected individuals with hbv vertical transmission reside . the present study aims to evaluate the impact of hepatitis co - infection on all - cause mortality in haart - naive hiv - infected individuals in northern thailand . to address the current research question , we re - analysed our previously conducted natural history cohort of hiv - infected patients in northern thailand [ 20 , 21 ] . this patient cohort was assembled from volunteers at the hiv centre of a government referral hospital with about 800 beds situated in the centre of lampang province in upper northern thailand . the centre was established in october 1995 as an outpatient clinic providing treatment , care and support for hiv - infected patients . the recruitment of this cohort was from 1 july 2000 to 15 october 2002 before the national antiretroviral treatment programme was launched . all adult ( aged > 18 years ) hiv - infected individuals attending the hiv clinic who were haart - naive at the first visit were approached by the research team and enrolled if written consent were obtained . all participants were requested to visit the clinic at least once every 3 months regardless of the presence of clinical symptoms and were followed up from the date of study enrolment until 15 october , 2004 . this study was approved by the thai government ethics committee in december 1999 and december 2005 . demographic ( gender and age at enrolment ) data and medical history [ hiv - related symptoms , history of antiretroviral therapy ( art ) and mode of transmission ] of patients were obtained at the study enrolment by well - trained research staff through face - to - face interviews based upon a structured questionnaire . in addition to physical examination by two research physicians , complete blood count ( cbc ) , platelet count , cd4 cell count and hiv viral load ( copies / ml ) were measured . cd4 cell count was determined by flow cytometric technique facscan ( bd biosciences , usa ) and hiv viral load was measured using a commercial kit ( amplicor hiv-1 monitor test , roche molecular systems inc . the remaining freeze - stored plasma samples from our previous study were retrospectively tested for hepatitis b surface antigen ( hbsag ) , anti - hepatitis b core antibody ( anti - hbcab ) and antibody to hepatitis c virus ( anti - hcv ) were retrospectively tested using commercially kits : cobas core hbsag ii eia , anti - hbc eia , and eti - ab - hcvk-4 ( diasorin s.p.a . , italy ) . hbsag positivity and anti - hcv positivity were determined to define hbv and hcv co - infection , respectively . survival status for each patient was ascertained by hospital records , death certificates , mailing letters , and contacting families or relatives . causes of death in hiv / hepatitis co - infected patients were investigated by reviewing hospital records . in survival analysis , patients who started haart before 15 october 2004 were regarded as censored on the date of starting haart . meier survival analysis was performed to estimate survival in relation to the existence of hiv / hbv or hcv co - infection . hbv co - infected patients were divided into two subgroups according to the existence of anti - hbcab . additionally , cox 's proportional hazard model was conducted to evaluate the influence of hbv or hcv co - infection on survival adjusted by several factors . in multivariate models , other than hepatitis co - infection status and existence of anti - hbcab , we included all variables with p < 01 in univariate analysis . results were presented as hazard ratios ( hrs ) with 95% confidence intervals ( cis ) . this study was conducted as part of the lampang hiv cohort phase i and lampang & phayao hiv cohort phase ii , which were approved by research ethics committee of the thai ministry of public health . seven hundred and fifty - five patients ( over 95% of patients who attended the clinic during the targeted period ) were enrolled in the study . of 755 hiv - infected persons , 55 patients were excluded ( 32 patients had received haart in private clinic or other clinical trial before recruitment , 22 patients had incomplete testing for hepatitis co - infection , and four patients had hbv / hcv dual co - infection ) . median age at enrolment was 33 years ( 95% ci 2937 ) , and 218% had received mono or dual art . the major transmission route was heterosexual ( 967% ) and half of participants were asymptomatic . the cd4 cell count was > 200 cells/l in 299 ( 415% ) patients and < 50 cells/l in 261 ( 363% ) patients . the prevalence of hbv and hcv co - infection was 119% and 33% , respectively . of 700 patients , 681 ( 973% ) had both hbsag and anti - hbcab status . hcv co - infection was strongly associated with idu ( p < 0001 ) . the baseline cd4 cell count was significantly lower in hbv vs. hcv co - infected patients whereas there was no difference between these patient groups in their baseline viral load . patients with hbv co - infection were more likely to have aids symptoms compared to hiv mono - infected patients ( p = 002 ) . platelet counts were lowest in hcv co - infected individuals followed by hbv co - infected patients ( p = 003 ) . of 86 hbv co - infected patients , 78 ( 907% ) had anti - hbcab . interestingly , eight ( 93% ) patients did not have anti - hbcab despite being hbsag positive . table 1.baseline characteristics of hiv mono - infected patients and hepatitis co - infected patientscharacteristics ( n = 700)hiv mono - infection(n = 594)hbv co - infection(n = 83)hcv co - infection(n = 23)age ( median , iqr)*33 ( 2937)32 ( 2935)30 ( 2634)male gender ( % ) 244 ( 411)42 ( 506)14 ( 509)mode of transmission ( % ) * heterosexual583 ( 981)79 ( 952)15 ( 652 ) homosexual6 ( 10)1 ( 12)1 ( 43 ) idu0 ( 00)0 ( 00)5 ( 217 ) others or multiple5 ( 08)2 ( 24)2 ( 86 ) unknown0 ( 00)1 ( 12)0 ( 00)previous art ( % ) 129 ( 217)17 ( 205)4 ( 174 ) clinical status ( % ) || asymptomatic312 ( 525)32 ( 386)8 ( 348 ) non - aids symptomatic115 ( 194)15 ( 181)7 ( 304 ) aids symptomatic167 ( 281)36 ( 434)8 ( 348)baseline cd4 cell count ( cells/l ) ( median , iqr)*150 ( 22351)64 ( 19219)334 ( 31459)baseline vial load ( copies / ml ) ( median , iqr)157431 ( 34891446396)169773 ( 69806437116)112334 ( 21415515515)baseline platelet count ( median , iqr)261000 ( 215000319000)237000 ( 191000300000)213000 ( 170000328000)iqr , interquartile range ; idu , injecting drug user ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome.*p < 005 , hiv mono - infection vs. hcv co-infection.p < 005 , hiv mono - infection vs. hbv co-infection.p < 005 , hbv co - infection vs. hcv co-infection.experience with antiretroviral therapy is limited to monotherapy or dual therapy.||definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . table 2.status of hbsag and anti - hbcabanti - hbcab(+)anti - hbcab()totalhbsag(+)78886hbsag()318277595total396285681hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody.both hbsag and anti - hbcab status data were available for 681 patients . baseline characteristics of hiv mono - infected patients and hepatitis co - infected patients iqr , interquartile range ; idu , injecting drug user ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome . p < 005 , hiv mono - infection vs. hcv co - infection . p < 005 , hiv mono - infection vs. hbv co - infection . p < 005 , hbv co - infection vs. hcv co - infection . experience with antiretroviral therapy is limited to monotherapy or dual therapy . definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . other than * , , there were not any significant differences between the groups . status of hbsag and anti - hbcab hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody . both hbsag and anti - hbcab status data were available for 681 patients . complete follow - up data were available for 694 ( 991% ) patients in the evaluated cohort . total follow - up time was 11667 person - years of observation ( pyo ) with a median patient follow - up of 588 days [ interquartile range ( iqr ) , 317967 ] . during the observation period , 258 ( 369% ) patients died , resulting in a mortality rate of 221/100 pyo ( 95% ci 167278 ) . when stratified according to baseline cd4 cell count , mortality was 646/100 pyo ( 95% ci 591709 ) in patients with cd4 < 50 cells/l compared to 229/100 pyo ( 95% ci 163297 ) in the group with cd4 < 50200 cells/l and 517/100 pyo ( 95% ci 257763 ) in patients with cd4 > 200 cells/l . when stratified with baseline clinical status , mortality was 871/100 pyo ( 95% ci 576116 ) in asymptomatic patients , 275/100 pyo ( 95% ci 196344 ) in symptomatic but non - aids patients and 606/100 pyo ( 95% ci 544667 ) in symptomatic aids patients . 1 ) revealed that hbv co - infection significantly increased mortality ( p < 0001 , log - rank test ) . hcv co - infection also tended to increase mortality , but the statistical significance was marginal . the curves for hiv / hbv and hcv / hiv co - infection converge at about 500 days . the likelihood ratio test for interaction by time band with a cut - point at 500 days revealed a p value of 02 , confirming lack of evidence for a relevant violation of the proportional hazard assumption , allowing the use of cox regression analysis . the influence of hepatitis co - infection on survival analysed by the cox proportional hazard model is presented in table 3a . in univariate analysis , factors associated with death were male gender , previous art treatment , clinical symptom at enrolment , baseline cd4 cell count , baseline viral load , hbv co - infection and existence of anti - hbcab . patients with a low platelet count ( < 150 000 ) were more likely to die than those with a higher platelet count . 1.kaplanmeier survival probability estimate of co - infected and mono - infected individuals showing that hbv co - infection significantly increased mortality . table 3 ( a).influence of hbv or hcv co - infection on survivalvariableshr ( 95% ci)p valueahr ( 95% ci)p valuemale sex259 ( 202333)<0001123 ( 094160)014age < 30 years*079 ( 061101)006081 ( 063105)011previous art067 ( 049092)001116 ( 084161)037transmission mode iduref. homosexual036 ( 005258)031 heterosexual068 ( 017272)058 others112 ( 021613)089clinical symptom* asymptomaticref . ref. symptomatic , non - aids306 ( 216433)<0001142 ( 096208)008 aids , symptomatic665 ( 493898)<0001205 ( 144293)<0001baseline cd4 cell count ( cells/l ) 200ref . ref. 50199446 ( 291684)<0001298 ( 185479)<0001 < 50127 ( 872186)<0001644 ( 404103)<0001baseline viral load ( copies / ml ) < 10000ref . ref. 1000049999244 ( 104575)004120 ( 048301)070 5000099999330 ( 139785)0007167 ( 070400)025 100000879 ( 413187)<0001219 ( 096500)006baseline platelet count < 150000152 ( 100229)005116 ( 076178)050hbv co - infection205 ( 149282)<0001181 ( 130253)<0001hcv co - infection126 ( 067238)047190 ( 098369)006hr , hazard ratio ; ci , confidence interval ; ahr , adjusted hazard ratio ; art , antiretroviral therapy ; idu , injecting drug user ; aids , acquired immunodeficiency syndrome.*at the time of enrolment.experience with antiretroviral therapy is limited to monotherapy or dual therapy.definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . kaplan meier survival probability estimate of co - infected and mono - infected individuals showing that hbv co - infection significantly increased mortality . influence of hbv or hcv co - infection on survival hr , hazard ratio ; ci , confidence interval ; ahr , adjusted hazard ratio ; art , antiretroviral therapy ; idu , injecting drug user ; aids , acquired immunodeficiency syndrome . at the time of enrolment . definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . survival estimates focused on hbv serology interestingly showed that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab ( p < 00001 by log - rank test , fig . 2 ) . in multivariate analysis ( table 3b ) , in addition to symptomatic aids and low cd4 cell count , hbv co - infection without anti - hbcab was a strong risk factor for death ( adjusted hr 634 , 95% ci 399103 ) . fig . 2.kaplanmeier survival probability estimate showing that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab . table 3 ( b).impact of hbs ag and anti - hbc ab status on survivalvariableshr ( 95% ci)p valueahr ( 95% ci)p valuemale sex259 ( 202333)<0001123 ( 096163)011age < 35 years*079 ( 061101)006079 ( 061102)008previous art067 ( 049092)001110 ( 079153)059transmission mode iduref. homosexual036 ( 005258)031 heterosexual068 ( 017272)058 others112 ( 021613)089clinical symptom* asymptomaticref.ref. symptomatic , non aids306 ( 216433)<0001147 ( 110215)005 aids , symptomatic665 ( 493898)<0001203 ( 142290)<0001baseline cd4 cell count ( cells/l ) 200ref . ref. 50199446 ( 291684)<0001203 ( 142290)<0001 < 50127 ( 872186)<0001634 ( 399103)<0001baseline viral load ( copies / ml ) < 10000ref . ref. 1000049999244 ( 104575)004173 ( 072413)022 5000099999330 ( 139785)0007117 ( 047293)074 100000879 ( 413187)<0001209 ( 091478)008baseline platelet count < 150000152 ( 100229)005128 ( 084194)025hbv serology hbsag()ref . ref. hbsag(+)anti - hbcab()634 ( 399103)<0001 hbsag(+ ) anti - hbcab(+)162 ( 114229)0007iqr , interquartile range ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome ; hr , hazard ratio ; ahr , adjusted hazard ratio ; ci , confidence interval ; hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody.*at the time of enrolment.experience with antiretroviral therapy is limited to mono- or dual-therapy.definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . meier survival probability estimate showing that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab . impact of hbs ag and anti - hbc ab status on survival iqr , interquartile range ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome ; hr , hazard ratio ; ahr , adjusted hazard ratio ; ci , confidence interval ; hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody . at the time of enrolment . definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . although primary causes of death were unknown for nine out of 56 hiv / hepatitis co - infected patients , the majority of deaths ( 46/56 , 821% ) were attributed to aids - related diseases . no patients were diagnosed with liver failure before death except for one patient who was hospitalized for 6 days due to newly diagnosed cirrhosis with portal hypertension . this patient was lost to follow - up after discharge with a cd4 cell count of 22 cells/l and died 3 months later . in the present study with a substantial sample size , we have clearly demonstrated that hbv co - infection significantly increased all - cause mortality of haart - naive hiv patients . it was only after the advent of haart substantially increased life expectancy of hiv - infected individuals that the importance of liver - related death due to hepatitis co - infection was highlighted . our data indicate that the influence of hepatitis co - infection on the natural history of haart - naive hiv patients should not be ignored . we also discovered an increased mortality of hcv co - infected patients compared to hiv mono - infected patients . the results of multivariate analysis showed only a trend , but this is probably due to the small number of hcv co - infection in this study . first , the lack of data on hbv dna and hcv rna viral load is a limitation . the absence of hcv viraemia in anti - hcv - positive patients might explain why hcv co - infection did not show the strong association with death . second , cause of death was determined only by reviewing medical charts while the information of survival status was ascertained by contacting families and relatives in addition to reviewing hospital records . however , we focused on all - cause mortality and these limitations do not alter the main results of the study . to our knowledge , this is the first study from a resource - limited country to address the adverse influence of hepatitis co - infection on survival in haart - naive hiv patients . we also found a higher mortality rate in this thai population compared to white patients in new york in the 1980s . our finding implies that the high prevalence of hepatitis co - infection is at least partially responsible for the high mortality observed in hiv - infected individuals in resource - limited countries . several studies have investigated the influence of hepatitis co - infection on the natural course of hiv infection but these studies often include patients receiving haart and show conflicting findings [ 1214 , 16 , 17 , 23 ] . one of the reasons for this discrepancy appears to be the sample sizes in previous studies [ 16 , 23 ] . in addition , none of the previous studies has analysed the association between hiv and hbv or hcv co - infection after adjusting for clinical status , viral load , and cd4 cell count in a cohort not receiving haart with substantial sample size . our investigation of hospital records did not identify any patients with liver failure , with one exception . however , we believe that the majority of chronic hbv or hcv co - infected hiv patients died from aids - defined illness rather than liver failure , since they all had significant opportunistic infections with a very low cd4 cell count . these results suggest that hepatitis co - infection accelerates the natural course of hiv infection itself . an in vitro study has demonstrated that hbv - x protein super - induces ongoing hiv replication and hiv-1 long - terminal repeat transcription . however , according to our results , hbv co - infection increased the mortality independent of hiv viral load . thus , the increased hiv viral load per se does not fully explain the higher mortality in hbv co - infected patients . we also considered the possibility that the higher mortality in hbv co - infected patients might have been due to confounding factors . however , our multivariate analysis demonstrated that the association with hbv co - infection was independent of age , gender , transmission route , clinical symptoms and immunological status such as cd4 cell count . nevertheless , as with most multivariate regression models , residual confounding can not be fully ruled out . it is striking that hbv co - infected patients without anti - hbcab had the poorest prognosis . even after adjustment for demographic and clinical factors , the impact on death remained substantially high . avettand - fenoel et al . suggested three circumstances leading to failure to elicit anti - hbcab during hbv infection . it is hypothesized that infants born to hbeag - positive carrier mothers may result in the lack of anti - hbcab production as they have helper t - cell tolerance to hbv core ag and hbeag induced by transplacental maternal hbvag . another reason for lack of anti - hbcab production is due to immunocompromised condition like uncontrolled hiv infection . if the former circumstance is true , these patients should be hbve ag - positive but such data is not available in this study . clinical implication of the absence of anti - hbcab during chronic hbv infection remain largely unknown except that it is not linked to severe hepatic disease course although its impact on hiv progression has never been reported . we found that the frequency of hbv patients without anti - hbcab is not uncommon in our hiv - infected population . together with the poor prognosis , our observation suggests that more attention should be given to this group . however , the results should be interpreted with caution because of the small number of hbv patients without anti - hbcab . after the initiation of haart , in both wealthy and resource - limited countries , the proportion of liver - related mortality increased in hepatitis and hiv co - infected patients [ 2730 ] . unfortunately , adequate treatment for chronic hepatitis is not available in resource - limited countries . a tenofovir - based first - line regimen is now recommended by who and is being adopted in many countries . however , the price of tenofovir needs to fall to allow more widespread access to this drug . currently , the prevailing regimen for chronic hepatitis infection includes lamivudine alone in the most resource - limited countries . thus , most hiv patients with hbv co - infection are inevitably receiving lamivudine monotherapy for hbv infection . the choice of antiretrovirals for such patients should include at least two drugs effective against hbv such as tenofovir . last , screening for hepatitis co - infection at the same time as hiv diagnosis should be urgently implemented . in summary , whereas hcv co - infection showed marginal association with the survival , hbv co - infection , especially without anti - hbcab , increased the all - cause mortality in haart - naive hiv patients in resource - limited countries . in this setting , clinicians and healthcare providers should prioritize hiv / chronic hepatitis co - infected individuals .

summarya total of 755 highly active antiretroviral therapy ( haart)-naive hiv - infected patients were enrolled at a government hospital in thailand from 1 june 2000 to 15 october 2002 . census date of survival was on 31 october 2004 or the date of haart initiation . of 700 ( 926% ) patients with complete data , the prevalence of hepatitis b virus ( hbv ) surface antigen and anti - hepatitis c virus ( hcv ) antibody positivity was 119% and 33% , respectively . eight ( 96% ) hbv co - infected patients did not have anti - hbv core antibody ( anti - hbcab ) . during 11667 person - years of observation ( pyo ) , 258 ( 369% ) patients died [ 221/100 pyo , 95% confidence interval ( ci ) 167278 ] . hbv and probably hcv co - infection was associated with a higher mortality with adjusted hazard ratios ( ahrs ) of 181 ( 95% ci 130253 ) and 190 ( 95% ci 098369 ) , respectively . interestingly , hbv co - infection without anti - hbc ab was strongly associated with death ( ahr 634 , 95% ci 399103 ) . the influence of hepatitis co - infection on the natural history of haart - naive hiv patients requires greater attention .

INTRODUCTION METHODS RESULTS DISCUSSION DECLARATION OF INTEREST

in resource - limited countries , the prevalence of chronic hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) infection is often high , and populations with a high prevalence of hbv and hcv usually overlap with those seriously affected by hiv . in a study from northern india , the reported prevalence of hbv and hcv co - infection in hiv - infected patients was 53% and 24% , respectively . a study in tanzania reported 173% and 181% of hiv - infected patients were co - infected with hbv and hcv , respectively , and an earlier report from thailand showed that the prevalence of hbv infection was 87% and hcv infection 78% in hiv - infected patients . accumulating evidence suggests that hiv co - infection adversely affects the clinical course of hepatitis . increased hbv carriage rates , greater levels of hbv viraemia , more rapid decline in hbv surface antibody , increased reactivation episodes , and faster progression to liver cirrhosis are all characteristic of hiv / hbv co - infected patients [ 5 , 6 ] . in hiv / hcv co - infected patients , faster progression of fibrosis resulting in decompensated cirrhosis have been shown in previous studies [ 7 , 8 ] . as patients on highly active antiretroviral therapy ( haart ) survive much longer , liver failure is becoming the major cause of death in patients with hepatitis co - infection . current international aids society guidelines for the management of hiv recommend that hiv / viral hepatitis co - infected patients should start haart , the same as hiv mono - infected patients . thus , we assume that there are still large groups of patients with hiv / chronic hepatitis co - infection who are not receiving haart in resource - limited countries . for better management of these patients , it is important to know the association between hepatitis co - infection and the natural history of hiv infection . the effect of viral hepatitis co - infection on hiv progression and all - cause mortality before initiating haart remains uncertain . in the majority of previous studies examining hbv and hcv co - infection , the main transmission mode of hiv in participants was homosexual intercourse or injecting drug use ( idu ) and none were conducted with a substantial sample size in asian or african countries where the majority of hiv - infected individuals with hbv vertical transmission reside . the present study aims to evaluate the impact of hepatitis co - infection on all - cause mortality in haart - naive hiv - infected individuals in northern thailand . to address the current research question , we re - analysed our previously conducted natural history cohort of hiv - infected patients in northern thailand [ 20 , 21 ] . the centre was established in october 1995 as an outpatient clinic providing treatment , care and support for hiv - infected patients . the recruitment of this cohort was from 1 july 2000 to 15 october 2002 before the national antiretroviral treatment programme was launched . all adult ( aged > 18 years ) hiv - infected individuals attending the hiv clinic who were haart - naive at the first visit were approached by the research team and enrolled if written consent were obtained . all participants were requested to visit the clinic at least once every 3 months regardless of the presence of clinical symptoms and were followed up from the date of study enrolment until 15 october , 2004 . demographic ( gender and age at enrolment ) data and medical history [ hiv - related symptoms , history of antiretroviral therapy ( art ) and mode of transmission ] of patients were obtained at the study enrolment by well - trained research staff through face - to - face interviews based upon a structured questionnaire . the remaining freeze - stored plasma samples from our previous study were retrospectively tested for hepatitis b surface antigen ( hbsag ) , anti - hepatitis b core antibody ( anti - hbcab ) and antibody to hepatitis c virus ( anti - hcv ) were retrospectively tested using commercially kits : cobas core hbsag ii eia , anti - hbc eia , and eti - ab - hcvk-4 ( diasorin s.p.a . hbsag positivity and anti - hcv positivity were determined to define hbv and hcv co - infection , respectively . causes of death in hiv / hepatitis co - infected patients were investigated by reviewing hospital records . in survival analysis , patients who started haart before 15 october 2004 were regarded as censored on the date of starting haart . meier survival analysis was performed to estimate survival in relation to the existence of hiv / hbv or hcv co - infection . hbv co - infected patients were divided into two subgroups according to the existence of anti - hbcab . additionally , cox 's proportional hazard model was conducted to evaluate the influence of hbv or hcv co - infection on survival adjusted by several factors . in multivariate models , other than hepatitis co - infection status and existence of anti - hbcab , we included all variables with p < 01 in univariate analysis . results were presented as hazard ratios ( hrs ) with 95% confidence intervals ( cis ) . of 755 hiv - infected persons , 55 patients were excluded ( 32 patients had received haart in private clinic or other clinical trial before recruitment , 22 patients had incomplete testing for hepatitis co - infection , and four patients had hbv / hcv dual co - infection ) . the prevalence of hbv and hcv co - infection was 119% and 33% , respectively . of 700 patients , 681 ( 973% ) had both hbsag and anti - hbcab status . hcv co - infection was strongly associated with idu ( p < 0001 ) . the baseline cd4 cell count was significantly lower in hbv vs. hcv co - infected patients whereas there was no difference between these patient groups in their baseline viral load . patients with hbv co - infection were more likely to have aids symptoms compared to hiv mono - infected patients ( p = 002 ) . platelet counts were lowest in hcv co - infected individuals followed by hbv co - infected patients ( p = 003 ) . of 86 hbv co - infected patients , 78 ( 907% ) had anti - hbcab . interestingly , eight ( 93% ) patients did not have anti - hbcab despite being hbsag positive . table 1.baseline characteristics of hiv mono - infected patients and hepatitis co - infected patientscharacteristics ( n = 700)hiv mono - infection(n = 594)hbv co - infection(n = 83)hcv co - infection(n = 23)age ( median , iqr)*33 ( 2937)32 ( 2935)30 ( 2634)male gender ( % ) 244 ( 411)42 ( 506)14 ( 509)mode of transmission ( % ) * heterosexual583 ( 981)79 ( 952)15 ( 652 ) homosexual6 ( 10)1 ( 12)1 ( 43 ) idu0 ( 00)0 ( 00)5 ( 217 ) others or multiple5 ( 08)2 ( 24)2 ( 86 ) unknown0 ( 00)1 ( 12)0 ( 00)previous art ( % ) 129 ( 217)17 ( 205)4 ( 174 ) clinical status ( % ) || asymptomatic312 ( 525)32 ( 386)8 ( 348 ) non - aids symptomatic115 ( 194)15 ( 181)7 ( 304 ) aids symptomatic167 ( 281)36 ( 434)8 ( 348)baseline cd4 cell count ( cells/l ) ( median , iqr)*150 ( 22351)64 ( 19219)334 ( 31459)baseline vial load ( copies / ml ) ( median , iqr)157431 ( 34891446396)169773 ( 69806437116)112334 ( 21415515515)baseline platelet count ( median , iqr)261000 ( 215000319000)237000 ( 191000300000)213000 ( 170000328000)iqr , interquartile range ; idu , injecting drug user ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome. *p < 005 , hiv mono - infection vs. hcv co-infection.p < 005 , hiv mono - infection vs. hbv co-infection.p < 005 , hbv co - infection vs. hcv co-infection.experience with antiretroviral therapy is limited to monotherapy or dual therapy.||definition of aids according to the national guidelines for the clinical management of hiv infection in children and adults , 6th edn . table 2.status of hbsag and anti - hbcabanti - hbcab(+)anti - hbcab()totalhbsag(+)78886hbsag()318277595total396285681hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody.both hbsag and anti - hbcab status data were available for 681 patients . baseline characteristics of hiv mono - infected patients and hepatitis co - infected patients iqr , interquartile range ; idu , injecting drug user ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome . p < 005 , hiv mono - infection vs. hcv co - infection . p < 005 , hiv mono - infection vs. hbv co - infection . p < 005 , hbv co - infection vs. hcv co - infection . status of hbsag and anti - hbcab hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody . total follow - up time was 11667 person - years of observation ( pyo ) with a median patient follow - up of 588 days [ interquartile range ( iqr ) , 317967 ] . during the observation period , 258 ( 369% ) patients died , resulting in a mortality rate of 221/100 pyo ( 95% ci 167278 ) . when stratified according to baseline cd4 cell count , mortality was 646/100 pyo ( 95% ci 591709 ) in patients with cd4 < 50 cells/l compared to 229/100 pyo ( 95% ci 163297 ) in the group with cd4 < 50200 cells/l and 517/100 pyo ( 95% ci 257763 ) in patients with cd4 > 200 cells/l . 1 ) revealed that hbv co - infection significantly increased mortality ( p < 0001 , log - rank test ) . hcv co - infection also tended to increase mortality , but the statistical significance was marginal . the curves for hiv / hbv and hcv / hiv co - infection converge at about 500 days . the influence of hepatitis co - infection on survival analysed by the cox proportional hazard model is presented in table 3a . in univariate analysis , factors associated with death were male gender , previous art treatment , clinical symptom at enrolment , baseline cd4 cell count , baseline viral load , hbv co - infection and existence of anti - hbcab . patients with a low platelet count ( < 150 000 ) were more likely to die than those with a higher platelet count . 1.kaplanmeier survival probability estimate of co - infected and mono - infected individuals showing that hbv co - infection significantly increased mortality . table 3 ( a).influence of hbv or hcv co - infection on survivalvariableshr ( 95% ci)p valueahr ( 95% ci)p valuemale sex259 ( 202333)<0001123 ( 094160)014age < 30 years*079 ( 061101)006081 ( 063105)011previous art067 ( 049092)001116 ( 084161)037transmission mode iduref. 1000049999244 ( 104575)004120 ( 048301)070 5000099999330 ( 139785)0007167 ( 070400)025 100000879 ( 413187)<0001219 ( 096500)006baseline platelet count < 150000152 ( 100229)005116 ( 076178)050hbv co - infection205 ( 149282)<0001181 ( 130253)<0001hcv co - infection126 ( 067238)047190 ( 098369)006hr , hazard ratio ; ci , confidence interval ; ahr , adjusted hazard ratio ; art , antiretroviral therapy ; idu , injecting drug user ; aids , acquired immunodeficiency syndrome. kaplan meier survival probability estimate of co - infected and mono - infected individuals showing that hbv co - infection significantly increased mortality . influence of hbv or hcv co - infection on survival hr , hazard ratio ; ci , confidence interval ; ahr , adjusted hazard ratio ; art , antiretroviral therapy ; idu , injecting drug user ; aids , acquired immunodeficiency syndrome . survival estimates focused on hbv serology interestingly showed that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab ( p < 00001 by log - rank test , fig . in multivariate analysis ( table 3b ) , in addition to symptomatic aids and low cd4 cell count , hbv co - infection without anti - hbcab was a strong risk factor for death ( adjusted hr 634 , 95% ci 399103 ) . 2.kaplanmeier survival probability estimate showing that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab . table 3 ( b).impact of hbs ag and anti - hbc ab status on survivalvariableshr ( 95% ci)p valueahr ( 95% ci)p valuemale sex259 ( 202333)<0001123 ( 096163)011age < 35 years*079 ( 061101)006079 ( 061102)008previous art067 ( 049092)001110 ( 079153)059transmission mode iduref. hbsag(+)anti - hbcab()634 ( 399103)<0001 hbsag(+ ) anti - hbcab(+)162 ( 114229)0007iqr , interquartile range ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome ; hr , hazard ratio ; ahr , adjusted hazard ratio ; ci , confidence interval ; hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody. meier survival probability estimate showing that hbv co - infected individuals without anti - hbcab had the poorest survival compared to hiv mono - infected or hbv co - infected patients with anti - hbcab . impact of hbs ag and anti - hbc ab status on survival iqr , interquartile range ; art , antiretroviral therapy ; aids , acquired immunodeficiency syndrome ; hr , hazard ratio ; ahr , adjusted hazard ratio ; ci , confidence interval ; hbsag , hepatitis b surface antigen ; anti - hbcab , anti - hepatitis b core antibody . although primary causes of death were unknown for nine out of 56 hiv / hepatitis co - infected patients , the majority of deaths ( 46/56 , 821% ) were attributed to aids - related diseases . in the present study with a substantial sample size , we have clearly demonstrated that hbv co - infection significantly increased all - cause mortality of haart - naive hiv patients . it was only after the advent of haart substantially increased life expectancy of hiv - infected individuals that the importance of liver - related death due to hepatitis co - infection was highlighted . our data indicate that the influence of hepatitis co - infection on the natural history of haart - naive hiv patients should not be ignored . we also discovered an increased mortality of hcv co - infected patients compared to hiv mono - infected patients . the absence of hcv viraemia in anti - hcv - positive patients might explain why hcv co - infection did not show the strong association with death . to our knowledge , this is the first study from a resource - limited country to address the adverse influence of hepatitis co - infection on survival in haart - naive hiv patients . our finding implies that the high prevalence of hepatitis co - infection is at least partially responsible for the high mortality observed in hiv - infected individuals in resource - limited countries . several studies have investigated the influence of hepatitis co - infection on the natural course of hiv infection but these studies often include patients receiving haart and show conflicting findings [ 1214 , 16 , 17 , 23 ] . in addition , none of the previous studies has analysed the association between hiv and hbv or hcv co - infection after adjusting for clinical status , viral load , and cd4 cell count in a cohort not receiving haart with substantial sample size . however , we believe that the majority of chronic hbv or hcv co - infected hiv patients died from aids - defined illness rather than liver failure , since they all had significant opportunistic infections with a very low cd4 cell count . these results suggest that hepatitis co - infection accelerates the natural course of hiv infection itself . however , according to our results , hbv co - infection increased the mortality independent of hiv viral load . thus , the increased hiv viral load per se does not fully explain the higher mortality in hbv co - infected patients . we also considered the possibility that the higher mortality in hbv co - infected patients might have been due to confounding factors . however , our multivariate analysis demonstrated that the association with hbv co - infection was independent of age , gender , transmission route , clinical symptoms and immunological status such as cd4 cell count . it is striking that hbv co - infected patients without anti - hbcab had the poorest prognosis . it is hypothesized that infants born to hbeag - positive carrier mothers may result in the lack of anti - hbcab production as they have helper t - cell tolerance to hbv core ag and hbeag induced by transplacental maternal hbvag . we found that the frequency of hbv patients without anti - hbcab is not uncommon in our hiv - infected population . however , the results should be interpreted with caution because of the small number of hbv patients without anti - hbcab . after the initiation of haart , in both wealthy and resource - limited countries , the proportion of liver - related mortality increased in hepatitis and hiv co - infected patients [ 2730 ] . thus , most hiv patients with hbv co - infection are inevitably receiving lamivudine monotherapy for hbv infection . last , screening for hepatitis co - infection at the same time as hiv diagnosis should be urgently implemented . in summary , whereas hcv co - infection showed marginal association with the survival , hbv co - infection , especially without anti - hbcab , increased the all - cause mortality in haart - naive hiv patients in resource - limited countries .

polycyclic aromatic hydrocarbons ( pahs ) are a group of highly toxic and persistent organic pollutants that are widely distributed in the environment worldwide . they are potentially dangerous due to their carcinogenicity and mutagenicity ; thus , their removal is an issue of great interest . the pahs degradation can be quantified by luminescence techniques such as fluorescence , due to their high quantum yield and relatively easy analysis , since fluorescence methods do not require extensive pretreatment of the sample and can be used with conventional instrumentation ( spectrofluorometer ) . one of the compounds used as model in studies of pahs degradation by microorganisms is anthracene , a polycyclic aromatic hydrocarbon of three rings , which has been listed as one of the priority environmental pollutants by the united states environmental protection agency . there is insufficient information to classify the anthracene as a substance that causes cancer ( http://www.epa.gov/osw/hazard/wastemin/priority.htm ) but causes concern due to the fact that their structure is resemblant to carcinogenic pahs such as benzo[a]pyrene and benzo[a]anthracene . therefore , anthracene is usually used as a pah model in studies of degradation [ 68 ] . traditional fluorescence collects a spectrum by scanning the excitation wavelength ( exc ) while the emission wavelength is fixed ( em ) ( excitation spectrum ) . the emission spectrum is obtained in a similar manner ; in this case , the excitation wavelength is constant ( exc ) , and the emission wavelength is scanned ( em ) . this method is known as excitation - emission fluorescence or excitation fluorescence and emission fluorescence . both techniques are extensively used for individual pahs analysis due to their high sensitivity , selectivity , speed , and relatively low cost [ 9 , 10 ] . this fluorescence method is not useful in the multicomponent analysis of pahs because these compounds have similar structures and there is overlapping of the spectral bands , which are generally wide . synchronous fluorescence is another method of fluorescence spectroscopy that emerged in 1971 . in one of its variants , it has been reported that this technique increases the selectivity ( the broad excitation - emission fluorescence bands are better defined ) and maintains the high sensitivity , resulting in the possibility of analysing multicomponent samples ( which surpasses conventional excitation - emission fluorescence method ) . the bioremediation of pahs contaminated environments is possible with halotolerant or alkalitolerant actinomycetes such as arthrobacter crystallopoietes , arthrobacter arilaitensis , micrococcus sp . , dietzia sp . , and rhodococcus sp . [ 13 , 14 ] . however , there are no reports about the use of haloalkalitolerant actinomycetes ( polyextremophile microorganisms able to live in either the absence or presence of salt , and in presence of ph values in the range 811 ) which were able to grow in pahs polluted environments . the use of these microorganisms in bioremediation processes is more convenient because unlike halophilic or alkalophilic ones ( which grow at a specific nacl and ph value ) , haloalkalitolerant grow within a range of nacl and ph . a general method for studying the pahs bioremediation capacity of haloalkalitolerant actinomycetes is by preparing culture media containing pahs and measuring the residual anthracene concentration in the cultures , by fluorescence , for instance , at different times . one of the culture media used to grow bacteria , when fluorescence measurements are going to be carried out , is the minimal salt medium ( msm ) because it is considered a transparent medium from the fluorescence point of view [ 1517 ] . however , these haloalkalitolerant actinomycetes require nacl concentration for growing , and it is already known that fluorescence signal is affected by the salinity of the sample regardless of the fluorescence method employed . the main question of this work is as follows : would it be adequate to use excitation fluorescence , emission fluorescence , or synchronous fluorescence to study the biodegrading capacity of haloalkalitolerant actinomycetes requiring high nacl concentration for their growth ( halotolerant bacteria ) ? and the objective is carrying out a comparative study based on the sensitivity , linearity , and detection limits of the excitation , emission , and synchronous fluorescence methods during the quantification of the residual anthracene concentration from haloalkalitolerant cultures in order to establish the most proper fluorescence method to study the pahs biodegrading capacity of these kind of bacteria . these strains had been already identified by the 16s rrna sequence . they tolerate % nacl and ph ranges as follow : kocuria palustris ( 025% ; ph 512 ) , kocuria rosea ( 010% ; ph 511 ) ; microbacterium testaceum ( 010% ; ph 511 ) ; and four strains of nocardia farcinica ( 03% ; ph 510 ) . however their optimal growth conditions are as follows : kocuria palustris ( 10% nacl ) ; kocuria rosea and microbacterium testaceum ( 3% nacl ) ; and four strains of nocardia farcinica ( 0.5% nacl ) . for all strains in this work the haloalkalitolerant actinomycetes were always cultivated at their optimal conditions for guaranteeing their best performance . nucleotide sequence data of these strains are available in genbank database under the accession numbers from kp100512 to kp100518 . minimal salt medium ( msm ) mg / l ; k2hpo4 , 200 mg / l ; mgso47h2o , 200 mg / l ; cacl22h2o , 100 mg / l ; fecl3h2o , 5 mg / l ; ( nh4)6mo7o24h2o , 1 mg / l . three variants of this medium were prepared with different nacl concentrations ( 0.5% , 3% , and 10% ) for optimal grow of each strain . each solution was adjusted to ph 8 and sterilised in an autoclave at 121c for 15 minutes . these msm media were divided in two sets ; one set was the three msm media already prepared and to the second one anthracene was added as follows . an initial stock solution of 54 g / ml of anthracene [ 2.7 mg of solid anthracene ( fluka ) dissolved in acetone ] was prepared in an amber - colour volumetric flask . an aliquot of 0.5 ml of this solution was transferred to three 100 ml of these flasks , and msm-0.5% , msm-3% , and msm-10% were , respectively , filled to the mark . these three 0.27 g / ml anthracene media were called anth-0.5% , anth-3% , and anth-10% , respectively . they were sterilised by membrane filtration ( millipore filter , millex with a 0.22 m durapore membrane , slgv033rs ) . from previous studies it is known that the exponential growth phase of these strains in msm media concludes at 7.5 h for all strains , except kocuria palustris , which concludes at 10 h. therefore , the inocula in this work were collected at 4 and 5 h , respectively . volumes of each inoculum equivalent to a od600 nm = 0.25 [ 0.5 nephelometric turbidity units ( ntu ) ] were transferred to 60 ml of the corresponding anth and msm media , contained in screw cap erlenmeyer flasks . in order to avoid the light , the inoculated media were incubated at room temperature and 150 rpm during 48 h. after 1 , 24 , and 48 h of incubation , 5 ml of each msm cultures was centrifuged ( 10,000 rpm , 5 minutes , room temperature ) for supernatant and biomass separation . these supernatants were analysed by excitation , emission , and synchronous fluorescence ( as described in section 2.7 ) to investigate whether the strains produced any metabolite that could interfere with the anthracene fluorescence signal . other samples also analysed by this three fluorescence method were empty cuvette , distilled water , msm-0.5% , msm-3% , and msm-10% . all these spectra make up the matrices signals ; therefore there is a matrix signal for each nacl concentration in each fluorescence method . five millilitres of the anth-0.5% , anth-3% , and anth-10% media without inoculation was analysed by the three fluorescence methods ( section 2.7 ) at 0 , 1 , 24 , and 48 h to monitor any possible anthracene loss by evaporation , sublimation , or photodegradation process . after 1 , 24 , and 48 h of incubation , 5 ml of each anth cultures was centrifuged ( 10,000 rpm , 5 minutes , room temperature ) for supernatant and biomass separation . the residual anthracene concentration was determined in each supernatant by excitation , emission , and synchronous fluorescence ( as described in section 2.7 ) . the initial concentration of anthracene ( t = 0 ) corresponds to the anthracene concentration of anth media without inoculation ( control samples ) . one ml of the 54 g / ml anthracene stock solution was transferred to three 100 ml amber - colour volumetric flasks covered , and msm-0.5% , msm-3% , and msm-10% were , respectively , filled to the mark . these 540 ng / ml anthracene standard solutions were called cc-0.5% , cc-3% , and cc-10% , respectively . they were later used in the constructions of the anthracene calibration curves . from the cc-0.5% , cc-3% , and cc-10% standard solutions , the volumes necessary to prepare 3 ml of 360 , 270 , 180 , 90 , 45 , and 22.5 ng / ml anthracene standard solutions were taken , respectively . each solution was completed to 3 ml volume using the corresponding msm-0.5% , msm-3% , and msm-10% media , transferred to the spectrofluorometer cuvette followed by the acquisition of the excitation , emission , and synchronous fluorescence spectra . each fluorescence intensity was corrected by the inner - filter effect using the absorbance - based approach ( aba ) [ 18 , 19 ] . this approach uses the measured absorbance ( a ) at each pair of excitation ( exc ) and emission ( em ) wavelengths to convert the observed fluorescence intensity ( fobs ) into the corrected fluorescence intensity ( fcorr ) according to the following equation:(1)fcorr = fobsantilogaexc+aem2.the absorbance measurements were carried out in a spectrophotometer ( perkin - elmer uv - vis , model 551s ) , using 1 cm quartz cuvette . the absorbance spectrum was recorded from 300 to 500 nm . from these corrected fluorescence intensities ( fcorr ) , three calibration curves ( fluorescence intensity versus anthracene concentration ) these nine curves were fitted to a straight line using the weighted least - squares model , and the slopes of these lines are the sensitivities of the methods for determining anthracene under the three different % nacl employed . the linearity was evaluated by the determination coefficient ( r ) and the regression coefficient ( r ) . the detection limits for each % nacl condition in each fluorescence method was calculated according to the equation : dl ( ng / ml ) = [ ( maximal background signal ) + 3 ( standard deviation of the background signal)/sensitivity ] . the maximal background signal for each fluorescence method and nacl concentration was taken from the set of spectra that makes up each matrix signal ( section 2.4.1 ) . in section 2.4.1 it was described that each measurement was repeated 5 times ; therefore the standard deviation of the background signal was calculated from there . the fluorescence spectra of the empty cuvette , distilled water , msm media , standard solution of each cc calibration curve , inoculated msm media ( matrix ) at 1 , 24 , and 48 h , inoculated anth media ( samples ) at 1 , 24 , and 48 h , and the anth media at 0 , 1 , 24 , and 48 h ( control samples ) were scanned by a spectrofluorometer ( horiba , fluoromax-3 ) at room temperature . the fluorescence measurements were performed using a 1 1 cm standard quartz cell . the conditions for spectra collection in the three methods ( excitation , emission , and synchronous ) were the following:(i)excitation fluorescence spectra ( 230390 ) nm : emission wavelength of 400 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 0.5 mm.(ii)emission fluorescence spectra ( 365500 ) nm : excitation wavelength of 340 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm.(iii)synchronous fluorescence spectra ( 200450 ) nm : = 150 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm.from the fluorescence signal of the matrix and cc standard solutions , the emission wavelength , excitation wavelength , and synchronous fluorescence wavelength of anthracene were selected for each % nacl employed . excitation fluorescence spectra ( 230390 ) nm : emission wavelength of 400 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 0.5 mm . emission fluorescence spectra ( 365500 ) nm : excitation wavelength of 340 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm . synchronous fluorescence spectra ( 200450 ) nm : = 150 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm . the relative concentrations of residual anthracene from the anth media determined by the three methods ( excitation , emission , and synchronous ) after 1 h of strain inoculation were statistically compared by a bivariate analysis . the two studied variables were strains ( 7 levels ) and fluorescence method ( 3 levels ) . figure 1 shows the excitation spectra of the net background signal , and of the three diluted anthracene standard solutions ( 22.5 ng / ml ) . the emission wavelength was set at 400 nm because the literature reports that anthracene has an emission band around this value . the net background signal in this figure is composed by those fluorescence signals from the matrix which have a maximal signal in some region of the whole scan . the ideal excitation wavelength is the one that selectively excites the analyte , maximally reducing the excitation of the matrix components ( background signal ) . the figure shows that 340 nm , 352 nm , 358 nm , and 374378 nm bands could be employed as excitation wavelength . two of the excitation s of anthracene ( 352 and 358 nm ) are very close or coincide with the 352 nm excitation of the matrix components ( background signals ) ; consequently , this is not adequate for excitation of the sample because it also excites the matrix . a similar finding is obtained with the excitation that appears at 374 nm , 376 nm , and 378 nm ; some matrix components are excited at these wavelengths . the ideal excitation situation only occurs with the excitation of 340 nm for the three anthracene solutions prepared in different % nacl . therefore , this excitation was used to obtain the emission fluorescence spectra of all analysed samples in this work . figure 2 shows the emission spectra of the net background signal , and of the three diluted anthracene standard solutions ( 22.5 ng / ml ) . the net background signal in this figure is composed of those fluorescence signals from the matrix which have a maximal signal in some region of the whole scan . the ideal emission wavelength is defined as the wavelength where the analyte emission spectrum has the band with lowest contribution of the matrix components ( background signal ) . the figure shows that 380 nm , 401403 nm , and 419425 nm bands could be employed as emission wavelength . from them the 401403 nm is the wavelength range where the anthracene has an intense emission band while the matrix has a minimal emission . therefore , the band at 401 nm was selected for obtaining the subsequent excitation spectra . in this work , = 150 nm was used to acquire the synchronous fluorescence spectra of anthracene because cai et al . experimentally demonstrated that this value of is optimal for obtaining good sensitivity and selectivity . figure 3 shows the synchronous fluorescence spectra of the net background and the 180 ng / ml anthracene standard solutions under this condition . as in the previous cases , the net background signal in this figure is composed of those fluorescence signals from the matrix which have a maximal signal in some region of the whole scan spectra . figure 1 also displays the signals of the 22.5 ng / ml anthracene standard solutions prepared from anth-0.5% , anth-3% , and anth-10% . if there is no effect of the nacl concentration on the excitation fluorescence signal , it is expected that there are no differences in the ( i ) shape , ( ii ) position , and ( iii ) intensity of the bands from these solutions . ( ii ) position : the lower wavelength bands ( 252 nm , 294 nm , 324 nm , and 340 nm ) coincide in position for the three solutions . the bands at higher wavelength are slightly shifted ( taking as reference the 0.5% spectrum ) ; 358 nm band is present in the 0.5% and 3% nacl media but is shifted to 352 nm in the 10% nacl medium . the 376 nm band appears in the 0.5% medium ; in the 3% medium , it is located at 378 nm , and in the 10% medium , it appears at 374 nm . ( iii ) intensity : anthracene bands in 0.5% are more intense than those in 3% , and both are much more intense than those in 10% ( 340 nm band in 10% nacl is 50% less intense than that in 0.5% nacl ) . to quantify anthracene in saline solutions the 340 nm band is very convenient because its position is not shifted with salinity . however , high % nacl reduces band intensity ; therefore the final selection depends on the concentration to measure and the background signal . in this work , to quantify anthracene by excitation fluorescence , the 340 nm band was used for samples in 0.5% and 3% of nacl and the 378 nm band was employed for samples in 10% nacl . figure 2 presents the emission spectra corresponding to the same samples presented in figure 1 , all excited at 340 nm . as in the previous case , the ( i ) shape , ( ii ) position , and ( iii ) intensity of the band spectra are analysed . ( i ) there are no changes in the shape of the emission spectra ; this means that the number of emission bands is the same in all spectra . ( ii ) there is not shifting in the anthracene emission bands in the 0.5% and 3% nacl media . the three emission maxima are at 381 nm , 401 nm , and 425 nm . the position of these maxima is shifted in the 10% nacl medium , the maxima are at 385 nm , 403 nm , and 419 nm . ( iii ) band intensities are similar for anthracene solutions prepared in 0.5% and 3% nacl media ( 425 nm 381 nm < 401 nm ) but different for the 10% media ( 385 nm < 403 nm 419 nm ) . the ideal situation for quantifying anthracene by emission fluorescence is to choose an emission at which the matrix components have little or no emission and the anthracene has an intense band . therefore , the quantification of anthracene in the anth-0.5% and anth-3% media was performed with the 401 nm band , which was only slightly affected by the matrix signal and had a good intensity . in the case of samples in anth-10% the band used was 419 nm . figure 3 displays the synchronous fluorescence spectra with = 150 nm of the 180 ng / ml anthracene standard solution prepared from cc-0.5% , cc-3% , and cc-10% , as well as the net background spectra . it can be observed that the shape of the spectrum of the anthracene from cc-0.5% and cc-3% standard solutions is very similar but varies in the cc-10% solution . band intensity in this fluorescence method behaves in contrast to fluorescence excitation and emission . in this case , in contrast with the excitation and emission fluorescence spectra of anthracene , which are well known , the synchronous fluorescence spectrum varies with the instrumental conditions used during acquisition ( ) , and some bands are influenced in a greater or lesser degree by the solvent . in order to determine the bands that proportionally change and do not vary their position with the anthracene concentration , the synchronous fluorescence spectra of the standard solutions prepared from cc-0.5% , cc-3% , and cc-10% were graphed in figure 4 . from figure 4 it can be noticed that the 22.5 ng / ml anthracene standard solutions in the three media under study have the 250 nm band , but it gradually decreases until disappearance with an increasing anthracene concentration , suggesting that it is associated with or very influenced by the medium . given that the only difference between the three anth media is the nacl concentration , then the intensity of this band is influenced by the medium salinity . our initial idea was to use this band for quantification , because cai et al . demonstrated that at = 150 nm it is optimal for obtaining good sensitivity and selectivity . in our case however , the 250 nm band was not useful probably because although our matrix is aqueous like the one they used , it is not exactly the same . a decision had to be made regarding which signal of the synchronous fluorescence spectrum to use for the anthracene quantification . figure 4(a ) shows bands at 296 nm , 300 nm , 306 nm , 310 nm , and 314 nm which correspond to anthracene concentrations ( ng / ml ) 22.5 , 45 , 90 , 180 , and 360 , respectively . they could be considered shifting of the same band , but there is not proportionality between band intensity and anthracene concentration ; therefore they should be associated with the solvent . this same effect is observed in figure 4(b ) with the 300 nm band corresponding to the 22.5 ng / ml concentration , which shifts with the increased anthracene concentration to reach a value of 316 nm for 360 ng / ml . this feature is also observed in figure 4(c ) with the 294 nm band , corresponding to the 22.5 ng / ml concentration , which shifts as the anthracene concentration increases up to 308 nm at a concentration of 360 ng / ml . figure 4 shows a band at 322324 nm in the three media under study ; this band does not shift with an increasing anthracene concentration , and its intensity varies proportionally with the concentration . therefore , this band is useful for anthracene quantification . in the spectra corresponding to the anth-0.5% and anth-3% media , the same behaviour described previously is observed with the bands at 340 nm , 358 nm , and 378 nm . the spectra of anthracene in the anth-10% medium do not present the bands at 340 nm and 378 nm , and the 358 nm band is shifted to 352 nm . this difference between the spectra is attributed to the different concentrations of nacl present in the three media . as the 340 nm band is most intense for the anth-0.5% and anth-3% media and the 322 nm band is most intense for the anth-10% medium these bands were chosen for anthracene quantification by synchronous fluorescence . the absorbance measured for all anthracene standard solutions at their respective excitation and emission wavelengths were in the range 0.0410.066 , and the ratio fobs / fcorr was 0.9 for the three fluorescence methods . we do not attribute this attenuation in the fluorescence signal to an inner - filter effect because the correction factor was the same in all solutions , and this value changes in the inner - filter effect , being higher at higher concentrations . the maxima absorbance value was slightly over 0.06 , being the inner - filter effect negligible . the corrected fluorescence values from the anthracene standard solutions give a calibration curve as linear as the one obtained from the observed values , demonstrating an attenuation effect but not inner - filter effect . since this attenuation effect is present in all samples equally , it does not affect quantification . table 1 shows the calibration curves obtained from a weighted regression using the least square model . the sensitivities are the slope of the curves , and ideally they should have the same value for the anth-0.5% , anth-3% , and anth-10% media , within the same fluorescence method . this result corroborates the salinity effect on the fluorescence mechanism . among the methods , excitation fluorescence is 1.11.3 times more sensitive than emission , and 2252 times more than synchronous fluorescence . these results agree with other studies reporting that excitation fluorescence and emission fluorescence are extremely sensitive and that synchronous fluorescence is less sensitive but more selective . in our case , however , synchronous fluorescence was not more selective , because band definition was better in excitation and emission fluorescence . from the sensitivities values , it seems that excitation fluorescence is the one less affected by salinity . the sensitivity reduction from anth-0.5% to anth-10% was 20% . in emission fluorescence , this reduction was 30% ; and unexpectedly in synchronous the sensitivity value was not reduced but increased in 188% . calibration curves for excitation and emission fluorescence do not have statistically significant intercept at 95% , meaning that there is not a matrix effect on the analyte fluorescence signal . this is a very important point for a quantification method . in synchronous fluorescence however , the intercept was always statistically significant , meaning that at zero analyte concentration there is a fluorescence signal attributed to the matrix . therefore in this study under our conditions the matrix , unlike excitation and emission fluorescence , has effect on the synchronous signal . the intercept was not constant for the three anth media , indicating that nacl concentration is also contributing to matrix effect . the first one indicates how well the model fits the data . in our case , it is a measure that allows us to determine how certain one can be in making predictions from a certain model / graph . the second one measures the strength and direction of the linear relationship between two variables . in this case , both coefficients are higher than 0.997 , indicating that a straight line is a very adequate model to fit our data ( anthracene concentration versus fluorescence intensity ) for the three fluorescence methods in the concentration range studied and that this linear relationship is strong and positive ( increasing the concentration increases the fluorescence signal ) . in addition , the dilution factor among the standard solutions employed in the calibration curves ( 1 : 16 , 1 : 8 , 1 : 4 , 1 : 2 , 1 : 1.3 ) coincides with the reduction in the fluorescence intensity . therefore , linearity is assured in the anthracene determination in all cases for all methods in the concentration range studied . table 1 also shows the detection limits for anthracene in the three media studied by the three fluorescence methods . the detection limit is an order of magnitude lower in excitation and emission fluorescence than in synchronous fluorescence because the net maximal matrix signal does not vary in intensity in the three methods at the wavelengths selected for quantification ( figures 13 ) , while the anthracene sensitivity decreases between 20 and 50 times from excitation - emission fluorescence to synchronous fluorescence . thus , for ensuring the best accuracy , all solutions were prepared in the same manner , using the same reagents and matrix solution and the same preparation techniques , and measured at the same temperature after the same amount of time . the fluorescence intensity of the anth solutions ( control solutions ) measured at 0 , 1 , 24 , and 48 h by the three methods showed a relative standard deviation ( rsd ) less than 5% , indicating that the incubation time does not affect the fluorescence signal of anthracene and no evaporation , sublimation , or photolysis processes were involved during sampling handling . the capabilities for biodegrading pahs of the genera to which the strains analysed belong have been already reported [ 2023 ] . the residual anthracene values determined in this study are in the range of those reported for other pahs ( naphthalene , phenanthrene , and dihydrophenanthrene ) for the study genera [ 2022 ] . therefore , the decrease in the anthracene fluorescence signal of the samples studied in this work is attributed to the consumption of anthracene by the strains . table 2 lists the values of the initial anthracene concentration and the residual anthracene present in the actinomycetes culture at 0 , 1 , 24 , and 48 h by the three methods . according to the results shown in the table , all strains were capable of significantly transforming anthracene during the first hour . during the intervals 124 h and 2448 h , this transformation was lower . from table 2 the precision in the anthracene concentration a bivariate analysis showed statistical differences ( p < 0.05 ) for the fluorescence methods and for the strains . statistical differences among strains mean that the biodegrading capacity of all these strains is not alike . statistical differences among the methods indicate that the performance of the three fluorescence methods is not statistically similar . other reports have demonstrated that results obtained by excitation and emission fluorescence ( in the pahs determination ) do not differ from results obtained by other techniques such as gas chromatography or hplc . fluorescence is a very convenient method to study pahs biodegradation because it is simple , easy , fast , inexpensive , very sensitive , and reproducible . for these reasons fluorescence was our method of choice for studying the capabilities of haloalkalitolerant actinomycetes for degrading pahs . these bacteria require nacl for growing ; therefore our study was focussed to investigate the performance of excitation , emission , and synchronous fluorescence in the determination of anthracene in the presence of different nacl concentrations in order to stablish the most proper fluorescence method to study the pahs biodegrading properties of these bacteria . our results show that anthracene excitation fluorescence spectrum in msm ( called anth ) does not change in shape , relative band intensity , or band position with the increasing % nacl . the calibration curves for the three nacl concentrations indicate that there is not background signal in the fluorescence response ( equation does not have a statistically significant y intercept ) ; the sensitivity is very high , and it is only reduced 20% from the medium containing 10% nacl respective to the one with 0.5% nacl ; the straight line is a very good fitting model for the calibration curves ( r > 0.998 ) ; and fluorescence signal versus concentration is linear in the studied range ( 22.5360 ng / ml ) . the inner - filter effect present in the fluorescence signal of the three calibration curves is negligible . the detection limit obtained , by using culture without anthracene as matrix background , is 11 , 13 , and 20 ng / ml for the media with 0.5% , 3% , and 10% nacl , respectively . in the case of the anthracene emission fluorescence spectra in msm and different nacl concentration , most of the features found in excitation fluorescence are similar with the following differences : the spectrum does not change in shape or band position but the relative band intensity changes , and the sensitivity is reduced 30% when nacl concentration is increased from 0.5% to 10% . the synchronous fluorescence spectra features of anthracene contained in msm with different nacl concentrations quite differ from the previous cases . the y intercept in the calibration curves was statistically significant ; therefore at zero anthracene concentration there is a fluorescence signal most likely coming from the matrix . unlike excitation and emission spectra where the sensitivity was reduced with the increasing nacl concentration , in synchronous fluorescence the sensitivity this result clearly shows that , in this fluorescence method , under the conditions used in this work the anthracene signal is overestimated due to matrix contribution . detection limits are worse than those from excitation and emission methods , being one order higher . selectivity was not improved in the anthracene determination using = 150 nm , in comparison to excitation and emission fluorescence . despite the fact that the three fluorescence methods indicate anthracene biodegradation , the results described above demonstrated that synchronous fluorescence is not the most adequate method ( among fluorescence methods ) to determining anthracene in haloalkalitolerant actinomycetes cultures with nacl concentrations as high as 10% , when = 150 nm is used . although excitation and emission fluorescence behave very similarly , excitation fluorescence performs slightly better , being our proposed fluorescence method for determining the residual anthracene concentration present in cultures of haloalkalitolerant actinomycetes potentially degraders of pahs , as long as standards and samples are prepared exactly in the same manner .

polycyclic aromatic hydrocarbons ( pahs ) are compounds that can be quantified by fluorescence due to their high quantum yield . haloalkalitolerant bacteria tolerate wide concentration ranges of nacl and ph . they are potentially useful in the pahs bioremediation of saline environments . however , it is known that salinity of the sample affects fluorescence signal regardless of the method . the objective of this work was to carry out a comparative study based on the sensitivity , linearity , and detection limits of the excitation , emission , and synchronous fluorescence methods , during the quantification of the residual anthracene concentration from the following haloalkalitolerant actinomycetes cultures kocuria rosea , kocuria palustris , microbacterium testaceum , and 4 strains of nocardia farcinica , in order to establish the proper fluorescence method to study the pahs biodegrading capacity of haloalkalitolerant actinobacteria . the study demonstrated statistical differences among the strains and among the fluorescence methods regarding the anthracene residual concentration . the results showed that excitation and emission fluorescence methods performed very similarly but sensitivity in excitation fluorescence is slightly higher . synchronous fluorescence using = 150 nm is not the most convenient method . therefore we propose the excitation fluorescence as the fluorescence method to be used in the study of the pahs biodegrading capacity of haloalkalitolerant actinomycetes .

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

polycyclic aromatic hydrocarbons ( pahs ) are a group of highly toxic and persistent organic pollutants that are widely distributed in the environment worldwide . they are potentially dangerous due to their carcinogenicity and mutagenicity ; thus , their removal is an issue of great interest . the pahs degradation can be quantified by luminescence techniques such as fluorescence , due to their high quantum yield and relatively easy analysis , since fluorescence methods do not require extensive pretreatment of the sample and can be used with conventional instrumentation ( spectrofluorometer ) . this method is known as excitation - emission fluorescence or excitation fluorescence and emission fluorescence . both techniques are extensively used for individual pahs analysis due to their high sensitivity , selectivity , speed , and relatively low cost [ 9 , 10 ] . this fluorescence method is not useful in the multicomponent analysis of pahs because these compounds have similar structures and there is overlapping of the spectral bands , which are generally wide . in one of its variants , it has been reported that this technique increases the selectivity ( the broad excitation - emission fluorescence bands are better defined ) and maintains the high sensitivity , resulting in the possibility of analysing multicomponent samples ( which surpasses conventional excitation - emission fluorescence method ) . however , there are no reports about the use of haloalkalitolerant actinomycetes ( polyextremophile microorganisms able to live in either the absence or presence of salt , and in presence of ph values in the range 811 ) which were able to grow in pahs polluted environments . the use of these microorganisms in bioremediation processes is more convenient because unlike halophilic or alkalophilic ones ( which grow at a specific nacl and ph value ) , haloalkalitolerant grow within a range of nacl and ph . a general method for studying the pahs bioremediation capacity of haloalkalitolerant actinomycetes is by preparing culture media containing pahs and measuring the residual anthracene concentration in the cultures , by fluorescence , for instance , at different times . one of the culture media used to grow bacteria , when fluorescence measurements are going to be carried out , is the minimal salt medium ( msm ) because it is considered a transparent medium from the fluorescence point of view [ 1517 ] . however , these haloalkalitolerant actinomycetes require nacl concentration for growing , and it is already known that fluorescence signal is affected by the salinity of the sample regardless of the fluorescence method employed . the main question of this work is as follows : would it be adequate to use excitation fluorescence , emission fluorescence , or synchronous fluorescence to study the biodegrading capacity of haloalkalitolerant actinomycetes requiring high nacl concentration for their growth ( halotolerant bacteria ) ? and the objective is carrying out a comparative study based on the sensitivity , linearity , and detection limits of the excitation , emission , and synchronous fluorescence methods during the quantification of the residual anthracene concentration from haloalkalitolerant cultures in order to establish the most proper fluorescence method to study the pahs biodegrading capacity of these kind of bacteria . they tolerate % nacl and ph ranges as follow : kocuria palustris ( 025% ; ph 512 ) , kocuria rosea ( 010% ; ph 511 ) ; microbacterium testaceum ( 010% ; ph 511 ) ; and four strains of nocardia farcinica ( 03% ; ph 510 ) . however their optimal growth conditions are as follows : kocuria palustris ( 10% nacl ) ; kocuria rosea and microbacterium testaceum ( 3% nacl ) ; and four strains of nocardia farcinica ( 0.5% nacl ) . from previous studies it is known that the exponential growth phase of these strains in msm media concludes at 7.5 h for all strains , except kocuria palustris , which concludes at 10 h. therefore , the inocula in this work were collected at 4 and 5 h , respectively . in order to avoid the light , the inoculated media were incubated at room temperature and 150 rpm during 48 h. after 1 , 24 , and 48 h of incubation , 5 ml of each msm cultures was centrifuged ( 10,000 rpm , 5 minutes , room temperature ) for supernatant and biomass separation . these supernatants were analysed by excitation , emission , and synchronous fluorescence ( as described in section 2.7 ) to investigate whether the strains produced any metabolite that could interfere with the anthracene fluorescence signal . five millilitres of the anth-0.5% , anth-3% , and anth-10% media without inoculation was analysed by the three fluorescence methods ( section 2.7 ) at 0 , 1 , 24 , and 48 h to monitor any possible anthracene loss by evaporation , sublimation , or photodegradation process . the residual anthracene concentration was determined in each supernatant by excitation , emission , and synchronous fluorescence ( as described in section 2.7 ) . they were later used in the constructions of the anthracene calibration curves . each solution was completed to 3 ml volume using the corresponding msm-0.5% , msm-3% , and msm-10% media , transferred to the spectrofluorometer cuvette followed by the acquisition of the excitation , emission , and synchronous fluorescence spectra . from these corrected fluorescence intensities ( fcorr ) , three calibration curves ( fluorescence intensity versus anthracene concentration ) these nine curves were fitted to a straight line using the weighted least - squares model , and the slopes of these lines are the sensitivities of the methods for determining anthracene under the three different % nacl employed . the detection limits for each % nacl condition in each fluorescence method was calculated according to the equation : dl ( ng / ml ) = [ ( maximal background signal ) + 3 ( standard deviation of the background signal)/sensitivity ] . the fluorescence spectra of the empty cuvette , distilled water , msm media , standard solution of each cc calibration curve , inoculated msm media ( matrix ) at 1 , 24 , and 48 h , inoculated anth media ( samples ) at 1 , 24 , and 48 h , and the anth media at 0 , 1 , 24 , and 48 h ( control samples ) were scanned by a spectrofluorometer ( horiba , fluoromax-3 ) at room temperature . the conditions for spectra collection in the three methods ( excitation , emission , and synchronous ) were the following:(i)excitation fluorescence spectra ( 230390 ) nm : emission wavelength of 400 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 0.5 mm. (iii)synchronous fluorescence spectra ( 200450 ) nm : = 150 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm.from the fluorescence signal of the matrix and cc standard solutions , the emission wavelength , excitation wavelength , and synchronous fluorescence wavelength of anthracene were selected for each % nacl employed . synchronous fluorescence spectra ( 200450 ) nm : = 150 nm , resolution of 2 nm , integration time = 0.5 s , and slits of 2 nm . the relative concentrations of residual anthracene from the anth media determined by the three methods ( excitation , emission , and synchronous ) after 1 h of strain inoculation were statistically compared by a bivariate analysis . two of the excitation s of anthracene ( 352 and 358 nm ) are very close or coincide with the 352 nm excitation of the matrix components ( background signals ) ; consequently , this is not adequate for excitation of the sample because it also excites the matrix . in this work , = 150 nm was used to acquire the synchronous fluorescence spectra of anthracene because cai et al . if there is no effect of the nacl concentration on the excitation fluorescence signal , it is expected that there are no differences in the ( i ) shape , ( ii ) position , and ( iii ) intensity of the bands from these solutions . the 376 nm band appears in the 0.5% medium ; in the 3% medium , it is located at 378 nm , and in the 10% medium , it appears at 374 nm . in this work , to quantify anthracene by excitation fluorescence , the 340 nm band was used for samples in 0.5% and 3% of nacl and the 378 nm band was employed for samples in 10% nacl . as in the previous case , the ( i ) shape , ( ii ) position , and ( iii ) intensity of the band spectra are analysed . ( ii ) there is not shifting in the anthracene emission bands in the 0.5% and 3% nacl media . the ideal situation for quantifying anthracene by emission fluorescence is to choose an emission at which the matrix components have little or no emission and the anthracene has an intense band . therefore , the quantification of anthracene in the anth-0.5% and anth-3% media was performed with the 401 nm band , which was only slightly affected by the matrix signal and had a good intensity . figure 3 displays the synchronous fluorescence spectra with = 150 nm of the 180 ng / ml anthracene standard solution prepared from cc-0.5% , cc-3% , and cc-10% , as well as the net background spectra . it can be observed that the shape of the spectrum of the anthracene from cc-0.5% and cc-3% standard solutions is very similar but varies in the cc-10% solution . band intensity in this fluorescence method behaves in contrast to fluorescence excitation and emission . in this case , in contrast with the excitation and emission fluorescence spectra of anthracene , which are well known , the synchronous fluorescence spectrum varies with the instrumental conditions used during acquisition ( ) , and some bands are influenced in a greater or lesser degree by the solvent . in order to determine the bands that proportionally change and do not vary their position with the anthracene concentration , the synchronous fluorescence spectra of the standard solutions prepared from cc-0.5% , cc-3% , and cc-10% were graphed in figure 4 . from figure 4 it can be noticed that the 22.5 ng / ml anthracene standard solutions in the three media under study have the 250 nm band , but it gradually decreases until disappearance with an increasing anthracene concentration , suggesting that it is associated with or very influenced by the medium . demonstrated that at = 150 nm it is optimal for obtaining good sensitivity and selectivity . in our case however , the 250 nm band was not useful probably because although our matrix is aqueous like the one they used , it is not exactly the same . a decision had to be made regarding which signal of the synchronous fluorescence spectrum to use for the anthracene quantification . they could be considered shifting of the same band , but there is not proportionality between band intensity and anthracene concentration ; therefore they should be associated with the solvent . this feature is also observed in figure 4(c ) with the 294 nm band , corresponding to the 22.5 ng / ml concentration , which shifts as the anthracene concentration increases up to 308 nm at a concentration of 360 ng / ml . figure 4 shows a band at 322324 nm in the three media under study ; this band does not shift with an increasing anthracene concentration , and its intensity varies proportionally with the concentration . the absorbance measured for all anthracene standard solutions at their respective excitation and emission wavelengths were in the range 0.0410.066 , and the ratio fobs / fcorr was 0.9 for the three fluorescence methods . we do not attribute this attenuation in the fluorescence signal to an inner - filter effect because the correction factor was the same in all solutions , and this value changes in the inner - filter effect , being higher at higher concentrations . the corrected fluorescence values from the anthracene standard solutions give a calibration curve as linear as the one obtained from the observed values , demonstrating an attenuation effect but not inner - filter effect . among the methods , excitation fluorescence is 1.11.3 times more sensitive than emission , and 2252 times more than synchronous fluorescence . these results agree with other studies reporting that excitation fluorescence and emission fluorescence are extremely sensitive and that synchronous fluorescence is less sensitive but more selective . in our case , however , synchronous fluorescence was not more selective , because band definition was better in excitation and emission fluorescence . from the sensitivities values , it seems that excitation fluorescence is the one less affected by salinity . calibration curves for excitation and emission fluorescence do not have statistically significant intercept at 95% , meaning that there is not a matrix effect on the analyte fluorescence signal . in synchronous fluorescence however , the intercept was always statistically significant , meaning that at zero analyte concentration there is a fluorescence signal attributed to the matrix . therefore in this study under our conditions the matrix , unlike excitation and emission fluorescence , has effect on the synchronous signal . in our case , it is a measure that allows us to determine how certain one can be in making predictions from a certain model / graph . in this case , both coefficients are higher than 0.997 , indicating that a straight line is a very adequate model to fit our data ( anthracene concentration versus fluorescence intensity ) for the three fluorescence methods in the concentration range studied and that this linear relationship is strong and positive ( increasing the concentration increases the fluorescence signal ) . in addition , the dilution factor among the standard solutions employed in the calibration curves ( 1 : 16 , 1 : 8 , 1 : 4 , 1 : 2 , 1 : 1.3 ) coincides with the reduction in the fluorescence intensity . therefore , linearity is assured in the anthracene determination in all cases for all methods in the concentration range studied . table 1 also shows the detection limits for anthracene in the three media studied by the three fluorescence methods . the detection limit is an order of magnitude lower in excitation and emission fluorescence than in synchronous fluorescence because the net maximal matrix signal does not vary in intensity in the three methods at the wavelengths selected for quantification ( figures 13 ) , while the anthracene sensitivity decreases between 20 and 50 times from excitation - emission fluorescence to synchronous fluorescence . the fluorescence intensity of the anth solutions ( control solutions ) measured at 0 , 1 , 24 , and 48 h by the three methods showed a relative standard deviation ( rsd ) less than 5% , indicating that the incubation time does not affect the fluorescence signal of anthracene and no evaporation , sublimation , or photolysis processes were involved during sampling handling . the residual anthracene values determined in this study are in the range of those reported for other pahs ( naphthalene , phenanthrene , and dihydrophenanthrene ) for the study genera [ 2022 ] . therefore , the decrease in the anthracene fluorescence signal of the samples studied in this work is attributed to the consumption of anthracene by the strains . table 2 lists the values of the initial anthracene concentration and the residual anthracene present in the actinomycetes culture at 0 , 1 , 24 , and 48 h by the three methods . according to the results shown in the table , all strains were capable of significantly transforming anthracene during the first hour . from table 2 the precision in the anthracene concentration a bivariate analysis showed statistical differences ( p < 0.05 ) for the fluorescence methods and for the strains . statistical differences among strains mean that the biodegrading capacity of all these strains is not alike . statistical differences among the methods indicate that the performance of the three fluorescence methods is not statistically similar . other reports have demonstrated that results obtained by excitation and emission fluorescence ( in the pahs determination ) do not differ from results obtained by other techniques such as gas chromatography or hplc . fluorescence is a very convenient method to study pahs biodegradation because it is simple , easy , fast , inexpensive , very sensitive , and reproducible . these bacteria require nacl for growing ; therefore our study was focussed to investigate the performance of excitation , emission , and synchronous fluorescence in the determination of anthracene in the presence of different nacl concentrations in order to stablish the most proper fluorescence method to study the pahs biodegrading properties of these bacteria . the calibration curves for the three nacl concentrations indicate that there is not background signal in the fluorescence response ( equation does not have a statistically significant y intercept ) ; the sensitivity is very high , and it is only reduced 20% from the medium containing 10% nacl respective to the one with 0.5% nacl ; the straight line is a very good fitting model for the calibration curves ( r > 0.998 ) ; and fluorescence signal versus concentration is linear in the studied range ( 22.5360 ng / ml ) . the inner - filter effect present in the fluorescence signal of the three calibration curves is negligible . in the case of the anthracene emission fluorescence spectra in msm and different nacl concentration , most of the features found in excitation fluorescence are similar with the following differences : the spectrum does not change in shape or band position but the relative band intensity changes , and the sensitivity is reduced 30% when nacl concentration is increased from 0.5% to 10% . the y intercept in the calibration curves was statistically significant ; therefore at zero anthracene concentration there is a fluorescence signal most likely coming from the matrix . unlike excitation and emission spectra where the sensitivity was reduced with the increasing nacl concentration , in synchronous fluorescence the sensitivity this result clearly shows that , in this fluorescence method , under the conditions used in this work the anthracene signal is overestimated due to matrix contribution . detection limits are worse than those from excitation and emission methods , being one order higher . selectivity was not improved in the anthracene determination using = 150 nm , in comparison to excitation and emission fluorescence . despite the fact that the three fluorescence methods indicate anthracene biodegradation , the results described above demonstrated that synchronous fluorescence is not the most adequate method ( among fluorescence methods ) to determining anthracene in haloalkalitolerant actinomycetes cultures with nacl concentrations as high as 10% , when = 150 nm is used . although excitation and emission fluorescence behave very similarly , excitation fluorescence performs slightly better , being our proposed fluorescence method for determining the residual anthracene concentration present in cultures of haloalkalitolerant actinomycetes potentially degraders of pahs , as long as standards and samples are prepared exactly in the same manner .

while inhaled corticosteroid ( ics)-containing regimens benefit certain patients with chronic obstructive pulmonary disease ( copd ) , concerns about the inappropriate overuse of ics in real - life practice , their clinical benefit and long - term risks , and considerable waste of health care resources that could be better used on other more appropriate management strategies prompted studies to evaluate whether certain patients already on ics therapy fare better without it.1 this perspective paper explores the evidence available on ics withdrawal in patients with copd and the potential phenotypes that can be withdrawn from ics therapy . furthermore , an algorithm for clinical practice is proposed to address the following critical questions : 1 ) in which patients is ics withdrawal safe ? and 2 ) how to withdraw ics in appropriate patients ? although ics have been long used in the management of copd , evidence supporting their use has been considered equivocal and their positioning in guidelines as controversial . according to the latest 2015 update of the global initiative for chronic obstructive lung disease ( gold ) report , ics in combination with a long - acting 2-agonist ( laba ) and/or long - acting muscarinic antagonist ( lama ) is the first recommended choice for patients with a high - risk of exacerbations ( groups c and d ; figure 1).2 in addition to this international gold standard guideline , a number of national and multinational guidelines exist ; however , their recommendations pertaining to the use of ics are inconsistent with those of the gold report , in part , due to the varying criteria for categorizing patients with copd and different interpretations of the equivocal evidence supporting ics use.1,37 despite this , there is consensus that ics are indicated in combination with long - acting bronchodilators ( labds ) for patients with copd at risk of exacerbations and/or in those with asthma copd overlap syndrome ( acos ) , but never as monotherapy . it is not surprising that there is a tremendous amount of confusion surrounding the use of ics for the management of copd in clinical practice . several studies in multiple countries found discrepancies between guideline recommendations and real - life practice regarding ics use in patients with copd.810 in fact , ics are being widely prescribed to the majority of patients with copd , many of whom do not meet the recommendation criteria for ics use ( ie , 38.8% and 51.8% of gold groups a and b , respectively).10 market research estimates suggest that ics are used by > 70% of patients with copd , and given as initial therapy to > 50% of newly diagnosed patients , mostly in combination with a laba.11 these high real - world utilization rates are also corroborated by the treatment profiles of patients entering recent randomized controlled trials ( rcts ) , where ~35% of patients classified into either gold group a or b were receiving ics at baseline.12,13 the introduction of ics in the management of copd was rather unorthodox and unsubstantiated for nearly 20 years ( summarized in table 1 and previously reviewed in detail elsewhere).1,11,14,15 briefly , in the 1980s , ics were adopted in copd on the basis of their effectiveness in asthma rather than scientific evidence.11,16 the earliest rcts of ics therapy in mild copd were conducted in the late 1990s ; however , their outcomes were negative.11,15,17,18 isolde ( inhaled steroids in obstructive lung disease in europe ) was the first trial to demonstrate a beneficial effect of ics on the occurrence of exacerbations in patients with moderate - to - severe copd ; albeit , no benefit on the rate of lung function decline was observed.19 in 2001 , a large population - based cohort study of 22,620 patients with copd and a very recent hospitalization found that those who received ics within 90 days postdischarge had 24% fewer rehospitalizations and a 29% risk reduction for mortality during a 1-year follow - up.20 although these findings are not definitive due to their observational nature , they suggest that patients who have had a hospitalization in the past year should continue receiving ics . from 2002 onward , the subsequent cohort of rcts began evaluating ics in combination with labds.11,21,22 in the landmark torch ( towards a revolution in copd health ) trial , it was found that ics plus a laba ( ics + laba ) resulted in significantly fewer exacerbations , slower rate of decline in lung function , and improved health status ; however , there was only a trend toward improved survival.22 these findings formed the initial basis for the inclusion of ics - containing treatment regimens in clinical guidelines.2,11 at this time , the importance of exacerbations as a key determinant of health status and its association with a faster decline in lung function was being recognized , and the prevention of exacerbations became a key objective of copd management.16 of note , a post hoc analysis of the torch data , which evaluated the independent contribution of ics and laba , found that the benefits of ics + laba were actually provided by the laba.1,15,23 soon after torch , the inspire ( investigating new standards for prophylaxis in reduction of exacerbations ) trial was the first to show that ics + laba was no more effective than a lama in preventing exacerbations in patients with severe copd , which initiated the discussion of whether adding ics to labds is beneficial.24 indeed , a recent cochrane meta - analysis questioned the superiority of ics + laba combinations over laba alone in preventing exacerbations.25 lastly , the findings of the summit ( study to understand mortality and morbidity in copd ) trial were recently presented at the 2015 european respiratory society congress.26 the summit trial to evaluate the impact of a once - daily ics + laba ( fluticasone furoate / vilanterol ) versus the monocomponents on the survival of 16,485 patients with moderate copd and either a history or increased risk of cardiovascular disease ( cvd ) . after a 44-month follow - up , it was found that the risk of mortality on ics + laba was 12.2% lower than on placebo over the study period ; however , this difference was not statistically significant ( p=0.137 ) . while the findings of the torch trial suggested that ics + laba may reduce mortality in patients with copd , particularly those with cvd risk , no study to date has been able to demonstrate a benefit.22,26 a number of local and systemic adverse events have been associated with ics , particularly with long - term and high - dose use.1,15 the local adverse events include pneumonia,27,28 oropharyngeal candidiasis,29 and tuberculosis,30,31 and the systemic adverse events include cataracts,32,33 glaucoma,15 osteoporosis and bone fractures,34 easy bruising,18 type 2 diabetes,35 and even cases of adrenal suppression.36 while there have always been concerns about the risks of developing osteoporosis , cataracts , and diabetes in patients with copd , risk of pneumonia is arguably the most notable and well - established adverse event associated with ics use in copd , as supported by consistent evidence from rcts.1,14,15,24,27,37 corroborating this , a recent canadian population - based cohort study of 103,386 patients with copd who were treated with ics demonstrated that discontinuation of ics was associated with a 37% reduction in the risk of serious pneumonia.38 indeed , in the latest update of the gold report , it is cautioned that long - term ics - containing treatment should not be prescribed outside their indication due to risk of pneumonia and the possibility of a slight increased risk of fractures following long - term exposure.2 as patients with copd are more likely to be older and often have several comorbidities for which they receive multiple medications , they are more susceptible to ics - associated adverse events , and their potential risks need to be weighed against the likely benefits on a case - by - case basis , even in those already on ics.1 given the concerns about the risk benefit profile of ics , there has been a renewed interest in reevaluating the role of ics in copd and identifying which patients can be managed as well with alternate therapies.37 early observational studies and rcts investigating the implications of ics withdrawal in patients with copd found that an abrupt cessation of ics therapy precipitates exacerbations , and results in a deterioration in lung function , symptoms , and health status ( summarized in table 2).3944 however , a meta - analysis of three of these rcts ( ie , cope , cosmic [ copd and seretide : a multi - center intervention and characterization ] , and wisp ) , the only trials deemed to be acceptable in terms of quality and level of bias , determined that withdrawal of ics was not associated with any statistically significant increase in the exacerbation rate , and that the effects on other outcomes , such as lung function and health status , were inconclusive.45 the contradictory findings of these studies may be due to a number of methodological issues , including heterogeneity in patients characteristics , disease severity , outcome definitions ( eg , exacerbations ) , concomitant treatments ( eg , run - in period treatment , maintenance with labds vs placebo ) , and setting ( ie , primary vs secondary care).45,46 of note , since patient inclusion in all trials was based on spirometry while receiving ics therapy , this does not rule out a diagnosis of concomitant asthma , even if spirometry was of no significant reversibility.4244 additionally , in cosmic , the forced expiratory volume in 1 second ( fev1)/forced vital capacity ratios used do not match the diagnostic criteria for copd ( ie , < 70% ) , suggesting that these patients may have had an asthma component , and thereby , cessation of ics could have been detrimental.2,43 according to the latest gold recommendations , patients with copd and a low risk of exacerbations should not be prescribed ics - containing regimens.2 given that a large proportion of patients are already initiated on such regimens , it would be helpful to know if there are consequences associated with ics withdrawal in such populations ( ie , gold groups a and b ) . recently , in the first real - life prospective study ( optimo [ real - life study on the appropriateness of treatment in moderate copd patients ] ) , it was demonstrated that withdrawal of ics in patients with symptomatic , moderate copd ( ie , fev1 > 50% predicted ) at a low risk of exacerbations ( ie , < 2/year ) was not associated with any deterioration in lung function , symptoms , and exacerbation rate over a 6-month observation period.47 these findings were further confirmed by the recent instead ( indacaterol : switching non - exacerbating patients with moderate copd from salmeterol / fluticasone to indacaterol ) trial , the first rct with a clearly defined patient population with moderate copd ( ie , fev1 50%80% predicted ) and no prior exacerbation history , which found that switching from a fixed - dose combination of ics + laba to a laba was not associated with any differences in lung function , symptoms , health status , and exacerbations.48 in addition to supporting the current gold recommendations ( ie , no need for ics in groups a and b ) , the results from both studies suggest that ics therapy can be safely withdrawn from patients with moderate copd and a low risk of exacerbations provided that they are left on maintenance treatment with labds . in all studies to date , withdrawal of ics has been abrupt . it is well known that an abrupt cessation of chronic corticosteroid therapy precipitates rebound systemic effects due to steroid withdrawal symptoms.11,46 currently , there is no clear evidence on whether abrupt ics withdrawal is safe ; however , similar to the approach used to down - titrate ics in patients with asthma , a stepwise withdrawal of ics may minimize the potential risk of rebound steroid effects.11,46,49 adding to the growing clinical evidence - base on the impact of ics withdrawal against a background of labds , the wisdom ( withdrawal of inhaled steroids during optimized bronchodilator management ) trial , which is larger than all of the previous ics withdrawal rcts combined , was the first to assess the question of whether a stepwise withdrawal of ics on top of maintenance therapy with dual bronchodilation ( tiotropium / salmeterol ) had a similar risk of exacerbations in patients with copd and a history of exacerbations ( ie , gold groups c and d , in whom gold recommendations support the addition of ics to labd therapy).2,46,50 in this , 12-month , randomized , double - blind , parallel - group , active - controlled trial , 2,485 patients with copd and a history of exacerbations received triple therapy ( ie , fluticasone propionate / tiotropium / salmeterol ) during a 6-week run - in period . in the ics withdrawal group , the dose of ics was gradually reduced by approximately half in a stepwise fashion every 6 weeks , such that after 12 weeks , ics was completely withdrawn . it was found that there was no difference in the occurrence of moderate or severe exacerbations between the two groups.50 additionally , subgroup analyses revealed no notable differences in the occurrence of exacerbations based on age , sex , smoking status , body mass index , ics or -blocker therapy at screening , chronic bronchitis , gold stage and group , and prior therapy with antibiotics or systemic glucocorticoids . while there was a small , but significant , reduction in lung function when ics was completely withdrawn , the clinical importance of this is unclear . taken together , these findings suggest that even patients with severe copd and a high - risk of exacerbations can be safely withdrawn from ics therapy as long as they are clinically stable and maintained on a background of labds . even with ics use being in line with the gold recommendations , the wisdom results also indicate that not all patients with severe - to - very - severe copd seem to benefit from including ics in their treatment regimen . with the intent of identifying potential markers of ics responsiveness , a substudy on ~500 patients was also performed in the wisdom trial.46 these patients were evaluated for emphysema , bronchiectasis , diffusing capacity for carbon monoxide , and the following biomarkers : adiponectin , leptin , c - reactive protein , interleukin-6 , interleukin-8 , tumor necrosis factor- , fibrinogen , soluble interleukin adhesion molecule-1 , serum amyloid a , procalcitonin , and b - type natriuretic peptide . airway inflammation through differential cell count in sputum and fraction of exhaled nitric oxide ( feno ) was also assessed . unfortunately , no subgroups or biomarkers were found to be associated with an increased likelihood of exacerbations after ics withdrawal;46,51 albeit , certain phenotypes and potential biomarkers that have been previously shown to be associated with ics responsiveness were not evaluated ( discussed in the next section).16 because copd is a complex and heterogeneous disease with several different pathophysiological mechanisms , it is likely that ics may have an effect on some components of the disease , particularly when airway inflammation is present.11,15 increasing evidence suggests that patients with certain copd phenotypes appear to benefit from ics treatment , including patients with acos , frequent exacerbators , and those with eosinophilia.1,6,15,52 acos was only formally recognized by the global initiative for asthma ( gina ) and gold for the first time in 2014 . it has been estimated that acos occurs in up to 25% of patients with copd ; however , establishing its prevalence has been difficult because there is no universally accepted definition for this syndrome.1,5256 according to the gina / gold consensus statement , acos is characterized by persistent airflow limitation and identified by the features that it shares with both asthma and copd.56 in addition to having a history or clinical features of asthma , the presence of a large bronchodilator response ( ie , > 12% and 400 ml ) and eosinophilic inflammation may also point to acos in patients with persistent airway obstruction.15,56 due to the presence of an asthma component , patients with acos are likely to benefit from ics therapy . recently , a large observational study reported a survival benefit in patients with copd starting on ics + laba compared with laba alone ; however , this survival benefit was only observed in those who had a codiagnosis of asthma.57 frequent exacerbators account for approximately one - third of patients with copd.52,58 the definition of an exacerbator is based on expert opinion rather than rigorous phenotypic assessment , with a history of exacerbations being the best predictor of exacerbations across all gold stages.2 in the current gold report , exacerbation risk is based on either gold classification of airway limitation or history of exacerbations ( ie , high exacerbation risk is defined as 2 exacerbations per year or 1 hospitalization ) ; however , not all patients with moderate - to - severe copd ( ie , gold groups c and d ) are necessarily exacerbators , and therefore , require ics.2,59 indeed , in a 26-week , randomized , double - blind , parallel - group trial of patients with moderate - to - severe copd and no history of exacerbations , it was found that dual bronchodilation ( ie , laba + lama ) provided significantly better and clinically relevant improvements in lung function versus ics + laba.12 the findings from the energito trial , which were recently reported at the 2015 european respiratory society congress , further corroborate this point.60 in this randomized , double - blind , double - dummy , four - period crossover trial , lung function was evaluated in patients with moderate - to - severe copd after treatment with laba + lama ( ie , tiotropium / olodaterol ) versus ics + laba ( ie , fluticasone propionate / salmeterol ) . after 6 weeks of treatment , it was found that laba + lama significantly improved lung function compared with ics + laba . thus , subclassification within gold groups c and d may be warranted to take into account nonexacerbators . to complicate matters further , while both history of exacerbations and copd severity are risk factors for repeat exacerbations , there are a number of other risk factors associated with repeat exacerbations that should be considered , including eosinophilic inflammation , comorbidities and extrapulmonary manifestations ( eg , cvd , anxiety , depression , myopathy , reflux disease ) , chronic bronchitis , and increasing age.6 lastly , it has been suggested that up to 30% of patients with copd have eosinophilia.16,61 eosinophilia has been suggested to be predictive of exacerbations in patients with mild - to - moderate copd following withdrawal of ics,62 and both sputum and blood eosinophil levels have been shown to be promising biomarkers of ics responsiveness in patients with copd.63,64 in one rct , a management strategy for patients with copd that suppressed sputum eosinophil levels 3% was found to reduce severe exacerbations.64 although it has been suggested that sputum eosinophil levels may be the most reliable predictor of ics responsiveness in patients with copd to date , obtaining sputum samples is technically demanding and not feasible in routine clinical practice.15 since there is a reasonable correlation between sputum and blood levels , blood eosinophil levels may offer a more practical alternative.63 consequently , blood eosinophil levels are currently a topical issue and defining the appropriate cutoff for determining ics responsiveness in patients with copd is a subject of intense discussion . in a recent post hoc analysis of two replicate , 12-month , double - blind rcts comparing a laba with ics + laba in a total of 3,177 patients with moderate - to - severe copd and a history of 1 exacerbation , it was found that across all doses of ics , ics + laba significantly reduced exacerbations by 29% versus laba alone in patients with blood eosinophil levels of 2%.63 conversely , in another post hoc analysis of two replicate , 26-week , double - blind , double - dummy , parallel - group rcts , an absolute blood eosinophil cutoff of > 300 cells / mm at baseline as opposed to a percent cutoff appeared to best differentiate patients with moderate - to - severe copd who benefited from ics + laba versus laba + lama therapy in terms of reduction in exacerbation risk.65 this observation was corroborated by another post hoc analysis of the randomized , double - blind , parallel - group forward ( foster 48-week trial to reduce exacerbations in copd ) trial , which concluded that a greater reduction in exacerbations was observed when ics was added to a laba in patients with severe copd and a history of exacerbations who had an eosinophil count 279.8 cells / mm.66 using an absolute as opposed to a percent cutoff was further supported by the findings of the copenhagen general population study , which evaluated 7,225 patients with copd over a median follow - up of 3.3 years and found that an absolute blood eosinophil count of 340 cells / mm was a better predictor of both moderate and severe exacerbation risk than a percent cutoff of 2%.67 accordingly , 300 cells / mm can be tentatively used as a cutoff until this value is further validated in prospective trials . meanwhile , it will suffice to recognize that blood eosinophil counts can be easily measured in clinical practice as they are more accessible than sputum eosinophil levels . in addition to eosinophil levels , it has been suggested that feno levels can be used as a surrogate marker of eosinophilic inflammation in patients with copd exacerbations.68,69 a recent review on acos suggested the following feno levels for comparing between patients with asthma , acos , and copd : > 50 , 2550 , and < 25 ppb , respectively.55 these cutoffs are in line with the 2011 american thoracic society feno guidelines , which suggest that feno < 25 ppb provides a strong indication for an unlikely ics response and feno > 50 ppb provides a strong indication for a likely ics response , but feno between 25 and 50 ppb should be interpreted with caution.70,71 since there is an overall paucity of evidence about when and how ics can be safely withdrawn , the criteria for continuing or withdrawing ics in patients with copd are uncertain and more studies are warranted to characterize patients in whom withdrawal is safe . in a recent spanish consensus document on the appropriate use of ics in copd , a panel of 25 experts voted on statements developed by a coordinator group that systematically reviewed scientific evidence on the efficacy and safety of ics , and criteria for ics withdrawal.52 consensus was reached on the following statements concerning ics withdrawal : withdrawal of ics in copd is feasible , patients who discontinue ics should be evaluated in the short - term , and ics withdrawal should be tapered . ics withdrawal was thought to be possible if there is no evidence of acos , no exacerbations in the previous 2 years , and , with a lower level of agreement , in the absence of a positive bronchodilator test and decline after switching from high to low ics doses . however , it should be kept in mind that these criteria are based on expert opinion and need to be validated in rcts . of note , there were also a significant number of statements for which it was difficult to reach a consensus , thereby identifying several areas of uncertainty . currently , no national or international clinical guidelines advocate withdrawal of ics nor do they provide recommendations regarding the safe withdrawal of ics in patients with copd . for reasons previously described , there is an urgent need for a step - by - step algorithm that can be applied in real - life clinical practice . figure 2 proposes such an algorithm and attempts to address the following questions : 1 ) in which patients is ics withdrawal safe ? and 2 ) how to withdraw ics in appropriate patients ? this algorithm takes into account not only exacerbation risk , as per gold , but also the emerging , neglected acos phenotype , as per the gina / gold consensus statement . potential markers of eosinophilia are also considered and noted as optional , as these are still theoretical and need to be tested in rcts . furthermore , the stepwise ics withdrawal protocol on top of maintenance therapy with dual bronchodilation is primarily based on the wisdom trial , although , instead of down - titrating ics every 6 weeks , it is proposed that physicians consider stepping down ics dose every 612 weeks . the rationale for this is to ensure that the effects of ics in the inflammation cascade of copd have been optimized on a physiological level,72 and from a practical perspective , permit enough time to monitor for potential exacerbations . in regards to optimizing bronchodilation following ics withdrawal , we are now more equipped than ever before with a larger armamentarium of novel laba + lama combinations that clinicians can consider for more effective bronchodilation maintenance ( table 3 ) . lastly , it is fully acknowledged that the proposed algorithm will need to be validated , particularly in the real - life setting . additionally , there are a number of potential barriers to using the proposed algorithm that need to be considered . health care resources , such as physician time , access to spirometry , and measurement of eosinophil and feno levels , may be limited or unavailable ; certain countries may not have multiple doses of ics approved to allow for a stepwise reduction ; and patients and physicians may be reluctant to change therapy if it is sufficient in terms of current disease control as opposed to avoiding future risks , such as side effects . patients may also be resistant to changing therapy , as it will require more effort on their part and there is a potential risk of symptom worsening , especially if they end up having a phenotype that should be receiving ics therapy . it is now becoming evident that maintaining or initiating certain patients with copd on ics therapy solely on the basis of reducing their risk of exacerbations may not be necessary , particularly if they are maintained on effective dual bronchodilation with a laba and lama . clinicians need to carefully tailor therapy on a case - by - case basis , and determine who is an appropriate patient for ics therapy , and if a patient is already on ics therapy , how to carefully step down ics therapy without doing harm . currently , no clinical guidelines provide any recommendations regarding the safe withdrawal of ics in patients with copd . accordingly , until this need is met , this perspective article proposes an algorithm for the stepwise withdrawal of ics in real - life clinical practice based on the evidence available to date .

current guidelines for the management of chronic obstructive pulmonary disease ( copd ) recommend limiting the use of inhaled corticosteroids ( ics ) to patients with more severe disease and/or increased exacerbation risk . however , there are discrepancies between guidelines and real - life practice , as ics are being overprescribed . in light of the increasing concerns about the clinical benefit and long - term risks associated with ics use , therapy needs to be carefully weighed on a case - by - case basis , including in patients already on ics . several studies sought out to determine the effects of withdrawing ics in patients with copd . early studies have deterred clinicians from reducing ics in patients with copd as they reported that an abrupt withdrawal of ics precipitates exacerbations , and results in a deterioration in lung function and symptoms . however , these studies were fraught with numerous methodological limitations . recently , two randomized controlled trials and a real - life prospective study revealed that ics can be safely withdrawn in certain patients . of these , the wisdom ( withdrawal of inhaled steroids during optimized bronchodilator management ) trial was the largest and first to examine stepwise withdrawal of ics in patients with copd receiving maintenance therapy of long - acting bronchodilators ( ie , tiotropium and salmeterol ) . even with therapy being in line with the current guidelines , the findings of the wisdom trial indicate that not all patients benefit from including ics in their treatment regimen . indeed , only certain copd phenotypes seem to benefit from ics therapy , and validated markers that predict ics response are urgently warranted in clinical practice . furthermore , we are now better equipped with a larger armamentarium of novel and more effective long - acting 2-agonist / long - acting muscarinic antagonist combinations that can be considered by clinicians to optimize bronchodilation and allow for safer ics withdrawal . in addition to providing a review of the aforementioned , this perspective article proposes an algorithm for the stepwise withdrawal of ics in real - life clinical practice .

Introduction Current guideline recommendations and real-life utilization of ICS in COPD Evidence for the use of ICS in COPD: is there a benefit? What are the risks associated with ICS? Evidence regarding ICS withdrawal COPD phenotypes: which patients benefit from ICS? Safely withdrawing ICS from patients with COPD: a proposed algorithm for clinical practice Conclusion

while inhaled corticosteroid ( ics)-containing regimens benefit certain patients with chronic obstructive pulmonary disease ( copd ) , concerns about the inappropriate overuse of ics in real - life practice , their clinical benefit and long - term risks , and considerable waste of health care resources that could be better used on other more appropriate management strategies prompted studies to evaluate whether certain patients already on ics therapy fare better without it.1 this perspective paper explores the evidence available on ics withdrawal in patients with copd and the potential phenotypes that can be withdrawn from ics therapy . according to the latest 2015 update of the global initiative for chronic obstructive lung disease ( gold ) report , ics in combination with a long - acting 2-agonist ( laba ) and/or long - acting muscarinic antagonist ( lama ) is the first recommended choice for patients with a high - risk of exacerbations ( groups c and d ; figure 1).2 in addition to this international gold standard guideline , a number of national and multinational guidelines exist ; however , their recommendations pertaining to the use of ics are inconsistent with those of the gold report , in part , due to the varying criteria for categorizing patients with copd and different interpretations of the equivocal evidence supporting ics use.1,37 despite this , there is consensus that ics are indicated in combination with long - acting bronchodilators ( labds ) for patients with copd at risk of exacerbations and/or in those with asthma copd overlap syndrome ( acos ) , but never as monotherapy . several studies in multiple countries found discrepancies between guideline recommendations and real - life practice regarding ics use in patients with copd.810 in fact , ics are being widely prescribed to the majority of patients with copd , many of whom do not meet the recommendation criteria for ics use ( ie , 38.8% and 51.8% of gold groups a and b , respectively).10 market research estimates suggest that ics are used by > 70% of patients with copd , and given as initial therapy to > 50% of newly diagnosed patients , mostly in combination with a laba.11 these high real - world utilization rates are also corroborated by the treatment profiles of patients entering recent randomized controlled trials ( rcts ) , where ~35% of patients classified into either gold group a or b were receiving ics at baseline.12,13 the introduction of ics in the management of copd was rather unorthodox and unsubstantiated for nearly 20 years ( summarized in table 1 and previously reviewed in detail elsewhere).1,11,14,15 briefly , in the 1980s , ics were adopted in copd on the basis of their effectiveness in asthma rather than scientific evidence.11,16 the earliest rcts of ics therapy in mild copd were conducted in the late 1990s ; however , their outcomes were negative.11,15,17,18 isolde ( inhaled steroids in obstructive lung disease in europe ) was the first trial to demonstrate a beneficial effect of ics on the occurrence of exacerbations in patients with moderate - to - severe copd ; albeit , no benefit on the rate of lung function decline was observed.19 in 2001 , a large population - based cohort study of 22,620 patients with copd and a very recent hospitalization found that those who received ics within 90 days postdischarge had 24% fewer rehospitalizations and a 29% risk reduction for mortality during a 1-year follow - up.20 although these findings are not definitive due to their observational nature , they suggest that patients who have had a hospitalization in the past year should continue receiving ics . from 2002 onward , the subsequent cohort of rcts began evaluating ics in combination with labds.11,21,22 in the landmark torch ( towards a revolution in copd health ) trial , it was found that ics plus a laba ( ics + laba ) resulted in significantly fewer exacerbations , slower rate of decline in lung function , and improved health status ; however , there was only a trend toward improved survival.22 these findings formed the initial basis for the inclusion of ics - containing treatment regimens in clinical guidelines.2,11 at this time , the importance of exacerbations as a key determinant of health status and its association with a faster decline in lung function was being recognized , and the prevention of exacerbations became a key objective of copd management.16 of note , a post hoc analysis of the torch data , which evaluated the independent contribution of ics and laba , found that the benefits of ics + laba were actually provided by the laba.1,15,23 soon after torch , the inspire ( investigating new standards for prophylaxis in reduction of exacerbations ) trial was the first to show that ics + laba was no more effective than a lama in preventing exacerbations in patients with severe copd , which initiated the discussion of whether adding ics to labds is beneficial.24 indeed , a recent cochrane meta - analysis questioned the superiority of ics + laba combinations over laba alone in preventing exacerbations.25 lastly , the findings of the summit ( study to understand mortality and morbidity in copd ) trial were recently presented at the 2015 european respiratory society congress.26 the summit trial to evaluate the impact of a once - daily ics + laba ( fluticasone furoate / vilanterol ) versus the monocomponents on the survival of 16,485 patients with moderate copd and either a history or increased risk of cardiovascular disease ( cvd ) . while the findings of the torch trial suggested that ics + laba may reduce mortality in patients with copd , particularly those with cvd risk , no study to date has been able to demonstrate a benefit.22,26 a number of local and systemic adverse events have been associated with ics , particularly with long - term and high - dose use.1,15 the local adverse events include pneumonia,27,28 oropharyngeal candidiasis,29 and tuberculosis,30,31 and the systemic adverse events include cataracts,32,33 glaucoma,15 osteoporosis and bone fractures,34 easy bruising,18 type 2 diabetes,35 and even cases of adrenal suppression.36 while there have always been concerns about the risks of developing osteoporosis , cataracts , and diabetes in patients with copd , risk of pneumonia is arguably the most notable and well - established adverse event associated with ics use in copd , as supported by consistent evidence from rcts.1,14,15,24,27,37 corroborating this , a recent canadian population - based cohort study of 103,386 patients with copd who were treated with ics demonstrated that discontinuation of ics was associated with a 37% reduction in the risk of serious pneumonia.38 indeed , in the latest update of the gold report , it is cautioned that long - term ics - containing treatment should not be prescribed outside their indication due to risk of pneumonia and the possibility of a slight increased risk of fractures following long - term exposure.2 as patients with copd are more likely to be older and often have several comorbidities for which they receive multiple medications , they are more susceptible to ics - associated adverse events , and their potential risks need to be weighed against the likely benefits on a case - by - case basis , even in those already on ics.1 given the concerns about the risk benefit profile of ics , there has been a renewed interest in reevaluating the role of ics in copd and identifying which patients can be managed as well with alternate therapies.37 early observational studies and rcts investigating the implications of ics withdrawal in patients with copd found that an abrupt cessation of ics therapy precipitates exacerbations , and results in a deterioration in lung function , symptoms , and health status ( summarized in table 2).3944 however , a meta - analysis of three of these rcts ( ie , cope , cosmic [ copd and seretide : a multi - center intervention and characterization ] , and wisp ) , the only trials deemed to be acceptable in terms of quality and level of bias , determined that withdrawal of ics was not associated with any statistically significant increase in the exacerbation rate , and that the effects on other outcomes , such as lung function and health status , were inconclusive.45 the contradictory findings of these studies may be due to a number of methodological issues , including heterogeneity in patients characteristics , disease severity , outcome definitions ( eg , exacerbations ) , concomitant treatments ( eg , run - in period treatment , maintenance with labds vs placebo ) , and setting ( ie , primary vs secondary care).45,46 of note , since patient inclusion in all trials was based on spirometry while receiving ics therapy , this does not rule out a diagnosis of concomitant asthma , even if spirometry was of no significant reversibility.4244 additionally , in cosmic , the forced expiratory volume in 1 second ( fev1)/forced vital capacity ratios used do not match the diagnostic criteria for copd ( ie , < 70% ) , suggesting that these patients may have had an asthma component , and thereby , cessation of ics could have been detrimental.2,43 according to the latest gold recommendations , patients with copd and a low risk of exacerbations should not be prescribed ics - containing regimens.2 given that a large proportion of patients are already initiated on such regimens , it would be helpful to know if there are consequences associated with ics withdrawal in such populations ( ie , gold groups a and b ) . recently , in the first real - life prospective study ( optimo [ real - life study on the appropriateness of treatment in moderate copd patients ] ) , it was demonstrated that withdrawal of ics in patients with symptomatic , moderate copd ( ie , fev1 > 50% predicted ) at a low risk of exacerbations ( ie , < 2/year ) was not associated with any deterioration in lung function , symptoms , and exacerbation rate over a 6-month observation period.47 these findings were further confirmed by the recent instead ( indacaterol : switching non - exacerbating patients with moderate copd from salmeterol / fluticasone to indacaterol ) trial , the first rct with a clearly defined patient population with moderate copd ( ie , fev1 50%80% predicted ) and no prior exacerbation history , which found that switching from a fixed - dose combination of ics + laba to a laba was not associated with any differences in lung function , symptoms , health status , and exacerbations.48 in addition to supporting the current gold recommendations ( ie , no need for ics in groups a and b ) , the results from both studies suggest that ics therapy can be safely withdrawn from patients with moderate copd and a low risk of exacerbations provided that they are left on maintenance treatment with labds . it is well known that an abrupt cessation of chronic corticosteroid therapy precipitates rebound systemic effects due to steroid withdrawal symptoms.11,46 currently , there is no clear evidence on whether abrupt ics withdrawal is safe ; however , similar to the approach used to down - titrate ics in patients with asthma , a stepwise withdrawal of ics may minimize the potential risk of rebound steroid effects.11,46,49 adding to the growing clinical evidence - base on the impact of ics withdrawal against a background of labds , the wisdom ( withdrawal of inhaled steroids during optimized bronchodilator management ) trial , which is larger than all of the previous ics withdrawal rcts combined , was the first to assess the question of whether a stepwise withdrawal of ics on top of maintenance therapy with dual bronchodilation ( tiotropium / salmeterol ) had a similar risk of exacerbations in patients with copd and a history of exacerbations ( ie , gold groups c and d , in whom gold recommendations support the addition of ics to labd therapy).2,46,50 in this , 12-month , randomized , double - blind , parallel - group , active - controlled trial , 2,485 patients with copd and a history of exacerbations received triple therapy ( ie , fluticasone propionate / tiotropium / salmeterol ) during a 6-week run - in period . even with ics use being in line with the gold recommendations , the wisdom results also indicate that not all patients with severe - to - very - severe copd seem to benefit from including ics in their treatment regimen . unfortunately , no subgroups or biomarkers were found to be associated with an increased likelihood of exacerbations after ics withdrawal;46,51 albeit , certain phenotypes and potential biomarkers that have been previously shown to be associated with ics responsiveness were not evaluated ( discussed in the next section).16 because copd is a complex and heterogeneous disease with several different pathophysiological mechanisms , it is likely that ics may have an effect on some components of the disease , particularly when airway inflammation is present.11,15 increasing evidence suggests that patients with certain copd phenotypes appear to benefit from ics treatment , including patients with acos , frequent exacerbators , and those with eosinophilia.1,6,15,52 acos was only formally recognized by the global initiative for asthma ( gina ) and gold for the first time in 2014 . recently , a large observational study reported a survival benefit in patients with copd starting on ics + laba compared with laba alone ; however , this survival benefit was only observed in those who had a codiagnosis of asthma.57 frequent exacerbators account for approximately one - third of patients with copd.52,58 the definition of an exacerbator is based on expert opinion rather than rigorous phenotypic assessment , with a history of exacerbations being the best predictor of exacerbations across all gold stages.2 in the current gold report , exacerbation risk is based on either gold classification of airway limitation or history of exacerbations ( ie , high exacerbation risk is defined as 2 exacerbations per year or 1 hospitalization ) ; however , not all patients with moderate - to - severe copd ( ie , gold groups c and d ) are necessarily exacerbators , and therefore , require ics.2,59 indeed , in a 26-week , randomized , double - blind , parallel - group trial of patients with moderate - to - severe copd and no history of exacerbations , it was found that dual bronchodilation ( ie , laba + lama ) provided significantly better and clinically relevant improvements in lung function versus ics + laba.12 the findings from the energito trial , which were recently reported at the 2015 european respiratory society congress , further corroborate this point.60 in this randomized , double - blind , double - dummy , four - period crossover trial , lung function was evaluated in patients with moderate - to - severe copd after treatment with laba + lama ( ie , tiotropium / olodaterol ) versus ics + laba ( ie , fluticasone propionate / salmeterol ) . to complicate matters further , while both history of exacerbations and copd severity are risk factors for repeat exacerbations , there are a number of other risk factors associated with repeat exacerbations that should be considered , including eosinophilic inflammation , comorbidities and extrapulmonary manifestations ( eg , cvd , anxiety , depression , myopathy , reflux disease ) , chronic bronchitis , and increasing age.6 lastly , it has been suggested that up to 30% of patients with copd have eosinophilia.16,61 eosinophilia has been suggested to be predictive of exacerbations in patients with mild - to - moderate copd following withdrawal of ics,62 and both sputum and blood eosinophil levels have been shown to be promising biomarkers of ics responsiveness in patients with copd.63,64 in one rct , a management strategy for patients with copd that suppressed sputum eosinophil levels 3% was found to reduce severe exacerbations.64 although it has been suggested that sputum eosinophil levels may be the most reliable predictor of ics responsiveness in patients with copd to date , obtaining sputum samples is technically demanding and not feasible in routine clinical practice.15 since there is a reasonable correlation between sputum and blood levels , blood eosinophil levels may offer a more practical alternative.63 consequently , blood eosinophil levels are currently a topical issue and defining the appropriate cutoff for determining ics responsiveness in patients with copd is a subject of intense discussion . in a recent post hoc analysis of two replicate , 12-month , double - blind rcts comparing a laba with ics + laba in a total of 3,177 patients with moderate - to - severe copd and a history of 1 exacerbation , it was found that across all doses of ics , ics + laba significantly reduced exacerbations by 29% versus laba alone in patients with blood eosinophil levels of 2%.63 conversely , in another post hoc analysis of two replicate , 26-week , double - blind , double - dummy , parallel - group rcts , an absolute blood eosinophil cutoff of > 300 cells / mm at baseline as opposed to a percent cutoff appeared to best differentiate patients with moderate - to - severe copd who benefited from ics + laba versus laba + lama therapy in terms of reduction in exacerbation risk.65 this observation was corroborated by another post hoc analysis of the randomized , double - blind , parallel - group forward ( foster 48-week trial to reduce exacerbations in copd ) trial , which concluded that a greater reduction in exacerbations was observed when ics was added to a laba in patients with severe copd and a history of exacerbations who had an eosinophil count 279.8 cells / mm.66 using an absolute as opposed to a percent cutoff was further supported by the findings of the copenhagen general population study , which evaluated 7,225 patients with copd over a median follow - up of 3.3 years and found that an absolute blood eosinophil count of 340 cells / mm was a better predictor of both moderate and severe exacerbation risk than a percent cutoff of 2%.67 accordingly , 300 cells / mm can be tentatively used as a cutoff until this value is further validated in prospective trials . in addition to eosinophil levels , it has been suggested that feno levels can be used as a surrogate marker of eosinophilic inflammation in patients with copd exacerbations.68,69 a recent review on acos suggested the following feno levels for comparing between patients with asthma , acos , and copd : > 50 , 2550 , and < 25 ppb , respectively.55 these cutoffs are in line with the 2011 american thoracic society feno guidelines , which suggest that feno < 25 ppb provides a strong indication for an unlikely ics response and feno > 50 ppb provides a strong indication for a likely ics response , but feno between 25 and 50 ppb should be interpreted with caution.70,71 since there is an overall paucity of evidence about when and how ics can be safely withdrawn , the criteria for continuing or withdrawing ics in patients with copd are uncertain and more studies are warranted to characterize patients in whom withdrawal is safe . accordingly , until this need is met , this perspective article proposes an algorithm for the stepwise withdrawal of ics in real - life clinical practice based on the evidence available to date .

fifteen type 1 diabetic patients with proliferative retinopathy , an indicator of microangiopathy resulting from chronic hyperglycemia ( type 1 diabetes ) , 29 type 1 diabetic patients without manifest microvascular complications ( type 1 diabetes ) , and 26 healthy control subjects matched for sex and education level enrolled in this study . participants were recruited from the departments of endocrinology and ophthalmology of the vu university medical center , amsterdam , the netherlands ( 50 patients ) , the department of internal medicine , groene hart hospital , gouda , the netherlands ( 10 patients ) , and by advertisements in diabetes magazines and a national newspaper ( 10 patients ) . inclusion criteria were age range 1855 years , right - handedness , for type 1 diabetic patients a disease duration of at least 10 years , proliferative retinopathy as described below , or no signs of microvascular complications . participants were excluded if they had a bmi above 35 kg / m , current use of drugs affecting cerebral functioning , alcohol abuse ( more than 20 g of alcohol per day ) , psychiatric disorders , anemia , thyroid dysfunction , use of glucocorticoids , hepatitis , stroke , severe head trauma , epilepsy , pregnancy , or poor visual acuity below 0.3 . fundus photography ( topcon nw 100 , capelle aan den ijssel , the netherlands ) was performed to screen for retinopathy . for each eye , one photograph with the macula in the center and one with the optic disc in the center were taken ( e.v.d . ) . photographs were rated by an ophthalmologist ( a.m. ) according to the european diabetes ( eurodiab ) classification ( 20 ) . only those participants with either an eurodiab classification score of 0 ( no retinopathy ) or of 4 or 5 ( proliferative retinopathy or lasercoagulation ) twenty - four hour urine collections were performed to assess albumin - to - creatinine ratio ( acr ) . type 1 diabetes patients had normoalbuminuria ( acr < 2.5 mg / mmol in men and < 3.5 mg / mmol in women ) and no neuropathy . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or the use of antihypertensive drugs . the study protocol was approved by the medical ethics committee of the vu university medical center , and written informed consent was obtained from all participants . before eligible participants received written information on the study , their medical records were screened using the aforementioned criteria . those willing to participate were additionally interviewed over the telephone to collect background information including educational level , using a dutch scoring system ranging from 1 to 8 . one indicates unfinished primary school and 8 indicates a completed university study at masters level . as depression may affect cognitive performance , depressive symptoms were assessed using the center for epidemiological studies scale for depression ( ces - d ) . eligible participants were invited for the first visit . during this visit , which took place in the afternoon , fundus photography was performed , blood samples were collected , and meg registration was performed . approximately 6 weeks after the first visit participants returned for neuropsychological assessment during a morning visit . all visits took place at the vu university medical center . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . if hyperglycemia ( i.e. , blood glucose 15 mmol / l ) would persist , participants were instructed to inject another 2 units of insulin ; if hypoglycemia would persist , participants additionally had to eat 20 g of carbohydrates . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . proper glycemic status was restored for all patients after the first step of the above - mentioned protocol . data of the one participant who experienced hypoglycemia symptoms during neuropsychological testing were included in all analyses , as this did not change analysis outcomes . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . domains were based on an earlier principal component analysis using varimax rotation with kaiser normalization in a large group of healthy subjects and adjusted according to earlier research ( 5,21,23 ) . the domain memory was assessed by the dutch version of the rey auditory verbal learning test ( ralvt ) ( 24 ) , wechsler adult intelligence scale3rd edition revised ( wais - iii - r ) digit span forward and backward ( 25 ) , and the wais - iii - r symbol substitution incidental learning test ( 25 ) . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . assessment of the domain executive functions was conducted using the stroop color - word test part 3 ( 26 ) , the cst part c ( 27 ) , the d2-test total errors ( 29 ) , the wisconsin cart sorting test ( 30 ) , and the category word fluency task ( 31 ) . the domain attention was assessed using the d2-test range , with total correct answers and total span ( 29 ) . motor speed consisted of the tapping test ( 28 ) and the cst part 0 , administered three times ( 27 ) . general cognitive ability was constructed by averaging the above mentioned five domains . to enhance comparability and allow construction of these domains , z - scores for every test were created based on the means sd of the healthy control group . meg data were obtained using a 151-channel whole - head meg system ( ctf systems ; port coquitlam , bc , canada ) , while participants were in a supine position in a magnetically shielded room ( vacuumschmelze , hanau , germany ) . a third - order software gradient ( 32 ) was used with a recording passband of 0.25125 hz and a sample frequency of 625 hz . magnetic fields were recorded for 2 min in an eyes - open , 5 min in an eyes - closed , 10 min in a task , and then 3 min in another eyes - closed condition . total acquisition time was 20 min . at the beginning , middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . changes in head position of 1.5 cm during a recording condition were accepted . if head position changed more than 1.5 cm , recordings had to be repeated . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . for a technical description of synchronization likelihood see stam and van dijk ( 33 ) and montez et al . , interactions between two neural networks , for instance the frontal ( x ) and temporal ( y ) networks , are of interest . meg data are recorded for all sensors surrounding the head , including those in the frontal and temporal areas . these signals represent the time series xi and yi from the frontal ( x ) and temporal ( y ) areas ( fig . it is assumed that x and y more strongly interact when xi and yi more it has been shown , however , that x and y can also interact when xi and yi do not resemble each other ( square with the dashed line in fig . parameter settings used for computation of synchronization likelihood are based on the frequency content of the data ( for parameter settings see montez et al . ) . synchronization likelihood can range from pref ( low synchronization ) to one ( complete synchronization ) . pref is a value close to zero ( zero indicates no synchronization , which is not possible ) and was set on 0.01 for all frequency bands . the square with the continuous line indicates a time series showing high resemblance between both networks . the square with the dashed line is an example of a time series showing low resemblance . a : network x ( frontal ) . ten channels were deleted as their signals were distorted in some participants . this had no influence on the calculations . offline , recordings of the first eyes - closed condition were transformed into ascii - files and imported into the digeegxp software ( c.j . stam , vu university medical center , amsterdam , the netherlands ) . for each participant five artifact - free epochs ( 6.25 s ) of 4,096 samples were selected by two authors ( e.v.d . and total data used were 31.25 s. with digeegxp synchronization likelihood was calculated ( 33 ) . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . subsequently , meg sensors were clustered according to their anatomical location , respectively , right and left frontal , central , parietal , temporal , and occipital . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . short - distance group consisted of 10 areas , left and right hemisphere central , frontal , parietal , occipital , and temporal regions . for each of these 10 areas , the synchronization likelihood between all possible pairs of sensors belonging to that area was averaged . average synchronization likelihood for long distances was obtained by averaging all pair wise synchronization likelihood values of sensors belonging to two different areas . intrahemispheric long - distance group consisted of eight pairs , left and right hemisphere frontoparietal , frontotemporal , parietoocciptal , and temporooccipital . interhemispheric long - distance group consisted of five pairs , left to right hemisphere central , parietal , temporal , occipital , and frontal ( 12,13 ) . now , synchronization likelihood resembles the likelihood that the data obtained from all sensors in the above - defined short - distance and long - distance groups are synchronized . schematic brain , with left and right frontal ( lf and rf , respectively ) , central ( lc and rc , respectively ) , parietal ( rp and lp , respectively ) , occipital ( lo and ro , respectively ) , and temporal ( lt and rt , respectively ) areas indicated as abbreviations . a and b : long - distance intrahemispheric pathways . eight long - distance intrahemispheric pathways ( a ) and five long - distance interhemispheric pathways ( b ) . differences between groups for demographical and medical variables were calculated using student 's t test for independent samples or one - way anova with tukey post hoc test ( for continuous variables ) and test ( for categorical variables ) . to determine group differences in neuropsychological performance mancova was used with all cognitive domains as dependent variables and group as independent variable . in case of a group difference on a domain , post synchronization likelihood data were normalized by means of a transformation ln10(x/[1-x ] ) ( 35 ) to allow the use of parametric statistical tests . to minimize statistical tests , for each meg frequency band ancova with repeated measures was used to determine group differences . for each frequency band , three ancovas with repeated measures repeated measures for intrahemispheric connections consisted of the 8 above - mentioned levels , interhemispheric connections of 5 levels , and local hemispheric connections of 10 levels . in case of significant interaction ( p values of greenhouse - geisser correction for degrees of freedom ) or main effects of group with frequency band , post hoc mancova analysis was used to determine effects of group and spatial location . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . and used in other meg research was implemented ( 11,12 ) . to reduce multiple comparisons , cognitive domains were added as covariates to the above - mentioned ancova with repeated measures method . effects of group on changing cognitive domains and meg regions were determined using mancova tests . positive or negative associations were further calculated using regression analysis . to correct for possible confounding influences , all statistical tests were corrected for age , sex , hypertension , neuropathy , bmi , education level , and depressive symptoms . partial is reported as a proportion of the total variance explained of the determinant by the independent factor corrected for the used covariates . all statistical analyses were performed using spss for windows version 15.0 ( spss , chicago , il ) . before eligible participants received written information on the study , their medical records were screened using the aforementioned criteria . those willing to participate were additionally interviewed over the telephone to collect background information including educational level , using a dutch scoring system ranging from 1 to 8 . one indicates unfinished primary school and 8 indicates a completed university study at masters level . as depression may affect cognitive performance , depressive symptoms were assessed using the center for epidemiological studies scale for depression ( ces - d ) . eligible participants were invited for the first visit . during this visit , which took place in the afternoon , fundus photography was performed , blood samples were collected , and meg registration was performed . approximately 6 weeks after the first visit participants returned for neuropsychological assessment during a morning visit . all visits took place at the vu university medical center . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . glycemic status was evaluated after 30 min . if hyperglycemia ( i.e. , blood glucose 15 mmol / l ) would persist , participants were instructed to inject another 2 units of insulin ; if hypoglycemia would persist , participants additionally had to eat 20 g of carbohydrates . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . proper glycemic status was restored for all patients after the first step of the above - mentioned protocol . data of the one participant who experienced hypoglycemia symptoms during neuropsychological testing were included in all analyses , as this did not change analysis outcomes . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . domains were based on an earlier principal component analysis using varimax rotation with kaiser normalization in a large group of healthy subjects and adjusted according to earlier research ( 5,21,23 ) . the domain memory was assessed by the dutch version of the rey auditory verbal learning test ( ralvt ) ( 24 ) , wechsler adult intelligence scale3rd edition revised ( wais - iii - r ) digit span forward and backward ( 25 ) , and the wais - iii - r symbol substitution incidental learning test ( 25 ) . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . assessment of the domain executive functions was conducted using the stroop color - word test part 3 ( 26 ) , the cst part c ( 27 ) , the d2-test total errors ( 29 ) , the wisconsin cart sorting test ( 30 ) , and the category word fluency task ( 31 ) . the domain attention was assessed using the d2-test range , with total correct answers and total span ( 29 ) . motor speed consisted of the tapping test ( 28 ) and the cst part 0 , administered three times ( 27 ) . general cognitive ability was constructed by averaging the above mentioned five domains . to enhance comparability and allow construction of these domains , z - scores for every test were created based on the means sd of the healthy control group . meg data were obtained using a 151-channel whole - head meg system ( ctf systems ; port coquitlam , bc , canada ) , while participants were in a supine position in a magnetically shielded room ( vacuumschmelze , hanau , germany ) . a third - order software gradient ( 32 ) was used with a recording passband of 0.25125 hz and a sample frequency of 625 hz . magnetic fields were recorded for 2 min in an eyes - open , 5 min in an eyes - closed , 10 min in a task , and then 3 min in another eyes - closed condition . middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . changes in head position of 1.5 cm during a recording condition were accepted . if head position changed more than 1.5 cm , recordings had to be repeated . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . for a technical description of synchronization likelihood see stam and van dijk ( 33 ) and montez et al . ( 34 ) . in short , interactions between two neural networks , for instance the frontal ( x ) and temporal ( y ) networks , are of interest . meg data are recorded for all sensors surrounding the head , including those in the frontal and temporal areas . these signals represent the time series xi and yi from the frontal ( x ) and temporal ( y ) areas ( fig . it is assumed that x and y more strongly interact when xi and yi more it has been shown , however , that x and y can also interact when xi and yi do not resemble each other ( square with the dashed line in fig . parameter settings used for computation of synchronization likelihood are based on the frequency content of the data ( for parameter settings see montez et al . ) . synchronization likelihood can range from pref ( low synchronization ) to one ( complete synchronization ) . pref is a value close to zero ( zero indicates no synchronization , which is not possible ) and was set on 0.01 for all frequency bands . the square with the continuous line indicates a time series showing high resemblance between both networks . the square with the dashed line is an example of a time series showing low resemblance . ten channels were deleted as their signals were distorted in some participants . this had no influence on the calculations . offline , recordings of the first eyes - closed condition were transformed into ascii - files and imported into the digeegxp software ( c.j . stam , vu university medical center , amsterdam , the netherlands ) . for each participant five artifact - free epochs ( 6.25 s ) of 4,096 samples were selected by two authors ( e.v.d . and total data used were 31.25 s. with digeegxp synchronization likelihood was calculated ( 33 ) . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . subsequently , meg sensors were clustered according to their anatomical location , respectively , right and left frontal , central , parietal , temporal , and occipital . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . short - distance group consisted of 10 areas , left and right hemisphere central , frontal , parietal , occipital , and temporal regions . for each of these 10 areas , the synchronization likelihood between all possible pairs of sensors belonging to that area was averaged . average synchronization likelihood for long distances was obtained by averaging all pair wise synchronization likelihood values of sensors belonging to two different areas . intrahemispheric long - distance group consisted of eight pairs , left and right hemisphere frontoparietal , frontotemporal , parietoocciptal , and temporooccipital . interhemispheric long - distance group consisted of five pairs , left to right hemisphere central , parietal , temporal , occipital , and frontal ( 12,13 ) . now , synchronization likelihood resembles the likelihood that the data obtained from all sensors in the above - defined short - distance and long - distance groups are synchronized . schematic brain , with left and right frontal ( lf and rf , respectively ) , central ( lc and rc , respectively ) , parietal ( rp and lp , respectively ) , occipital ( lo and ro , respectively ) , and temporal ( lt and rt , respectively ) areas indicated as abbreviations . a and b : long - distance intrahemispheric pathways . eight long - distance intrahemispheric pathways ( a ) and five long - distance interhemispheric pathways ( b ) . differences between groups for demographical and medical variables were calculated using student 's t test for independent samples or one - way anova with tukey post hoc test ( for continuous variables ) and test ( for categorical variables ) . to determine group differences in neuropsychological performance mancova was used with all cognitive domains as dependent variables and group as independent variable . in case of a group difference on a domain , post synchronization likelihood data were normalized by means of a transformation ln10(x/[1-x ] ) ( 35 ) to allow the use of parametric statistical tests . to minimize statistical tests , for each meg frequency band ancova with repeated measures was used to determine group differences . for each frequency band , three ancovas with repeated measures were performed for intra- , inter- , and local hemispheric connections . repeated measures for intrahemispheric connections consisted of the 8 above - mentioned levels , interhemispheric connections of 5 levels , and local hemispheric connections of 10 levels . in case of significant interaction ( p values of greenhouse - geisser correction for degrees of freedom ) or main effects of group with frequency band , post hoc mancova analysis was used to determine effects of group and spatial location . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . and used in other meg research was implemented ( 11,12 ) . to reduce multiple comparisons , cognitive domains were added as covariates to the above - mentioned ancova with repeated measures method . effects of group on changing cognitive domains and meg regions were determined using mancova tests . positive or negative associations were further calculated using regression analysis . to correct for possible confounding influences , all statistical tests were corrected for age , sex , hypertension , neuropathy , bmi , education level , and depressive symptoms . partial is reported as a proportion of the total variance explained of the determinant by the independent factor corrected for the used covariates . all statistical analyses were performed using spss for windows version 15.0 ( spss , chicago , il ) . the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . * education level measured with a dutch scoring system from 1 to 8 , with 1 representing unfinished primary school and 8 representing university at masters level . risk of major depression was defined as a ces - d score of 16 or above . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or use of antihypertensive drugs . early disease onset is defined as onset below 7 years of age . * * averaged amount of self - reported severe hypoglycemic events per individual . severe hypoglycemic events are those events for which the participants need others ' assistance to cope with the effects of low blood glucose , loss of consciousness , or seizure . nephropathy was based on a 24-h urine sample and defined as an acr of 2.5 or above for men and 3.5 for women . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . meg results are shown in table 2 ( with p values and ) and visually represented in fig . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . raw synchronization likelihood values for significant group differences data are given as mean synchronization likelihood values per group sd . p value and ( percentage of total variance explained by the determinant ) are given . black and gray indicate lower synchronization likelihood ; black dashed arrows indicate higher synchronization likelihood . gray circles indicate lower short - distance local hemispheric differences with p < 0.05 ; black circles indicate lower short - distance local hemispheric differences with p < 0.01 ; gray arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.05 ; black arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.01 ; black dashed arrow indicates higher long - distance intrahemispheric differences with p < 0.05 . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . the lower local frequency band was related to attention ( p = 0.047 ) . for executive functions only , significant relations with anatomical areas were found . in the type 1 diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . motor speed was associated with local ( p = 0.028 ) and local ( p = 0.036 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . * education level measured with a dutch scoring system from 1 to 8 , with 1 representing unfinished primary school and 8 representing university at masters level . risk of major depression was defined as a ces - d score of 16 or above . hypertension was defined as a systolic blood pressure of 140 mmhg or above , a diastolic blood pressure of 90 mmhg or above , or use of antihypertensive drugs . early disease onset is defined as onset below 7 years of age . * * averaged amount of self - reported severe hypoglycemic events per individual . severe hypoglycemic events are those events for which the participants need others ' assistance to cope with the effects of low blood glucose , loss of consciousness , or seizure . nephropathy was based on a 24-h urine sample and defined as an acr of 2.5 or above for men and 3.5 for women . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . meg results are shown in table 2 ( with p values and ) and visually represented in fig . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . raw synchronization likelihood values for significant group differences data are given as mean synchronization likelihood values per group sd . p value and ( percentage of total variance explained by the determinant ) are given . black and gray indicate lower synchronization likelihood ; black dashed arrows indicate higher synchronization likelihood . gray circles indicate lower short - distance local hemispheric differences with p < 0.05 ; black circles indicate lower short - distance local hemispheric differences with p < 0.01 ; gray arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.05 ; black arrows indicate either lower long - distance intra- or interhemispheric differences with p < 0.01 ; black dashed arrow indicates higher long - distance intrahemispheric differences with p < 0.05 . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . the lower local frequency band was related to attention ( p = 0.047 ) . for executive functions diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . motor speed was associated with local ( p = 0.028 ) and local ( p = 0.036 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients this is , to the best of our knowledge , the first study using meg to assess functional brain connectivity in type 1 diabetes . we showed that , compared with sex- and education - matched type 1 diabetes patients and control subjects , type 1 diabetes had decreased functional connectivity . this decrease was most profound in the left and right central and parietal areas in the , lower and upper , and frequency bands . moreover , some intra- and interhemispheric differences were found in the lower and bands . in contrast , an increase in functional connectivity was found in the lower band when comparing the type 1 diabetes patients with control subjects . several frequency bands were positively related to cognitive domains , indicating these domains to be dependent of functional connectivity . on cognitive assessment , type 1 diabetic patients with and those without proliferative retinopathy performed significantly poorer on information processing speed , motor speed , and general cognitive ability . results are in line with the hypothesis that type 1 diabetes patients show cognitive and cerebral changes . when both patient groups were pooled and compared with control subjects , cognitive results did not change ; however , all differences in meg measurements , but those in the intrahemispheric band , lost statistical significance ( supplementary table 1a and b , available in an online appendix ) . this indicates that type 1 diabetes patients largely resemble the control subjects regarding functional connectivity . given the novelty of the technique used , caution is required when it comes to the interpretation of our connectivity findings . as there is currently only one study available that uses our method in metabolic disease in female obese adolescents , significant increases in functional connectivity in and frequency bands were found ( 13 ) , possibly because of an increase in white matter , which has been reported in mri studies of obese adolescents . in contrast , in multiple sclerosis a decrease in functional connectivity was reported ( 37 ) . this might be related to a loss of white matter , one of the core features of the disease . following this line of reasoning , our results for the type 1 diabetes patients could be a consequence of white matter loss , as has been found earlier in type 1 diabetes patients than type 1 diabetes patients and healthy control subjects ( 5 ) . conversely , the increase in functional connectivity in type 1 diabetes patients might reflect a compensatory mechanism , which fails in type 1 diabetes patients . because of large interindividual differences in cognitive performance , this compensation was not observed in cognitive functioning . therefore , larger samples are needed to enable the detection of significant differences between type 1 diabetes and type 1 diabetes patients . importantly , we found a significant positive relationship between cognitive functioning and functional connectivity , suggesting changes in functional connectivity are involved in cognitive dysfunction in type 1 diabetic patients . our data support the hypothesis that cerebral changes are related to the presence of microvascular complications . however , we also found indications that cerebral and cognitive changes may be present before microvascular complications become apparent . it could be hypothesized that chronic hyperglycemia , even in the absence of clinically detectable microvascular complications , can negatively affect cognitive functioning and cerebral communication . it is known that hyperglycemia as such leads to a cascade of changes , including increases in formation of reactive oxygen species and advanced glycation end products ( 38,39 ) , as well as activation of the hypothalamic - pituitary - adrenal axis ( 40 ) , which could have an effect on the brain . effects of hypoglycemia , particularly of asymptomatic hypoglycemic episodes because of hypoglycemia unawareness , can not be completely ruled out as a contributor . this did not change the results ( supplementary table 2a c , available in an online appendix ) . another possible contributor to the altered functional connectivity and cognitive functions observed in type 1 diabetic patients may be cerebrovascular , that is , macrovascular disease , the subclinical form of which can be estimated by ultrasound measurements of the carotid intima media thickness , which is known to be increased in asymptomatic type 1 diabetic patients relative to their nondiabetic peers ( 41 ) . furthermore , early diabetes onset , defined as disease onset before 7 years of age , has been shown previously to negatively affect intellectual and cognitive performance and cerebral structure ( 42,43 ) furthermore , the cognitive tests chosen had the highest sensitivity to detect cognitive changes . however , these tests may not be sufficiently sensitive to detect very subtle changes in type 1 diabetes associated cognitive decline . second , age of disease onset and subsequently disease duration differ between the patient groups . although the percentages of early disease onset do not statistically differ between groups , for the type 1 diabetes patients age of onset reflects childhood and early adolescence , whereas for the type 1 diabetes patients it reflects late adolescence and early adulthood . in a subsequent analysis we compared both patient groups , additionally correcting for age of diabetes onset and diabetes duration ( see supplementary table 3a c , available in an online appendix ) . taking into account the risk of emerging power problems when adding two more covariates to this small sample , these adjustments did not essentially change the conclusions regarding cognitive functioning and functional connectivity . disease duration might be less of a confounder here as the type 1 diabetes patients had an average disease duration of almost 20 years , which is sufficient for complications to develop . as stated before , an increase in functional connectivity although the possibility of a statistical aberration can not be completely ruled out , synchronization likelihood tended to be higher for 20 of the 23 anatomical locations of the lower band in type 1 diabetes patients than control subjects ( supplementary table 4 , available in an online appendix ) . moreover , we additionally repeated the post hoc mancova for the lower band correcting for multiple comparisons . as both age and depressive symptoms differed between groups , we statistically corrected for both variables in all analyses . supplementary table 5 , available in an online appendix , summarizes the relation between age and depressive symptoms and cognitive and meg variables . third , a1c values over the time period preceding meg recording and neuropsychological assessment unfortunately were not available . in conclusion , this study showed that changes in functional brain connectivity and cognitive function exist in type 1 diabetic patients with and without microvascular complications . longitudinal research in larger samples is required to determine the predictive clinical value of our findings for the progression of cognitive deterioration in type 1 diabetic patients as well as its potential to serve as clinical outcome parameter .

objectivehyperglycemia - associated microvascular disease may underlie changes in cerebral functioning and cognitive performance in patients with type 1 diabetes . functional connectivity , an indicator of functional interactions and information exchange between brain regions , provides a measure of cerebral functioning . this study addresses functional connectivity and cognition in type 1 diabetic patients with and without proliferative retinopathy , relative to healthy control subjects , using magnetoencephalography.research design and methodsfluctuations in magnetic field at scalp for , , lower and upper , , and lower and upper frequency bands were measured using magnetoencephalography . synchronization likelihood , a measure of functional connectivity , was computed . using neuropsychological tests , cognitive functioning was assessed and its associations with functional connectivity were determined.resultscompared with control subjects , type 1 diabetic patients performed poorer on general cognitive ability , information processing speed , and motor speed , irrespective of their microvascular complication status . functional connectivity , however , was lowest for type 1 diabetic patients with retinopathy , compared with type 1 diabetic patients without microvascular complications and control subjects , whereas type 1 diabetic patients without microvascular complications showed an increase relative to control subjects . positive associations were found between functional connectivity and executive functioning , memory , information processing speed , motor speed , and attention.conclusionscompared with healthy control subjects , functional connectivity and cognition differed in type 1 diabetic patients irrespective of microvascular complication status , indicating that chronic hyperglycemia , among other factors , may negatively affect brain functioning even before microvascular damage becomes manifest . the association found between synchronization likelihood and cognition suggests functional connectivity plays a significant role in cognitive functioning .

RESEARCH DESIGN AND METHODS Study design. Neuropsychological assessment. MEG protocol. Synchronization likelihood. Data analysis. Statistical analysis. RESULTS Patient characteristics. Neuropsychological assessment. MEG measurements. Correlation between MEG and cognitive performance. DISCUSSION

fifteen type 1 diabetic patients with proliferative retinopathy , an indicator of microangiopathy resulting from chronic hyperglycemia ( type 1 diabetes ) , 29 type 1 diabetic patients without manifest microvascular complications ( type 1 diabetes ) , and 26 healthy control subjects matched for sex and education level enrolled in this study . inclusion criteria were age range 1855 years , right - handedness , for type 1 diabetic patients a disease duration of at least 10 years , proliferative retinopathy as described below , or no signs of microvascular complications . participants were excluded if they had a bmi above 35 kg / m , current use of drugs affecting cerebral functioning , alcohol abuse ( more than 20 g of alcohol per day ) , psychiatric disorders , anemia , thyroid dysfunction , use of glucocorticoids , hepatitis , stroke , severe head trauma , epilepsy , pregnancy , or poor visual acuity below 0.3 . type 1 diabetes patients had normoalbuminuria ( acr < 2.5 mg / mmol in men and < 3.5 mg / mmol in women ) and no neuropathy . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . the domain memory was assessed by the dutch version of the rey auditory verbal learning test ( ralvt ) ( 24 ) , wechsler adult intelligence scale3rd edition revised ( wais - iii - r ) digit span forward and backward ( 25 ) , and the wais - iii - r symbol substitution incidental learning test ( 25 ) . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . general cognitive ability was constructed by averaging the above mentioned five domains . at the beginning , middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . it is assumed that x and y more strongly interact when xi and yi more it has been shown , however , that x and y can also interact when xi and yi do not resemble each other ( square with the dashed line in fig . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . to rule out confounding as a result of extreme blood glucose levels , levels were checked in the type 1 diabetes participants before the start of the neuropsychological assessment and meg registration and had to be between 4 and 15 , patients were instructed to inject 2 units of the participants ' current rapid - acting insulin analog when blood glucose levels were between 15 and 20 mmol / l ( 270362 mg / dl ) and 4 units when glucose levels exceeded 20 mmol / l ( 362 mg / dl ) . two type 1 diabetes participants were hyperglycemic before start of the neuropsychological assessment and one before meg acquisition . based on earlier type 1 diabetes cognitive research ( 5,21 ) , as well as clinical neuropsychological practice ( 22 ) , a battery of cognitive tests was chosen to measure potential differences in five major cognitive domains : memory , information processing speed , executive functions , attention , and motor speed . information processing speed was created using the wais - iii - r symbol substitution test ( 25 ) , the stroop color - word test parts 1 and 2 ( 26 ) , the concept shifting task ( cst ) parts a and b ( 27 ) , the simple auditory and visual reaction time tests ( 28 ) , and the computerized visual searching task ( cvst ) ( 28 ) . general cognitive ability was constructed by averaging the above mentioned five domains . middle , and end of each recording , the head position relative to the coordinate system of the helmet was determined by leading small alternating currents through three head position coils attached to the left and right preauricular points and the nasion on the subjects head . from the acquired meg data , information about functional connectivity was calculated by means of a mathematical construct , the synchronization likelihood . synchronization likelihood was calculated between signals recorded at all possible sensor pairs for ( 0.54 hz ) , ( 48 hz ) , lower ( 810 hz ) , upper ( 1013 hz ) , ( 1330 hz ) , lower ( 3045 hz ) , and upper ( 5580 hz ) frequency bands . average synchronization likelihood values were then obtained for short - distance , intrahemispheric long - distance , and interhemispheric long - distance groups . to determine associations between cognitive functioning and synchronization likelihood , a procedure proposed by stoffers et al . the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . in the type 1 diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients the control group was significantly younger than the type 1 diabetes patients and had a significantly lower mean score of depressive symptoms . type 1 diabetes patients had a significantly lower age of disease onset and longer disease duration . compared with control subjects , both patient groups had a significantly lower information processing speed ( type 1 diabetes : p = 0.003 , = 0.242 ; type 1 diabetes : p = 0.009 , = 0.135 ) and motor speed ( type 1 diabetes : p < 0.001 , = 0.311 ; type 1 diabetes : p = 0.014 , = 0.122 ) as well as general cognitive ability ( type 1 diabetes : p = 0.007 , = 0.198 ; type 1 diabetes : p = 0.004 , = 0.165 ) . z values were created based on the means sd of the healthy control subjects . asterisk indicates significant decline for type 1 diabetes and type 1 diabetes patients compared with healthy control subjects . significant interaction effects with group were found for the band local ( p = 0.001 ) , lower band local ( p = 0.004 ) , upper band local ( p = 0.008 ) , band interhemispheric ( p = 0.043 ) , and band local ( p = 0.020 ) . significant main effects of group were found for the lower band intrahemispheric ( p = 0.028 ) , lower band local hemispheric ( p = 0.005 ) , and upper band local ( p = 0.042 ) . post hoc mancova analysis revealed significantly higher synchronization likelihood in the lower right parietooccipital pathway for the type 1 diabetes compared with the healthy control subjects . comparing type 1 diabetes patients with control subjects revealed significantly lower synchronization likelihood in the and lower left and right central and parietal areas , in the upper left frontal and right frontal , central , and parietal areas , and in the interparietal , left parietal and right central , and parietal areas . comparisons of type 1 diabetes with type 1 diabetes patients yielded lower synchronization likelihood scores for type 1 diabetes patients in the left and right central and parietal areas , in the lower band left frontotemporal , left parietooccipital , right parietooccipital , and right temporooccipital pathways , in the left frontal and left and right central and parietal areas , upper left parietal and right central and parietal areas and in the -right parietal area . to summarize , type 1 diabetes patients showed lower synchronization likelihood in both higher and lower frequency bands compared with both type 1 diabetes patients and control subjects . type 1 diabetes patients showed increased synchronization likelihood in the lower band compared with control subjects . no statistically significant differences were found when the combined patient groups were compared with healthy control subjects . on the contrary , a significant difference in the intrahemispheric region was observed ( supplementary table 1a and b , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0425/dc1 ) . to test for associations between functional connectivity in different meg frequency bands and cognition , meg statistics were repeated with cognitive domains added as additional covariates for both type 1 diabetes groups . in type 1 diabetes patients significant associations were found between the domain of executive functions and intrahemispheric ( p = 0.041 ) , interhemispheric ( p = 0.008 ) , and local ( p = 0.043 ) frequency band . furthermore , interhemispheric was related to the domains of memory ( p = 0.030 ) , information processing speed ( p = 0.040 ) , and motor speed ( p = 0.014 ) . for executive functions diabetes patients significant relations were found between attention and intrahemispheric ( p = 0.048 ) , upper intrahemispheric ( p = 0.013 ) , and interhemispheric ( p = 0.043 ) . lastly , intrahemispheric was associated with information processing speed ( p = 0.040 ) . subsequently , regression analysis was used to determine positive or negative relations between synchronization likelihood and cognition . in both groups , associations between cognition and meg frequency bands for type 1 diabetes and type 1 diabetes patients this is , to the best of our knowledge , the first study using meg to assess functional brain connectivity in type 1 diabetes . we showed that , compared with sex- and education - matched type 1 diabetes patients and control subjects , type 1 diabetes had decreased functional connectivity . this decrease was most profound in the left and right central and parietal areas in the , lower and upper , and frequency bands . moreover , some intra- and interhemispheric differences were found in the lower and bands . in contrast , an increase in functional connectivity was found in the lower band when comparing the type 1 diabetes patients with control subjects . several frequency bands were positively related to cognitive domains , indicating these domains to be dependent of functional connectivity . on cognitive assessment , type 1 diabetic patients with and those without proliferative retinopathy performed significantly poorer on information processing speed , motor speed , and general cognitive ability . results are in line with the hypothesis that type 1 diabetes patients show cognitive and cerebral changes . when both patient groups were pooled and compared with control subjects , cognitive results did not change ; however , all differences in meg measurements , but those in the intrahemispheric band , lost statistical significance ( supplementary table 1a and b , available in an online appendix ) . this indicates that type 1 diabetes patients largely resemble the control subjects regarding functional connectivity . as there is currently only one study available that uses our method in metabolic disease in female obese adolescents , significant increases in functional connectivity in and frequency bands were found ( 13 ) , possibly because of an increase in white matter , which has been reported in mri studies of obese adolescents . following this line of reasoning , our results for the type 1 diabetes patients could be a consequence of white matter loss , as has been found earlier in type 1 diabetes patients than type 1 diabetes patients and healthy control subjects ( 5 ) . conversely , the increase in functional connectivity in type 1 diabetes patients might reflect a compensatory mechanism , which fails in type 1 diabetes patients . because of large interindividual differences in cognitive performance , this compensation was not observed in cognitive functioning . therefore , larger samples are needed to enable the detection of significant differences between type 1 diabetes and type 1 diabetes patients . importantly , we found a significant positive relationship between cognitive functioning and functional connectivity , suggesting changes in functional connectivity are involved in cognitive dysfunction in type 1 diabetic patients . our data support the hypothesis that cerebral changes are related to the presence of microvascular complications . however , we also found indications that cerebral and cognitive changes may be present before microvascular complications become apparent . it could be hypothesized that chronic hyperglycemia , even in the absence of clinically detectable microvascular complications , can negatively affect cognitive functioning and cerebral communication . another possible contributor to the altered functional connectivity and cognitive functions observed in type 1 diabetic patients may be cerebrovascular , that is , macrovascular disease , the subclinical form of which can be estimated by ultrasound measurements of the carotid intima media thickness , which is known to be increased in asymptomatic type 1 diabetic patients relative to their nondiabetic peers ( 41 ) . furthermore , early diabetes onset , defined as disease onset before 7 years of age , has been shown previously to negatively affect intellectual and cognitive performance and cerebral structure ( 42,43 ) furthermore , the cognitive tests chosen had the highest sensitivity to detect cognitive changes . however , these tests may not be sufficiently sensitive to detect very subtle changes in type 1 diabetes associated cognitive decline . although the percentages of early disease onset do not statistically differ between groups , for the type 1 diabetes patients age of onset reflects childhood and early adolescence , whereas for the type 1 diabetes patients it reflects late adolescence and early adulthood . taking into account the risk of emerging power problems when adding two more covariates to this small sample , these adjustments did not essentially change the conclusions regarding cognitive functioning and functional connectivity . disease duration might be less of a confounder here as the type 1 diabetes patients had an average disease duration of almost 20 years , which is sufficient for complications to develop . as stated before , an increase in functional connectivity although the possibility of a statistical aberration can not be completely ruled out , synchronization likelihood tended to be higher for 20 of the 23 anatomical locations of the lower band in type 1 diabetes patients than control subjects ( supplementary table 4 , available in an online appendix ) . in conclusion , this study showed that changes in functional brain connectivity and cognitive function exist in type 1 diabetic patients with and without microvascular complications . longitudinal research in larger samples is required to determine the predictive clinical value of our findings for the progression of cognitive deterioration in type 1 diabetic patients as well as its potential to serve as clinical outcome parameter .

mother - to - child transmission ( mtct ) of syphilis can result in a spectrum of adverse outcomes , including stillbirths , neonatal deaths , premature and low birth weight infants , as well as congenital deformities . in china , the reported incidence of congenital syphilis increased rapidly from 0.01 cases per 100 000 live births in 1991 to 64.41 cases in 2009 . in 2009 , 10 757 congenital syphilis cases were reported , compared with 57 cases of mtct of hiv . further , according to health economic estimations , averting a single case of mtct of syphilis in china costs approximately us $ 4391 , compared with us $ 7420 to avert an mtct of hiv case . despite evidence of a rapidly increasing burden , the cost - effectiveness of ( simple ) interventions , and the existence of promising pilot programs at a local level , the prevention of mtct ( pmtct ) of syphilis was not a health policy priority in china prior to 2010 . in contrast , pmtct of hiv was high on the health agenda despite the relatively small number of cases reported annually . in 2004 , the ministry of health ( moh ) issued the national working guidelines to prevent mother - to - child transmission of hiv , which set targets to screen 85% of pregnant women for hiv and provision of comprehensive care for 90% of mothers living with hiv and their infants . in 2006 , the provision of pmtct of hiv services was ratified by the regulations on prevention and treatment of hiv / aids the first state council decree directly aimed at controlling hiv . in june 2010 , the moh ( finally ) issued the china 20102020 plan for syphilis control and prevention , indicating the beginning of national government commitment to control mtct of syphilis . this plan set an overall target to reduce the incidence of mtct of syphilis through dual control with mtct of hiv to below 15 per 100 000 live births by 2020 , as well as several stepwise but ambitious benchmarks for antenatal syphilis screening coverage ( 80% by 2015 and 90% by 2020 ) and intervention uptake among infected mothers and their infants ( 90% by 2015 and 95% by 2020 ) . three months later , in the national guidelines for the management of hiv control programs , the moh further committed to providing integrated hiv , syphilis , and hepatitis b interventions for 80% of all pregnant women by 2015 . how and why did mtct of hiv rise more swiftly and resolutely on the health agenda , particularly given its comparatively far lower burden ? herein , we investigate the contrasting cases of issue agenda - setting ( i.e. generation of policy priority ) for the prevention of syphilis and hiv among pregnant women . by comparing the responses to these two infections lessons for political priority generation for mtct of syphilis , particularly in relation to effective implementation of dual control with mtct of hiv , are also explored . we used a policy framework developed by shiffman , which assesses political prioritization across nine factors , to explore facets of agenda - setting for both infections . through documentation review , stakeholder interviews , and nonparticipant observation of relevant meetings / trainings , policy - relevant data were collected and triangulated to minimize bias . we reviewed multiple types and sources of documentation , including government reports and policy documents , technical guidelines , epidemiological and implementation reports , training materials , published research , and mainstream media coverage . from september 2011 to august 2012 , we conducted 24 interviews at national ( n = 9 ) , provincial ( n = 3 ) , and city levels ( n = 12 ) . interviewees included heads and program directors in national institutions , representatives of international agencies , local health officials , heads and directors in implementing institutions / hospitals , and academics . seven policy - relevant events were observed , including meetings and trainings relevant to pmtct of hiv and syphilis at national ( n = 1 ) , provincial ( n = 1 ) , and city levels ( n = 4 ) , as well as national academic conferences ( n = 1 ) . interview and observation transcripts / notes and all documents were compared to verify the accuracy of information , coded for factors relevant to agenda - setting , and managed with nvivo 10 ( qsr international ) . factor codes were grouped into themes allowing revision of existing themes and inclusion of emerging themes during iterative data analysis . drawing on these themes , we modified shiffman s framework to capture china s unique health policy context . ethical approval for this study was obtained from the research ethics committee of university college london , united kingdom . as shown in table 1 , prior to 2010 , the levels of political priority received by and resources allocated to mtct of hiv and syphilis varied markedly . pmtct of hiv became one of the country s foremost health priorities around 2004 , when the state council aids working committee was established . financial inputs from central government increased from 6.43 million yuan per year in 2003 to 82.66 million yuan per year in 2009 , facilitating expansion of the national pmtct of hiv program from one pilot county in 2002 to 453 county - level divisions ( out of 2862 ) in 2009 . the rate of mtct of hiv fell significantly from 30% in the early 2000s to 9% in 2009 . for mtct of syphilis control , however , special funding was not allocated and technical guidelines were not developed before 2010 . intervention coverage data were not available during this period , but the rapid rise in incidence suggests coverage was probably low . several factors , as suggested by shiffman , may have accounted for the differences observed in health policy priorities of mtct of hiv and syphilis . international advocates and organizations draw national political attention to particular issues through strategies , such as the promotion of norms , to set expectations for appropriate behaviors of national decision - makers . from the early 2000s onwards , united nations ( un ) millennium development goal 6 ( to halt and reverse the spread of hiv ) , along with the un general assembly special session ( ungass ) on hiv / aids , committed countries to a reduction in the proportion of infants infected with hiv by 50% by 2010 by ensuring that 80% of pregnant women have access to pmtct of hiv services . the global aids reporting mechanism has had considerable impact on countries accountability for hiv and has thus served as a powerful instrument to exert influence over state norms for action . on the contrary , mtct of syphilis enjoyed no such high - level commitment from the international community or expectations for national action . it was not until 2007 that the who issued an action plan , the global elimination of congenital syphilis : rationale and strategy for action , and in contrast to pmtct of hiv , there was no global reporting instrument , let alone one that reported to the un general assembly . international financial and technical assistance can also influence national agenda - setting by enticing and enabling governments to undertake particular activities or reallocate national funds . aids has been a funding priority for china s international development partners since the late 1990s , and unaids established its office in beijing in 1996 . the central government s attention to hiv control was also motivated by participation in collaborative activities with international agencies and donors . from 2002 , international partners , particularly the unicef and the global fund to fight aids , tuberculosis and malaria , provided technical and financial support to pmtct programs . in contrast , the government received no financial support from any of the international agencies for pmtct of syphilis . policy communities are networks of actors from different types of organizations ( bureaucracies , legislatures , ngos , academia , etc ) committed to a common cause . policy communities for both pmtct of hiv and syphilis have existed in china for more than a decade , however , they have differed in their degree of cohesion and the effectiveness of leadership . in the early 2000s , a group composed of the division of women and children s health in the moh , unicef , and the national center for women and children s health ( ncwch ) , as well as clinicians and academics , was active in policymaking for pmtct of hiv . this community coalesced into a tight network , transforming international norms and knowledge - based authority into political influence and pushing the central government to act . the moh appointed the ncwch as the leading institution and assigned clear responsibilities and accountabilities for each participating sector / actor . experts had no formal mechanism through which to interact and around which to coalesce , and there was no clear institutional leadership . as a result , implementation of antenatal syphilis screening largely depended on single institutions and individual professionals and lacked a clear accountability mechanism . according to officials from the national center for std control ( ncstd ) and the ncwch , both institutions had tried to place pmtct of syphilis into the national health agenda . nevertheless , since the two centers are under the authority of different divisions of the moh and had limited communication during policy advocacy , their efforts were neither unified nor influential . agenda - setting scholars have demonstrated that the availability of clear indicators constitutes an important factor that shapes whether an issue will generate the attention of decision - makers . in 1997 , the first hiv sentinel site for pregnant women was established , followed by the establishment of a network of national and provincial sentinel sites . additional attention to mtct of hiv was generated by studies in henan province , which suggested a higher than expected prevalence ( 30% to 38% ) of hiv among infants born to women living with hiv . using this evidence , pmtct of hiv policy community compelled the moh to take action and , in 2002 , the first pmtct of hiv program was piloted in henan . however , despite the national sexually transmitted diseases ( stds ) surveillance program reporting a significant increase in incidence of syphilis from 1991 onward , pmtct - syphilis was neither publicized nor highlighted in any report from the moh or central government . it was not until 2007 , with the publication of national surveillance data in the lancet , that china s significant burden of mtct of syphilis became widely known to national officials . focusing events , such as crises , major scientific discoveries , or conferences can be influential in setting agendas by attracting visibility and attention from broad audiences to hitherto relatively hidden issues . the most important focusing event for hiv in china is known internationally as the blood selling scandal , during which hundreds of thousands of rural chinese in the central provinces were infected with hiv through blood selling in the 1990s . from 2000 onward , an increase in international media attention toward the scandal and the resultant aids villages , combined with pressure from national scientists and health workers , resulted in hiv being placed higher on the national health agenda . the 2001 ungass declaration of commitment on hiv / aids was the first major global focusing event for pmtct of hiv . by signing the declaration , china s central government deemed the international commitments , including commitment to pmtct of hiv , as legitimate and worthy of compliance . soon thereafter , the state council issued the china action plan on hiv / aids containment and prevention ( 20012005 ) , which indicated the national government s commitment to delivering pmtct - hiv services . the publication of national surveillance data in the lancet may have helped place pmtct of syphilis on china s health policy agenda . from 2007 to 2009 , a plan of action for the elimination of mtct of syphilis in china was prepared by the ncwch , with technical support from the who and inputs from external experts ; nevertheless , this was not approved by the moh . in 2010 , a publication in the new england journal of medicine revealed the country s increasing incidence of mtct of syphilis and stated that in 2008 , an average of more than one baby per hour was born with congenital syphilis in china , for a total of 9,480 cases . , national health officials believed that publication in a leading medical journal and western mainstream media would largely affect china s image in international society specifically because it was released on the eve of the opening of expo 2010 in shanghai . this may have provided the impetus for the two relevant moh divisions to join forces to push the timetable forward so as to finalize the china 20102020 plan for syphilis control and prevention in june 2010 . policymakers are more likely to commit to tackling a problem if they are convinced by proposals that demonstrate the problem to be readily solvable . a large body of international and national evidence regarding the feasibility of pmtct of hiv programs was available to chinese policymakers . on the basis of experiences gained from the henan pilot , the central government perceived that pmtct of hiv was achievable in china , and made the relevant provisions in its annual budget planning . on the contrary , according to several informants , since most pmtct of syphilis pilot programs were initiated in economically developed areas where both financial support from local governments and human resources were relatively sufficient , the interventions were not seen as generalizable to other ( less economically developed ) areas of the country . major political transitions and reforms alter political priorities by giving new leadership agenda - setting power and by changing the policymaking process . in 2003 , a change of national leadership and its shift of focus from purely economic growth to socioeconomic equality helped facilitate the generation of political priority for aids in china . the outbreak of sars in the winter / spring 20022003 precipitated a critical review for the newly installed government of the effectiveness of the state s apparatus to control life- and economy - threatening infectious diseases , including hiv . according to one informant , the review painted aids as a potential obstacle in the face of china s plan to achieve an all - round moderately prosperous ( xiaokang ) society . consequently , the national response to aids became significantly more public and aggressive , and was accompanied by increased government investment in infectious disease control . former premier wen jiabao s visit to aids patients at ditan hospital , beijing , on world aids day in december 2003 exemplified the intense political attention from the highest level of government . not long thereafter , in february 2004 , the state council working group on aids was established and included 20 ministry members . in contrast , the new leadership did not use the opportunity to include mtct of syphilis on the health agenda . beyond shiffman s framework , we found evidence of an important dynamic facilitating political attention : issue framing . in china , aids politics were introduced during the first national conference on hiv prevention in 2001 . hiv was described as an epidemic with severe impacts on public health , economic development , social stability , and national security , and thus could only be solved politically . one interviewee from the ncwch expressed the view that , in the early 2000s , the central government started to shift its attention to aids because a large proportion of people infected , although seriously stigmatized , were deemed the return of syphilis from the late 1970s onward was interpreted as a consequence of increased levels of extramarital sex , commercial sex , homosexuality , and drug use , all of which are illegal and/or immoral in china . according to an informant from a local std institution , because stis are closely related to sex work and drug use , which are linked with immorality, self - abuse, and guilt due to the restrictions of china s traditional morals , significant stigma is attached to std patients and officials tend to think of them as condemnable and punishable and therefore have been reluctant to admit and respond to the resurgent epidemic . several informants further attributed the low policy priority to the framing of syphilis as a disease without severe symptoms and international advocates and organizations draw national political attention to particular issues through strategies , such as the promotion of norms , to set expectations for appropriate behaviors of national decision - makers . from the early 2000s onwards , united nations ( un ) millennium development goal 6 ( to halt and reverse the spread of hiv ) , along with the un general assembly special session ( ungass ) on hiv / aids , committed countries to a reduction in the proportion of infants infected with hiv by 50% by 2010 by ensuring that 80% of pregnant women have access to pmtct of hiv services . the global aids reporting mechanism has had considerable impact on countries accountability for hiv and has thus served as a powerful instrument to exert influence over state norms for action . on the contrary , mtct of syphilis enjoyed no such high - level commitment from the international community or expectations for national action . it was not until 2007 that the who issued an action plan , the global elimination of congenital syphilis : rationale and strategy for action , and in contrast to pmtct of hiv , there was no global reporting instrument , let alone one that reported to the un general assembly . international financial and technical assistance can also influence national agenda - setting by enticing and enabling governments to undertake particular activities or reallocate national funds . aids has been a funding priority for china s international development partners since the late 1990s , and unaids established its office in beijing in 1996 . the central government s attention to hiv control was also motivated by participation in collaborative activities with international agencies and donors . from 2002 , international partners , particularly the unicef and the global fund to fight aids , tuberculosis and malaria , provided technical and financial support to pmtct programs . in contrast , the government received no financial support from any of the international agencies for pmtct of syphilis . policy communities are networks of actors from different types of organizations ( bureaucracies , legislatures , ngos , academia , etc ) committed to a common cause . policy communities for both pmtct of hiv and syphilis have existed in china for more than a decade , however , they have differed in their degree of cohesion and the effectiveness of leadership . in the early 2000s , a group composed of the division of women and children s health in the moh , unicef , and the national center for women and children s health ( ncwch ) , as well as clinicians and academics , was active in policymaking for pmtct of hiv . this community coalesced into a tight network , transforming international norms and knowledge - based authority into political influence and pushing the central government to act . the moh appointed the ncwch as the leading institution and assigned clear responsibilities and accountabilities for each participating sector / actor . experts had no formal mechanism through which to interact and around which to coalesce , and there was no clear institutional leadership . as a result , implementation of antenatal syphilis screening largely depended on single institutions and individual professionals and lacked a clear accountability mechanism . according to officials from the national center for std control ( ncstd ) and the ncwch , both institutions had tried to place pmtct of syphilis into the national health agenda . nevertheless , since the two centers are under the authority of different divisions of the moh and had limited communication during policy advocacy , their efforts were neither unified nor influential . agenda - setting scholars have demonstrated that the availability of clear indicators constitutes an important factor that shapes whether an issue will generate the attention of decision - makers . in 1997 , the first hiv sentinel site for pregnant women was established , followed by the establishment of a network of national and provincial sentinel sites . additional attention to mtct of hiv was generated by studies in henan province , which suggested a higher than expected prevalence ( 30% to 38% ) of hiv among infants born to women living with hiv . using this evidence , pmtct of hiv policy community compelled the moh to take action and , in 2002 , the first pmtct of hiv program was piloted in henan . however , despite the national sexually transmitted diseases ( stds ) surveillance program reporting a significant increase in incidence of syphilis from 1991 onward , pmtct - syphilis was neither publicized nor highlighted in any report from the moh or central government . it was not until 2007 , with the publication of national surveillance data in the lancet , that china s significant burden of mtct of syphilis became widely known to national officials . focusing events , such as crises , major scientific discoveries , or conferences can be influential in setting agendas by attracting visibility and attention from broad audiences to hitherto relatively hidden issues . the most important focusing event for hiv in china is known internationally as the blood selling scandal , during which hundreds of thousands of rural chinese in the central provinces were infected with hiv through blood selling in the 1990s . from 2000 onward , an increase in international media attention toward the scandal and the resultant aids villages , combined with pressure from national scientists and health workers , resulted in hiv being placed higher on the national health agenda . the 2001 ungass declaration of commitment on hiv / aids was the first major global focusing event for pmtct of hiv . by signing the declaration , china s central government deemed the international commitments , including commitment to pmtct of hiv , as legitimate and worthy of compliance . soon thereafter , the state council issued the china action plan on hiv / aids containment and prevention ( 20012005 ) , which indicated the national government s commitment to delivering pmtct - hiv services . the publication of national surveillance data in the lancet may have helped place pmtct of syphilis on china s health policy agenda . from 2007 to 2009 , a plan of action for the elimination of mtct of syphilis in china was prepared by the ncwch , with technical support from the who and inputs from external experts ; nevertheless , this was not approved by the moh . in 2010 , a publication in the new england journal of medicine revealed the country s increasing incidence of mtct of syphilis and stated that in 2008 , an average of more than one baby per hour was born with congenital syphilis in china , for a total of 9,480 cases . , national health officials believed that publication in a leading medical journal and western mainstream media would largely affect china s image in international society specifically because it was released on the eve of the opening of expo 2010 in shanghai . this may have provided the impetus for the two relevant moh divisions to join forces to push the timetable forward so as to finalize the china 20102020 plan for syphilis control and prevention in june 2010 . policymakers are more likely to commit to tackling a problem if they are convinced by proposals that demonstrate the problem to be readily solvable . a large body of international and national evidence regarding the feasibility of pmtct of hiv programs was available to chinese policymakers . on the basis of experiences gained from the henan pilot , the central government perceived that pmtct of hiv was achievable in china , and made the relevant provisions in its annual budget planning . on the contrary , according to several informants , since most pmtct of syphilis pilot programs were initiated in economically developed areas where both financial support from local governments and human resources were relatively sufficient , the interventions were not seen as generalizable to other ( less economically developed ) areas of the country . major political transitions and reforms alter political priorities by giving new leadership agenda - setting power and by changing the policymaking process . in 2003 , a change of national leadership and its shift of focus from purely economic growth to socioeconomic equality helped facilitate the generation of political priority for aids in china . the outbreak of sars in the winter / spring 20022003 precipitated a critical review for the newly installed government of the effectiveness of the state s apparatus to control life- and economy - threatening infectious diseases , including hiv . according to one informant , the review painted aids as a potential obstacle in the face of china s plan to achieve an all - round moderately prosperous ( xiaokang ) society . consequently , the national response to aids became significantly more public and aggressive , and was accompanied by increased government investment in infectious disease control . former premier wen jiabao s visit to aids patients at ditan hospital , beijing , on world aids day in december 2003 exemplified the intense political attention from the highest level of government . not long thereafter , in february 2004 , the state council working group on aids was established and included 20 ministry members . in contrast , the new leadership did not use the opportunity to include mtct of syphilis on the health agenda . beyond shiffman s framework , we found evidence of an important dynamic facilitating political attention : issue framing . in china , aids politics were introduced during the first national conference on hiv prevention in 2001 . hiv was described as an epidemic with severe impacts on public health , economic development , social stability , and national security , and thus could only be solved politically . one interviewee from the ncwch expressed the view that , in the early 2000s , the central government started to shift its attention to aids because a large proportion of people infected , although seriously stigmatized , were deemed the return of syphilis from the late 1970s onward was interpreted as a consequence of increased levels of extramarital sex , commercial sex , homosexuality , and drug use , all of which are illegal and/or immoral in china . according to an informant from a local std institution , because stis are closely related to sex work and drug use , which are linked with immorality, self - abuse, and guilt due to the restrictions of china s traditional morals , significant stigma is attached to std patients and officials tend to think of them as condemnable and punishable and therefore have been reluctant to admit and respond to the resurgent epidemic . several informants further attributed the low policy priority to the framing of syphilis as a disease without severe symptoms and can be easily cured by cheap drugs . in the 2000s , mtct of syphilis remained low on china s health policy agenda despite published evidence of high incidence and highly cost - effective interventions . in contrast , mtct of hiv , despite lower burden of disease , occupied a prominent position . our comparative analysis indicates that pressure from the international community , including establishment of specific and powerful norms , as well as provision of financial and technical support , had a major impact on the priority placed on mtct of hiv . the central government decided to combat the hiv epidemic as a response to both international expectations of state behavior and local evidence of the impact of infectious disease epidemics ( such as sars ) on population health and socioeconomic development . the shame brought by the blood selling scandal resulted in aids being considered a political disease , calling for action from highest political levels . decision - makers tended to portray hiv patients as victims of the selling blood scandal . a strong and cohesive policy community succeeded in lobbying for political attention and concomitant resource allocation to address mtct of hiv . credible indicators were employed to show the severity of its burden , focusing events generated political attention at both national and local levels , and a clear policy proposal convinced policymakers that the problem was surmountable . in contrast , the lack of national political attention on mtct of syphilis reflected the relative neglect of the disease at a global level . the absence of a lead institution and lack of a unified and vocal policy community further set back the cause of pmtct of syphilis . political efforts to tackle syphilis were restricted by chinese traditional ideas of linkage between stds and immorality and criminality . in 2007 , with public revelations of a shockingly high incidence of mtct of syphilis in the international academic press and the media , china s policymakers were prompted to take action . integrating syphilis screening into an existing antenatal hiv screening program in china can be substantially more cost - effective than hiv screening alone . in 2010 , china made a commitment to the dual elimination of mtct of hiv and syphilis , providing an important opportunity for the mtct of syphilis to be prioritized in policy and resource allocation at national and subnational levels . our findings demonstrate the importance of understanding political and policy contexts in moving toward goals of dual elimination . we found that empirical and technical evidence of the burden of disease associated with mtct of syphilis did not translate into political priority and resource allocation . national action on mtct of hiv , with its lower burden and less cost - effective intervention , was spurred by a combination of political framing , strong national policy communities , credible epidemiological indicators , clear policy proposals , media pressure , existence of a global institutional framework with resources for country support , and global monitoring and accountability mechanisms . maintaining political commitment to dual control at national and subnational levels will require a nuanced framing of these two infections as being essentially , linked in terms of underlying risk and vulnerability , as well as regarding the ease and feasibility of available solutions . a dual elimination goal will only be reached when policymakers and program managers at both national and subnational levels are convinced not only of the burden of preventable diseases , but also of the inherent imperative of conquering two infections simultaneously . this will require the mtct of syphilis policy community to have a stronger leadership , and also work closely with the mtct of hiv policy community at provincial level and below to correspondingly enhance prioritization of mtct of syphilis on the local health agendas .

objectivethe present study aims to identify reasons behind the lower political priority of mother - to - child transmission ( mtct ) of syphilis compared with hiv , despite the former presenting a much larger and growing burden than the latter , in china , over the 20 years prior to 2010.methodswe undertook a comparative policy analysis , based on informant interviews and documentation review of control of mtct of syphilis and hiv , as well as nonparticipant observation of relevant meetings / trainings to investigate agenda - setting prior to 2010.resultswe identified several factors contributing to the lower priority accorded to mtct of syphilis : relative neglect at a global level , dearth of international financial and technical support , poorly unified national policy community with weak accountability mechanisms , insufficient understanding of the epidemic and policy options , and a prevailing negative framing of syphilis that resulted in significant stigmatization.conclusiona dual elimination goal will only be reached when prioritization of mtct of syphilis is enhanced in both the international and national agendas .

Background Materials and methods Results Global shaping of national norms International support Policy community cohesion and leading institutions Credible indicators Focusing events Feasible policy options National policy environment Framing of issues Conclusions Conflict of interest

mother - to - child transmission ( mtct ) of syphilis can result in a spectrum of adverse outcomes , including stillbirths , neonatal deaths , premature and low birth weight infants , as well as congenital deformities . in china , the reported incidence of congenital syphilis increased rapidly from 0.01 cases per 100 000 live births in 1991 to 64.41 cases in 2009 . in 2009 , 10 757 congenital syphilis cases were reported , compared with 57 cases of mtct of hiv . further , according to health economic estimations , averting a single case of mtct of syphilis in china costs approximately us $ 4391 , compared with us $ 7420 to avert an mtct of hiv case . despite evidence of a rapidly increasing burden , the cost - effectiveness of ( simple ) interventions , and the existence of promising pilot programs at a local level , the prevention of mtct ( pmtct ) of syphilis was not a health policy priority in china prior to 2010 . in 2004 , the ministry of health ( moh ) issued the national working guidelines to prevent mother - to - child transmission of hiv , which set targets to screen 85% of pregnant women for hiv and provision of comprehensive care for 90% of mothers living with hiv and their infants . in june 2010 , the moh ( finally ) issued the china 20102020 plan for syphilis control and prevention , indicating the beginning of national government commitment to control mtct of syphilis . this plan set an overall target to reduce the incidence of mtct of syphilis through dual control with mtct of hiv to below 15 per 100 000 live births by 2020 , as well as several stepwise but ambitious benchmarks for antenatal syphilis screening coverage ( 80% by 2015 and 90% by 2020 ) and intervention uptake among infected mothers and their infants ( 90% by 2015 and 95% by 2020 ) . three months later , in the national guidelines for the management of hiv control programs , the moh further committed to providing integrated hiv , syphilis , and hepatitis b interventions for 80% of all pregnant women by 2015 . herein , we investigate the contrasting cases of issue agenda - setting ( i.e. generation of policy priority ) for the prevention of syphilis and hiv among pregnant women . by comparing the responses to these two infections lessons for political priority generation for mtct of syphilis , particularly in relation to effective implementation of dual control with mtct of hiv , are also explored . we used a policy framework developed by shiffman , which assesses political prioritization across nine factors , to explore facets of agenda - setting for both infections . through documentation review , stakeholder interviews , and nonparticipant observation of relevant meetings / trainings , policy - relevant data were collected and triangulated to minimize bias . we reviewed multiple types and sources of documentation , including government reports and policy documents , technical guidelines , epidemiological and implementation reports , training materials , published research , and mainstream media coverage . from september 2011 to august 2012 , we conducted 24 interviews at national ( n = 9 ) , provincial ( n = 3 ) , and city levels ( n = 12 ) . interviewees included heads and program directors in national institutions , representatives of international agencies , local health officials , heads and directors in implementing institutions / hospitals , and academics . seven policy - relevant events were observed , including meetings and trainings relevant to pmtct of hiv and syphilis at national ( n = 1 ) , provincial ( n = 1 ) , and city levels ( n = 4 ) , as well as national academic conferences ( n = 1 ) . interview and observation transcripts / notes and all documents were compared to verify the accuracy of information , coded for factors relevant to agenda - setting , and managed with nvivo 10 ( qsr international ) . as shown in table 1 , prior to 2010 , the levels of political priority received by and resources allocated to mtct of hiv and syphilis varied markedly . financial inputs from central government increased from 6.43 million yuan per year in 2003 to 82.66 million yuan per year in 2009 , facilitating expansion of the national pmtct of hiv program from one pilot county in 2002 to 453 county - level divisions ( out of 2862 ) in 2009 . the rate of mtct of hiv fell significantly from 30% in the early 2000s to 9% in 2009 . for mtct of syphilis control , however , special funding was not allocated and technical guidelines were not developed before 2010 . several factors , as suggested by shiffman , may have accounted for the differences observed in health policy priorities of mtct of hiv and syphilis . on the contrary , mtct of syphilis enjoyed no such high - level commitment from the international community or expectations for national action . it was not until 2007 that the who issued an action plan , the global elimination of congenital syphilis : rationale and strategy for action , and in contrast to pmtct of hiv , there was no global reporting instrument , let alone one that reported to the un general assembly . international financial and technical assistance can also influence national agenda - setting by enticing and enabling governments to undertake particular activities or reallocate national funds . aids has been a funding priority for china s international development partners since the late 1990s , and unaids established its office in beijing in 1996 . in contrast , the government received no financial support from any of the international agencies for pmtct of syphilis . in the early 2000s , a group composed of the division of women and children s health in the moh , unicef , and the national center for women and children s health ( ncwch ) , as well as clinicians and academics , was active in policymaking for pmtct of hiv . agenda - setting scholars have demonstrated that the availability of clear indicators constitutes an important factor that shapes whether an issue will generate the attention of decision - makers . additional attention to mtct of hiv was generated by studies in henan province , which suggested a higher than expected prevalence ( 30% to 38% ) of hiv among infants born to women living with hiv . using this evidence , pmtct of hiv policy community compelled the moh to take action and , in 2002 , the first pmtct of hiv program was piloted in henan . however , despite the national sexually transmitted diseases ( stds ) surveillance program reporting a significant increase in incidence of syphilis from 1991 onward , pmtct - syphilis was neither publicized nor highlighted in any report from the moh or central government . it was not until 2007 , with the publication of national surveillance data in the lancet , that china s significant burden of mtct of syphilis became widely known to national officials . the most important focusing event for hiv in china is known internationally as the blood selling scandal , during which hundreds of thousands of rural chinese in the central provinces were infected with hiv through blood selling in the 1990s . from 2000 onward , an increase in international media attention toward the scandal and the resultant aids villages , combined with pressure from national scientists and health workers , resulted in hiv being placed higher on the national health agenda . by signing the declaration , china s central government deemed the international commitments , including commitment to pmtct of hiv , as legitimate and worthy of compliance . the publication of national surveillance data in the lancet may have helped place pmtct of syphilis on china s health policy agenda . from 2007 to 2009 , a plan of action for the elimination of mtct of syphilis in china was prepared by the ncwch , with technical support from the who and inputs from external experts ; nevertheless , this was not approved by the moh . in 2010 , a publication in the new england journal of medicine revealed the country s increasing incidence of mtct of syphilis and stated that in 2008 , an average of more than one baby per hour was born with congenital syphilis in china , for a total of 9,480 cases . , national health officials believed that publication in a leading medical journal and western mainstream media would largely affect china s image in international society specifically because it was released on the eve of the opening of expo 2010 in shanghai . a large body of international and national evidence regarding the feasibility of pmtct of hiv programs was available to chinese policymakers . on the basis of experiences gained from the henan pilot , the central government perceived that pmtct of hiv was achievable in china , and made the relevant provisions in its annual budget planning . on the contrary , according to several informants , since most pmtct of syphilis pilot programs were initiated in economically developed areas where both financial support from local governments and human resources were relatively sufficient , the interventions were not seen as generalizable to other ( less economically developed ) areas of the country . major political transitions and reforms alter political priorities by giving new leadership agenda - setting power and by changing the policymaking process . in 2003 , a change of national leadership and its shift of focus from purely economic growth to socioeconomic equality helped facilitate the generation of political priority for aids in china . the outbreak of sars in the winter / spring 20022003 precipitated a critical review for the newly installed government of the effectiveness of the state s apparatus to control life- and economy - threatening infectious diseases , including hiv . consequently , the national response to aids became significantly more public and aggressive , and was accompanied by increased government investment in infectious disease control . in contrast , the new leadership did not use the opportunity to include mtct of syphilis on the health agenda . in china , aids politics were introduced during the first national conference on hiv prevention in 2001 . hiv was described as an epidemic with severe impacts on public health , economic development , social stability , and national security , and thus could only be solved politically . one interviewee from the ncwch expressed the view that , in the early 2000s , the central government started to shift its attention to aids because a large proportion of people infected , although seriously stigmatized , were deemed the return of syphilis from the late 1970s onward was interpreted as a consequence of increased levels of extramarital sex , commercial sex , homosexuality , and drug use , all of which are illegal and/or immoral in china . according to an informant from a local std institution , because stis are closely related to sex work and drug use , which are linked with immorality, self - abuse, and guilt due to the restrictions of china s traditional morals , significant stigma is attached to std patients and officials tend to think of them as condemnable and punishable and therefore have been reluctant to admit and respond to the resurgent epidemic . several informants further attributed the low policy priority to the framing of syphilis as a disease without severe symptoms and international advocates and organizations draw national political attention to particular issues through strategies , such as the promotion of norms , to set expectations for appropriate behaviors of national decision - makers . from the early 2000s onwards , united nations ( un ) millennium development goal 6 ( to halt and reverse the spread of hiv ) , along with the un general assembly special session ( ungass ) on hiv / aids , committed countries to a reduction in the proportion of infants infected with hiv by 50% by 2010 by ensuring that 80% of pregnant women have access to pmtct of hiv services . on the contrary , mtct of syphilis enjoyed no such high - level commitment from the international community or expectations for national action . it was not until 2007 that the who issued an action plan , the global elimination of congenital syphilis : rationale and strategy for action , and in contrast to pmtct of hiv , there was no global reporting instrument , let alone one that reported to the un general assembly . international financial and technical assistance can also influence national agenda - setting by enticing and enabling governments to undertake particular activities or reallocate national funds . in contrast , the government received no financial support from any of the international agencies for pmtct of syphilis . in the early 2000s , a group composed of the division of women and children s health in the moh , unicef , and the national center for women and children s health ( ncwch ) , as well as clinicians and academics , was active in policymaking for pmtct of hiv . agenda - setting scholars have demonstrated that the availability of clear indicators constitutes an important factor that shapes whether an issue will generate the attention of decision - makers . additional attention to mtct of hiv was generated by studies in henan province , which suggested a higher than expected prevalence ( 30% to 38% ) of hiv among infants born to women living with hiv . using this evidence , pmtct of hiv policy community compelled the moh to take action and , in 2002 , the first pmtct of hiv program was piloted in henan . however , despite the national sexually transmitted diseases ( stds ) surveillance program reporting a significant increase in incidence of syphilis from 1991 onward , pmtct - syphilis was neither publicized nor highlighted in any report from the moh or central government . it was not until 2007 , with the publication of national surveillance data in the lancet , that china s significant burden of mtct of syphilis became widely known to national officials . the most important focusing event for hiv in china is known internationally as the blood selling scandal , during which hundreds of thousands of rural chinese in the central provinces were infected with hiv through blood selling in the 1990s . from 2000 onward , an increase in international media attention toward the scandal and the resultant aids villages , combined with pressure from national scientists and health workers , resulted in hiv being placed higher on the national health agenda . by signing the declaration , china s central government deemed the international commitments , including commitment to pmtct of hiv , as legitimate and worthy of compliance . from 2007 to 2009 , a plan of action for the elimination of mtct of syphilis in china was prepared by the ncwch , with technical support from the who and inputs from external experts ; nevertheless , this was not approved by the moh . in 2010 , a publication in the new england journal of medicine revealed the country s increasing incidence of mtct of syphilis and stated that in 2008 , an average of more than one baby per hour was born with congenital syphilis in china , for a total of 9,480 cases . a large body of international and national evidence regarding the feasibility of pmtct of hiv programs was available to chinese policymakers . on the basis of experiences gained from the henan pilot , the central government perceived that pmtct of hiv was achievable in china , and made the relevant provisions in its annual budget planning . on the contrary , according to several informants , since most pmtct of syphilis pilot programs were initiated in economically developed areas where both financial support from local governments and human resources were relatively sufficient , the interventions were not seen as generalizable to other ( less economically developed ) areas of the country . major political transitions and reforms alter political priorities by giving new leadership agenda - setting power and by changing the policymaking process . in 2003 , a change of national leadership and its shift of focus from purely economic growth to socioeconomic equality helped facilitate the generation of political priority for aids in china . the outbreak of sars in the winter / spring 20022003 precipitated a critical review for the newly installed government of the effectiveness of the state s apparatus to control life- and economy - threatening infectious diseases , including hiv . consequently , the national response to aids became significantly more public and aggressive , and was accompanied by increased government investment in infectious disease control . not long thereafter , in february 2004 , the state council working group on aids was established and included 20 ministry members . in contrast , the new leadership did not use the opportunity to include mtct of syphilis on the health agenda . in china , aids politics were introduced during the first national conference on hiv prevention in 2001 . hiv was described as an epidemic with severe impacts on public health , economic development , social stability , and national security , and thus could only be solved politically . one interviewee from the ncwch expressed the view that , in the early 2000s , the central government started to shift its attention to aids because a large proportion of people infected , although seriously stigmatized , were deemed the return of syphilis from the late 1970s onward was interpreted as a consequence of increased levels of extramarital sex , commercial sex , homosexuality , and drug use , all of which are illegal and/or immoral in china . several informants further attributed the low policy priority to the framing of syphilis as a disease without severe symptoms and can be easily cured by cheap drugs . in the 2000s , mtct of syphilis remained low on china s health policy agenda despite published evidence of high incidence and highly cost - effective interventions . in contrast , mtct of hiv , despite lower burden of disease , occupied a prominent position . our comparative analysis indicates that pressure from the international community , including establishment of specific and powerful norms , as well as provision of financial and technical support , had a major impact on the priority placed on mtct of hiv . the shame brought by the blood selling scandal resulted in aids being considered a political disease , calling for action from highest political levels . a strong and cohesive policy community succeeded in lobbying for political attention and concomitant resource allocation to address mtct of hiv . credible indicators were employed to show the severity of its burden , focusing events generated political attention at both national and local levels , and a clear policy proposal convinced policymakers that the problem was surmountable . in contrast , the lack of national political attention on mtct of syphilis reflected the relative neglect of the disease at a global level . the absence of a lead institution and lack of a unified and vocal policy community further set back the cause of pmtct of syphilis . in 2007 , with public revelations of a shockingly high incidence of mtct of syphilis in the international academic press and the media , china s policymakers were prompted to take action . in 2010 , china made a commitment to the dual elimination of mtct of hiv and syphilis , providing an important opportunity for the mtct of syphilis to be prioritized in policy and resource allocation at national and subnational levels . our findings demonstrate the importance of understanding political and policy contexts in moving toward goals of dual elimination . we found that empirical and technical evidence of the burden of disease associated with mtct of syphilis did not translate into political priority and resource allocation . national action on mtct of hiv , with its lower burden and less cost - effective intervention , was spurred by a combination of political framing , strong national policy communities , credible epidemiological indicators , clear policy proposals , media pressure , existence of a global institutional framework with resources for country support , and global monitoring and accountability mechanisms . maintaining political commitment to dual control at national and subnational levels will require a nuanced framing of these two infections as being essentially , linked in terms of underlying risk and vulnerability , as well as regarding the ease and feasibility of available solutions . a dual elimination goal will only be reached when policymakers and program managers at both national and subnational levels are convinced not only of the burden of preventable diseases , but also of the inherent imperative of conquering two infections simultaneously . this will require the mtct of syphilis policy community to have a stronger leadership , and also work closely with the mtct of hiv policy community at provincial level and below to correspondingly enhance prioritization of mtct of syphilis on the local health agendas .

a survey of more than 500 total syntheses shows that lithium diisopropylamide ( lda ) is the most frequently used reagent in organic synthesis . it is this prominence that piqued our interest in structural and mechanistic studies of lda studies that have now spanned more than 25 years . for several practical reasons , we focused mechanistic studies on reactions that could be monitored at temperatures ranging from 55 to + 25. despite a large number of mechanistic variations arising from dozens of substrate solvent combinations , the aggregate equilibrations were rapid on the time scales of the rate - limiting substrate lithiation . the opposite limiting behavior has been the focus of reich and co - workers using rapid - injection nmr spectroscopy . lda - mediated enolizations of reactive ketones that are observable on short time scales below 135 c are rapid on the time scales of aggregate exchanges . there is necessarily a window of substrate reactivity a critical temperature range in which readily observed lithiations and lda aggregate exchanges occur at comparable rates . in this regime , the chemistry would certainly become complex . in an irony that will be lost on few organic chemists , this twilight zone for lda / tetrahydrofuran ( thf)-mediated lithiations is centered at 78 c : any lda / thf - mediated lithiation that proceeds at observable rates at 78c is occurring under nonlimiting conditions in which aggregation events and reactions with substrate vie to be rate limiting . under such nonlimiting conditions , the rules governing reactivity change markedly : aggregates are no longer in full equilibrium ; the rate - limiting step shifts unpredictably with subtle changes in reaction conditions ; catalysis by traces of extraneous lithium salts ( especially lithium chloride ) and autocatalysis by the developing product become acute ; and substituting deuterium for protium can change the rate , mechanism , and rate law . in short , lda / thf - mediated lithiations observed at 78 c are complex even by the standards of organolithium chemistry.1 we describe herein mechanistic studies of the lda / thf - mediated lithiation of 1,4-difluorobenzene ( 1 ) ( eq 1 ) . spoiler alert : here is what we find . lithiation of arene 1 by dimeric lda occurs through the cascading deaggregation illustrated by the reaction coordinate diagram in scheme 1 . note that scheme 1 connotes qualitative relative barrier heights but lacks the implicit balancing to be called a free energy diagram . it is also a living , breathing diagram that changes consequentially with concentrations and , as we describe , isotopic substitution . in the absence of catalysis , autocatalysis by the resulting aryllithium via mixed - aggregate - based transition structure 5 circumvents 4 , revealing lda - tetramer - based transition state 11 lurking over the thermochemical horizon . perdeuteration of 1 , by virtue of the often large and highly variable isotope effects for lda - mediated ortholithiations , drops the zero - point energy ( zpe ) of the barrier corresponding to 4 to reveal two competing dimer - based proton transfers ( 13 and 16 ) as the highest remaining barriers . the most efficient deaggregation catalyst reported to date circumvents barriers corresponding to the aforementioned transition states altogether via mixed - aggregate - based transition state 6 delineated previously , affording trisolvated - monomer - based transition structure 10 for proton transfer . these conclusions emanate from a series of spectroscopic , kinetic , and computational studies of the reaction cascade involved in the lithiation of 1 and its perdeuterated analogue . the mechanistic changes accompanying subtle changes in conditions are legion , but we have not peered beyond rate - limiting steps with such clarity as described below . to simplify the results and discussion , we introduce the following shorthand used in scheme 1 : a = an lda subunit , s = thf , arh = arene 1 , ard = tetradeuterated arene 1-d4 , and arli refers to aryllithiums 2 or 2-d3 . we will denote general structures and their more specific counterparts ( arli 2 and 2a , for example ) interchangeably depending on the context . arh and ard are mechanistically so different that they demanded fully independent rate studies ; they are presented in separate sections within the following subsections : uncatalyzed , autocatalyzed , and licl - catalyzed lithiations . the key to understanding the results is that changes in conditions essentially any changes shift the rate - limiting steps and consequentially alter the mathematical form of the accompanying rate law . metalation of relatively high concentrations of arh by lda specifically at low thf concentrations promotes mixed aggregation . f nmr spectroscopy shows the time dependence of a number of species : a downwardly curving decay of arh , a nearly linear formation of arli , and sigmoidal growth of an lda the mixed aggregate is much less relevant than one might think , and the other decays are much more complex than most could imagine . the curves represent a best - fit numerical integration to a model described herein . time - dependent concentrations measured by f nmr spectroscopy using 0.10 m lda and 0.030 m arh in 3.05 m thf / hexanes at 65 c . the curves represent a best - fit numerical integration to the emergent model ( vide infra ) . previous studies of [ li , n]lda using li and n nmr spectroscopies have revealed exclusively disolvated dimer 3 . trisolvated mixed dimer 17 is observed at low levels and only at high lda and low thf concentrations . f nmr spectroscopy of a sample prepared from 2:1 lda / arh in 3.5 m thf reveals a pair of doublets owing to five - bond f f coupling with additional h f coupling discernible using window functions , consistent with 2a ( figure 2 ) . a c nmr spectrum shows the carbanionic carbon resonance of 2a as a triplet ( jli c = 6.0 hz ) further split by a large ( 130 hz ) two - bond f c coupling and a small ( 18.6 hz ) three - bond f f nmr spectrum of lda ( 0.10 m ) with arh ( 0.050 m ) and diisopropylamine ( 0.050 m ) in 3.5 m thf / hexanes at 78 c . f = 31.6 hz ) , 88.43 ( d , jf f = 31.6 hz ) . c{h } nmr spectrum of arli generated from arh ( 0.30 m ) with [ li]lda ( 0.40 m ) in 12.2 m thf - d8 at 105 c : 173.75 ( ddt , jc f = 130.2 hz , jc f = 18.6 hz , jc li = 6.0 hz ) , 167.81 ( d , jc f = 44.8 hz , jc f = 8.8 hz ) , 109.82 ( dd , jc f = 3.7 hz ) , 108.50 ( dd , jc f = 25.5 hz , jc f = 7.2 hz ) . we determined the solvation number of 2a using three independent methods : ( 1 ) we relied on the recently completed assignment of bis - trifluoromethylated aryllithium 18 as a trisolvated monomer . monitoring the equilibrium in eq 2 versus thf concentration shows no dependence ( 10% ) over a 10-fold thf concentration range , confirming 2a as a trisolvate.2 ( 2 ) lithiation using excess i - pr2nh and variable thf concentrations ( eq 3 ) , conditions in which arli and arh coexist at equilibrium , establishes the solvation number according to eq 4 ( figure 4 ) . monitoring the concentrations of arh and arli with f nmr spectroscopy and back calculating the concentrations of i - pr2nh and lda affords the solvation number of 3.6 0.2.34 ( 3 ) density functional theory ( dft ) computations at the b3lyp/6 - 31g(d ) level with single - point calculations at the mp2 level of theory of the serial solvation revealed that trisolvate 2a is 7.3 kcal more stable than the corresponding disolvate ( eq 5 ) ; no minimum was found for the tetrasolvate . hereafter , we draw arli 2 as trisolvate 2a.5 plot of y from eq 4 versus [ thf ] in hexanes cosolvent for the ortholithiation of arh ( 0.050 m ) with lda ( 0.10 m ) in the presence of added diisopropylamine ( 0.050 m ) measured by f nmr spectroscopy at 78 c . [ a = ( 0.03 0.01 ) 10 , n = 2.6 0.2 ] . in the presence of excess [ li , n]lda and low thf concentration , f nmr spectroscopy shows the two doublets of 2a along with two additional broad doublets corresponding to mixed dimer 17 that resolve into more complex multiplets owing to h f coupling with application of window functions . computations showed a 6.3 kcal / mol greater stability of trisolvated dimer 17b than disolvate 17a ( eq 6 ) . ( the computations show a distinct f li interaction in 17a , b ) . monitoring the mixed dimer equilibrium versus thf concentration ( eq 7 and figure 5 ) and fitting according to eq 8 implicates trisolvated mixed dimer 17 ( solvation number of 2.4 0.1 ) . the distinction is not germane to the rate and mechanistic studies.678 plot of y ( eq 8) versus [ thf ] in hexanes cosolvent for the ortholithiation of arh ( 0.050 m ) with lda ( 0.10 m ) in the presence of diisopropylamine ( 0.050 m ) measured with f nmr spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to y = a[thf ] . lithiation of arh using analytically pure ( recrystallized ) lda was monitored using in situ ir spectroscopy by following the disappearance of a strong arene stretch at 1510 cm . reactions that were carried out at low arh concentrations ( 0.0050 m arh ) and that were also clearly first order in arh were followed to > 5 half - lives , and the pseudo - first - order rate constants ( kobsd ) were determined with standard nonlinear fits . under non - pseudo - first - order conditions in situations in which rate - limiting deaggregation dominates or under conditions in which autocatalysis caused deviation from a first - order decay , the initial rates were determined by following the reaction to 5% conversion and extracting the rate at t = 0 from a polynomial fit as described . reaction orders in thf and lda were determined by plotting either kobsd or initial rate versus the respective concentrations . rate studies reveal a rate law described by eq 9 that is consistent with rate - limiting deaggregation of dimer 4 ( eq 10 ) . lithiation of arh at low concentrations ( 0.0050 m ) shows a linearity ( figure 6 ) that suggests either zeroth order in arene or an inherently upwardly curving decay being straightened by autocatalysis ; a plot of initial rate versus arh concentration shows a clear arh concentration independence consistent with a zeroth order in arh ( figure 6 inset ) . the initial rates are also zeroth - order in thf ( figure 7 ) ; the cosolvent dependence illustrates a standard control experiment confirming that the downward slope derives from small medium effects . an approximate first - order ( 1.12 0.06 order ) dependence on lda concentration ( figure 8) is consistent with a dimer - based transition structure . the slight upward curvature signifying an elevated lda order foreshadows mechanistic complexity . isotopic labeling studies confirm post - rate - limiting proton transfer , but the complexity demands that we describe perdeuterated arene in its own section . the overall idealized rate law ( eq 9 ) is consistent with a dominant disolvated - dimer - based rate - limiting deaggregation ( eq 10 ) as noted in previous studies.910 representative plot showing linear decay for the ortholithiation of arh ( 0.0050 m ) with lda ( 0.10 m ) in 12.2 m thf monitored using ir spectroscopy at 78 c . inset shows a plot of initial rate versus [ arh ] ( initial arene concentration ) for the ortholithiation of arh with lda ( 0.10 m ) in thf ( 12.2 m ) measured with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to y = a[arh ] b. [ a = ( 5 5 ) 10 , b = ( 3.1 0.2 ) 10 ] . plot of initial rate versus [ thf ] in et2o ( curve a ) and in hexanes ( curve b ) cosolvent for the ortholithiation of arh ( 0.050 m ) by lda ( 0.10 m ) at 78 c . the curves depict unweighted least - squares fits to y = a[thf ] b. curve a : a = ( 1.1 0.3 ) 10 , b = ( 3.3 0.3 ) 10 . curve b : a = ( 2.8 0.3 ) 10 , b = ( 4.7 0.3 ) 10 . the greater slope using hexanes as cosolvent compared with that using et2o as cosolvent illustrates the influence of long - range medium effects . plot of initial rate versus [ lda ] in thf ( 12.2 m ) for the ortholithiation of arh ( 0.0050 m ) measured with ir spectroscopy at 78 c . [ a = ( 3.5 0.3 ) 10 , n = 1.12 0.06 ] . we suspected that the subtle upward curvature in figure 6 is masked by downward curvature arising from autocatalysis . reveal the anticipated albeit subtle downward curvature ( figure 9 ) . to tease out the underlying mechanistic changes , we carried out the lithiations under pseudo - first - order conditions with varying concentrations of arli . figure 10 shows the rates versus arli concentration and reveals a sigmoid consistent with higher - order saturation kinetics but only a small ( 3-fold ) overall increase in rate . previous studies are fully consistent with such higher - order catalysis but showed only second - order arli dependencies . the analogous second - order curve is included to show the similarity . we suspect that the disagreement is not about the mechanism per se but rather due to the sensitivity of such determinations.11 representative plot showing sigmoidal decay for the ortholithiation of arh ( 0.020 m ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . the red dotted line depicts the time - dependent linear decay extrapolated from the initial rate in the absence of autocatalysis . plot of initial rate versus [ arli ] for the ortholithiation of arh ( 0.0050 m ) by 0.10 m lda in 12.2 m thf monitored with ir spectroscopy at 78 c . solid curve : a = 2 4 , b = ( 3 8) 10 , c = 2.94 10 , n = 3.0 0.5 . dotted curve : n is set at 2 ; a = ( 9 2 ) 10 ) , b = ( 1.7 0.3 ) 10 , c = 2.94 10 . for an alternative view of the autocatalysis , we applied the method of continuous variations ( a job plot ) . initial rates were monitored versus the mole fraction of arli while keeping the total normality of arli and lda constant ( figure 11 ) . the curve corresponds to a nonlinear least - squares fit to the generalized expression in eq 12 . equation 12 is an approximation because it corresponds to a fit of a statistical job plot . moreover , in contrast to normal job plots in which the curvature and position of the maximum provide insight into relative stoichiometries ( via parameters m and n in eq 12 ) , the shifting rate - limiting step precludes such a simple interpretation . had arli been a highly efficient catalyst , for example , the maximum would have been pressed against the left - hand y axis irrespective of stoichiometry . figure 11 does , however , offer a visually retrievable , qualitative view of the catalysis.12 plot of initial rates versus mole fraction of arli ( xarli ) for the serial injection of 0.010 m aliquots of arh to 0.10 m lda in 12.2 m thf monitored with ir spectroscopy at 78 c . [ k = ( 1.67 0.09 ) 10 , k = ( 1.93 0.05 ) 10 , although the saturation has the superficial appearance of michaelis menten kinetics in which an intermediate becomes the dominant observable form , no such form exists . instead , saturation corresponds to a shift in the rate - limiting step as described by eqs 13 and 14 . saturation occurs when rate - limiting deaggregation favored at zero or low arli concentration k1 + karli[arli ] k2 shifts to a new rate - limiting step at high arli concentration k1 + karli[arli ] k2. as the evidence shows , the new rate - limiting step still does not involve proton transfer.13 to ascertain the nature of the new rate - limiting step , we simply added sufficient arli at the outset of the reaction ( 0.020 m arli ) to establish full saturation ( plateau in figure 10 ) and determine a rate law . a zeroth - order in substrate , zeroth order in thf , and second order in lda ( figure 12 ) affords the idealized rate law in eq 15 and implicates a tetrasolvated tetramer - based lda aggregation as the rate - limiting step ( eq 16 ) . the roles of arli catalysis and mixed tetramer intermediates remain shrouded in mystery despite considerable experimental and computational probing . nonetheless , catalyzing the aggregate exchange of dimer 3 has revealed a rate - limiting tetramer pathway ( labeled 11 in scheme 1 ) lurking just beyond the first barrier . in theory , we could bring the proton transfer into view by slowing the trapping step in eq 14 through deuteration . in practice , it is not that simple.1516 plot of initial rate versus [ lda ] in thf ( 12.2 m ) for the ortholithiation of arh ( 0.0050 m ) in the presence of 0.020 m arli monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to y = a[lda ] . [ a = ( 7 1 ) 10 , n = 1.80 0.09 ] . previous studies have shown marked catalysis by traces of licl attributed in all instances to catalyzed deaggregations and monomer - based lithiations . licl ( 0.0010 m ) accelerates the lithiation of arh by lda / thf so much that rates can not be monitored at 78 c with technology available to us . notably , at full saturation using arli as a catalyst ( figure 10 ) , added licl causes a further rate spike ( figure 13 ) , confirming that arli and licl catalyze distinctly different processes . representative plot showing the absorbance of arh versus time for the ortholithiation of arh ( 0.0050 m ) with lda ( 0.10 m ) in thf ( 12.2 m ) at 78 c ( curve a ) . curve b shows the decay under the same conditions as in a but with 0.020 m arli . after the lithiation was complete , 0.0010 m licl was added and a second aliquot was injected into this mixture ( curve c ; see inset for expansion ) . reactions were monitored with ir spectroscopy . as noted in the introduction , lithiations of arh and perdeuterated arene 1-d4 ( ard ) are markedly different . we offer the reaction coordinate diagram in scheme 2 showing qualitative ( relative ) barrier heights for lithiation of ard to aid the discussion . of course , the arh and ard barriers in schemes 1 and 2 can be placed on the same diagram to fully display the influence of isotopic substitution , but the cost is a considerable increase in complexity ; we will do so in the discussion . as a reminder to the reader , the diagram is a static snapshot of a much more fluid picture in which the relative barriers vary markedly with changes in the concentrations of lda , thf , and ard . lda - mediated lithiation of ard at low concentration ( 0.0025 m ard ) affords a decay showing an upward curvature that is neither zeroth nor first order . the intermolecular kinetic isotope effect ( kie)the isotope effect obtained from independently measured initial rates for arh and ard is near unity ( kh / kd = 1.5 ) . the complementary competitive isotope effect , obtained by monitoring the relative rates within a single reaction vessel reveals biphasic kinetics ( figure 14 ) from which kh / kd = 40 was determined from the initial rates . the biphasic kinetics and large isotope effects are highly characteristic of a dominantly post - rate - limiting lithiation in which the less reactive ard does not react until arh is consumed . the fits in figure 14 derive from a numerical integration using the simplified model in scheme 3 . competitive ortholithiation of arh ( 0.0050 m ) and ard ( 0.0050 m ) with lda ( 0.10 m ) in thf ( 12.2 m ) at 78 c . the curves result from a best - fit numerical integration to the highly simplified model in scheme 3 and afford kh / kd = 30 ( supporting information ) . by contrast , measuring the initial slopes directly affords kh / kd = 40 . detailed rate studies reveal the origins of these odd behaviors and present a new view of the ortholithiation . in contrast to arh in which the lithiation occurs in a post - rate - limiting step , rate limitation for ard depends on concentration ( figure 15 ) . the rate studies are consistent with the rate law described by eq 17 and mechanisms described by eqs 1820 . the evidence is presented in the limits of high and low ard concentration as follows.17181920high ard concentration limit:2122low ard concentration limit:232425 plot of initial rate versus [ ard ] for the ortholithiation of ard with lda ( 0.10 m ) in thf ( 12.2 m ) monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to a first - order saturation function : d[arh]/dt = ( a[ard])/(1 b[ard ] ) . [ a = ( 1.5 0.3 ) 10 , b = ( 3.5 0.8 ) 10 ] . ( 1 ) at high ard concentration trapping of a fleeting ( dimeric ) intermediate a2s2 * is efficient ( k1 < k2[ard ] and k1 < k3[ard][s ] in eqs 1720 ) , rendering the reaction zeroth order in ard ( eqs 21 and 22 ) . the initial rates show a first - order dependence on lda concentration and a zeroth - order dependence on thf concentration . the rate law in eq 17 reduces to the much simpler rate law in eq 21 , which corresponds to the rate - limiting dimer fragmentation ( via 4 ) described by eq 10 . ( 2 ) at low ard concentration the trapping is inefficient ( k1 > k2[ard ] and k1 > k3[ard][s ] in eqs 1720 ) . a linear dependence on thf concentration with a significant nonzero intercept ( figure 16 ) and first - order lda dependencies at both low and high thf concentrations ( figure 17 ) reduce the rate law to that in eq 23 . the data are consistent with an a2s2a2s2 * dimer - based pre - equilibrium and an emergent superposition of rate - limiting di- and trisolvated - dimer - based lithiations ( eqs 24 and 25 ) . the failure to observe the saturation kinetics for arh stemmed from the high reactivity and consequent efficient trapping at both low and high concentrations , which may have obscured the trisolvated - dimer - based mechanism made visible by ard . evidence exists , however , that the high isotopic sensitivity diverts proton and deuterium transfers through distinctly different pathways ( vide infra ) . plot of initial rate versus [ thf ] in et2o for the ortholithiation of ard ( 0.0020 m ) by lda ( 0.10 m ) monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to y = a[thf ] b. [ a = ( 1.9 0.3 ) 10 , b = ( 7 2 ) 10 ] . plot of initial rate versus [ lda ] in 12.2 m thf ( curve a : 12.2 m , curve b : 2.03 m ) for the ortholithiation of ard ( 0.0020 m ) monitored with ir spectroscopy at 78 c . curve a depicts an unweighted least - squares fit to y = a[lda ] . [ a = ( 3.4 5 ) 10 , n = 1.08 0.08 ] . curve b depicts an unweighted least - squares fit to y = a[lda ] . [ a = ( 6.8 0.9 ) 10 , n = 0.92 0.07 ] . with stoichiometries of the rate - limiting transition structures in hand , we examined di- and trisolvated - dimer - based metalations computationally ( eq 26 ) . the energy difference is negligible.26 recall that metalations autocatalyzed by arli ( scheme 1 ) bypass the rate - limiting conversion of starting lda dimer 3 to putative open dimer a2s2 * 4 , revealing rate - limiting [ a4s4 ] transition structure 11 and a kinetically invisible post - rate - limiting metalation of arh . guided by previous studies implicating analogous tetramer - based pathways , we surmised that suppressing the rate of metalation using ard would bring either a tetramer- or a monomer - based metalation into view.as stated , this idea contains embedded flaws ; the story is considerably more nuanced . monitoring the initial rates for the metalation of ard versus arli concentration ( perdeuterated aryllithium 2-d3 to be more precise ) showed saturation kinetics ( figure 18 ) analogous to that for arh ( figure 10 ) with an attenuated acceleration but the same high - order dependence on arli concentration . plot of initial rate versus [ arli ] ( specifically , 2-d3 ) for the ortholithiation of ard ( 0.0020 m ) by 0.10 m lda in 12.2 m thf monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to d[arh]/dt = ( a[ard])/(1 b[ard ] ) c. solid curve : [ a = ( 6 2 ) , b = ( 4 1 ) 10 , c = 2.94 10 , n = 3 ] . dotted curve : [ a = ( 4 1 ) 10 ) , b = ( 2.5 0.9 ) 10 , c = 3.23 10 , n = 2 ] . as already described , the mechanism in the limit of low ( zero ) arli concentration is via a2s2-based transition structure 4 using arh . ascertaining the concentration dependencies at saturation ( 0.020 m arli ) a plot of rates versus ard reveals saturation kinetics , indicating an ard concentration dependence at low ard and ard concentration independence at high ard ( figure 19 ) . to be clear , this plot depicts saturation in substrate superimposed on saturation in arli . we treat the two limiting behaviors observed in the ard saturation kinetics separately.figure 19plot of initial rate versus [ ard ] for the ortholithiation of ard in the presence of 0.020 m arli ( 2-d3 ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to a first - order saturation function : d[ard]/dt = ( a[ard])/(1 b[ard ] ) . [ a = ( 1.6 0.3 ) 10 , b = 40 10 ] . plot of initial rate versus [ ard ] for the ortholithiation of ard in the presence of 0.020 m arli ( 2-d3 ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to a first - order saturation function : d[ard]/dt = ( a[ard])/(1 b[ard ] ) . [ a = ( 1.6 0.3 ) 10 , b = 40 10 ] . ( 1 ) in the limit of low ard with added arli , the dependence on ard concentration attests to ard participation in the rate - limiting step . the decays of ard also show curvatures consistent with significant contributions from a first - order dependence as expected for at least a partially rate - limiting metalation . plots of initial rates versus lda and thf show nearly linear dependencies ( with a small nonzero intercept with thf ) , implicating a transition structure of stoichiometry [ a2s3(ard ) ] . because the proton transfer for arh under arli - catalyzed conditions was not kinetically visible , we could not measure the intermolecular isotope effect . an arh / ard competition shows biphasic behavior ( figure 20 ) consistent with the trapping of a common intermediate in a post - rate - limiting step and a substantial kie ( kh / kd = 12 ) . this is consistent with the partial mechanism in scheme 3 in which arh is scavenging a2s2 * ( directly or via tetramer 14 , scheme 1 ) , largely precluding deuterium transfer . competitive ortholithiation of arh ( 0.0050 m ) and ard ( 0.0050 m ) with lda ( 0.10 m ) in the presence of 0.020 m arli in 12.2 m thf at 78 c . the curves result from a best - fit numerical integration to the highly simplified model in scheme 3 and afford kh / kd = 6.3 . fitting the initial rates of both decays ( linearly ) directly affords kh / kd = 12 . ( 2 ) in the limit of high ard with added arli , the decays of ard are decidedly linear ( zeroth order ) , and orders in lda and thf are both unity . the rate - limiting transition state is of stoichiometry [ a2s3]9 ( scheme 2 ) . the most unexpected aspect of the rate studies using arli as the catalyst is that the metalation of arh proceeds via a rate - limiting a4s4-tetramer - based aggregation event , whereas ard diverts to a2s2- and a2s3-dimer - based mechanisms . although one could infer the intermediacy of tetramer en route from one dimer to another , we believe there is a more rational explanation ( vide infra ) . in previous studies of lda / thf - mediated lithiations at 78 c , the dramatic effects of licl on rates were traced to monomer - based lithiations without exception . metalations of arh were too fast to test this thesis , but ard metalations proved highly tractable . monitoring the ard metalation versus licl shows a second - order dependence that saturates at very low ( > 0.0010 m ) licl concentrations , as expected from previous studies ( figure 21 ) . the threefold acceleration is small owing to a very large isotope effect ( kh / kd > 50 ) . the lithiations of ard at full saturation ( 0.0015 m licl ) follow a clean exponential decay consistent with rate - limiting ortholithiation . a half - order lda dependence and second - order thf dependence ( figure 22 ) are consistent with the rate law in eq 27 and the generic trisolvated - monomer - based mechanism described by eqs 28 and 29.27282930computational studies probing the relative efficacies of the open and closed trisolvated - monomer - based transition structures support the closed form presumably owing to a strong li f interaction ( eq 31).31 plot of initial rate versus [ licl ] for the ortholithiation of ard ( 0.0020 m ) by 0.10 m lda in 12.2 m thf monitored with ir spectroscopy at 78 c . [ ard ] = 0.0020 m , [ a2s2 ] = 0.050 m , c = 2.63 10 . [ k1 = ( 5 1 ) 10 , k1 = ( 6 2 ) 10 , k2 = 4.06 , n = 2.0 ] . plot of initial rate versus [ thf ] in et2o for the ortholithiation of ard ( 0.0020 m ) by lda ( 0.10 m ) in the presence of 1.5 mol % licl ( 1.5 mm ) monitored with ir spectroscopy at 78 c . the curve depicts an unweighted least - squares fit to y = a[thf ] . [ a = ( 5 1 ) 10 , n = 1.9 0.1 ] . the rate studies of various metalations have suggested that several lda aggregation events may be detectable using nmr spectroscopy : ( a ) lda subunit exchange should be observable on laboratory time scales at low temperatures , and ( b ) the subunit exchange might occur via a dissociative dimer - derived deaggregation or a tetramer - based associative mechanism . we examined these suppositions using two distinctly different probes of lda subunit exchange . let us first consider the two mechanisms : dissociative subunit exchange:32 associative subunit exchange:33 to facilitate the discussion , we use a2 and b2 as shorthand for [ li]lda and [ li , n]lda , respectively . the two mechanisms are highly simplified but easily distinguished nonetheless . in the dissociative mechanism ( eq 32 ) , the rate - limiting step for subunit exchange is necessarily dimer - based , affording an overall first - order dependence ; once a dimer dissociates it is committed to exchange albeit statistically weighted based on the probability of reaggregating in a mixed isotopic form . although this statistical factor within unequal populations of a2 and b2 can be accounted for , it is more expedient to eliminate it by maintaining equal relative proportions of a2 and b2 . the associative mechanism in eq 33 , by contrast , necessarily involves a tetramer - based rate - limiting step and would manifest an overall second - order dependence . we examined the time - dependent conversion of [ li]lda and [ li , n]lda to the mono - n - labeled isotopologue ( eq 34 ) at fixed 1:1 stoichiometry . the exchange was easily followed using li nmr spectroscopy on laboratory time scales at 60 c ( figure 23 ) . this is satisfyingly consistent with the rate - limiting aggregation events detected in the ortholithiation rate studies . the extended time scales of the exchange when compared with the metalations stems from the second - order conditions . a plot of initial rate versus total lda concentration in equimolar [ li]lda and [ li , n]lda mixtures ( figure 24 ) shows an upward curvature and an order of 1.7 consistent with a composite first- and second - order dependencies expected for competing dimer- and tetramer - based exchange . the rates are also independent of the thf concentration , implicating a2s2- and a4s4-based transition structures.34 li nmr spectra showing the li nuclear exchange of [ li]lda ( 0.10 m ) and doubly n - labeled [ li , n]lda ( 0.10 m ) in 12.2 m thf at 60 c ( eq 34 ) . plot of initial rate for the loss of [ li , n]lda in 1:1 mixtures of [ li]lda and [ li , n]lda versus total [ lda ] titer at 60 c in 12.2 m thf . the curve depicts an unweighted least - squares fit to y = a[lda ] . [ a = ( 1.9 0.3 ) 10 , n = 1.7 0.1 ] . we examined lda subunit exchange by monitoring the temperature - dependent coalescence of the li triplet of [ li , n]lda . if exchange of monomer subunits occurs by a unimolecular process the resulting rate constant , kexch , would be independent of the lda concentration . by contrast , an overall bimolecular exchange mechanism would manifest a linear dependence of kexch on lda concentration . in the event , kexch shows a distinct linear dependence and a substantial non - zero y - intercept ( eq 35 ) for all thf concentrations ( figure 25 ) , implicating competing unimolecular and bimolecular pathways ( eq 36 ) . moreover , the 12fold range of thf concentrations shows minor slope and intercept variations at both low and high [ li , n]lda consistent with zeroth - order thf dependencies as expected for a2s2- and a4s4-based rate - limiting transition structures.35 plot of li nuclear exchange rate at 35 c versus [ lda ] at varying [ li , n]lda and thf concentrations with hexanes as cosolvent . we had previously examined in considerable detail the dimer - based deaggregation of lda to monomer . we turned to dft computations to examine how a4s4 tetramers might be formed and how they might be involved in a metalation . this computational problem is extremely difficult ( in our hands ) ; we offer the artist s rendition of a reaction coordinate in scheme 4 . they provide energies , but we do not take them seriously . the role of bridging thfs as transitional substructures in critical deaggregation steps were detected in dimer - based deaggregation , whereas the higher aggregates appear to be too congested for such thf bridging . the computed solvation numbers come up short by one ( a4s3 rather than a4s4 observed kinetically ) . the most fundamental flaw and the origin of the highest energies ( 26 kcal / mol maximum ) is that two high energy forms monosolvated cyclic dimer and disolvated open dimer condense to form tetramers . ongoing studies of lda - mediated metalations under nonequilibrium conditions are , in essence , a study of lda deaggregation and rate limitation . paradoxical behaviors abound under these conditions in which aggregation exchanges and reactions with substrates battle to determine the rate - limiting step . studies of the ortholithiation of 1,4-difluorobenzene ( 1 ) and its perdeuterated analogue ( 1-d4 ) reveal the reaction coordinate illustrated in schemes 1 and 2 . for readers who bypassed the results , we reiterate a shorthand introduced to simplify the presentation : ( 1 ) the various lda - based fragments are reduced to amsn notations in which a and s connote the lda subunits and coordinated thf ligands , respectively ; ( 2 ) arene 1 and its perdeuterated analogue 1-d4 are represented as arh and ard , respectively ; ( 3 ) aryllithium 2 , perdeuterated aryllithium 2-d3 , and structurally most accurate trisolvated monomer 2a are collectively denoted as simply arli . a staggering number of kinetically detectable minima and maxima cluster in an energetically very narrow window a reaction coordinate approximating a metaphorical washboard . changing concentrations of arh , lda , and thf alter the relative dominance of the barriers , resulting in wild swings in concentration dependencies and rate laws . swapping ard for arh a simple experiment in most settings completely transforms the rate laws and observed mechanisms . autocatalysis by arli accelerates the metalation and shifts the rate - limiting steps , markedly changing the rate laws . adding traces of licl similarly accelerates the reaction but does so via catalysis on an altogether different portion of the reaction coordinate . saturation kinetics simultaneously superimposed saturation kinetics are legion owing to the relentlessly shifting rate - limiting steps . in short , the rules governing rates and mechanisms under conditions in which aggregates are in full equilibrium falter badly for nonequilibrium conditions . within this chaotic picture , however , are several critically important common denominators : ( 1 ) the complexity stems from coincident barriers to reaction with substrate and barriers corresponding to lda aggregation and solvation steps , and ( 2 ) the conditions under which this coincidence occurs lda / thf/78 c is the same for any substrate that reacts measurably . although the different substrates probe a single process the deaggregation of lda dimer 3each substrate provides a different perspective and different mechanistic insights . we begin the analysis with an overview of the mechanism in the context of the reaction coordinate diagram depicted in scheme 1 . it represents a snapshot of a living , breathing reaction coordinate in which the relative barrier heights depend on many parameters . because the equilibria are implicitly rather than explicitly balanced to minimize clutter fragments of lda and solvent molecules are inserted only where needed we opened the results with a reaction profile ( figure 1 ) showing the formation of arli and an lda - arli mixed aggregate characterized as 2a and 17 , respectively . mixed aggregate 17 is only observable under highly specialized circumstances low thf and high lda concentrations and is of little to no importance to our mechanistic thinking . odd curvatures , however , are a consequence of a zeroth - order dependence in substrate overlaid with low levels of autocatalysis . the model used to generate the curves in figure 1 stems from the rate studies . we hasten to add that the quality of the fit is satisfying and consistent with the conclusions but should not be construed as confirmation . the metalation of arh by lda dimer 3 ( a2s2 ) proceeds via the [ a2s2 ] rate - limiting transition structure 4 to give fleeting a2s2 * -dimer - based intermediate 7 . a chemically tangible , computationally viable depiction of this dimer to open dimer conversion is shown in eq 10 . ensuing autocatalysis accelerates the overall reaction via a2(arli)n transition structure 5 ( n = 2 or 3 ) . although the acceleration is moderate , it circumvents the [ a2s2 ] barrier , revealing a well - defined tetramer - based [ a4s4 ] barrier previously lurking over the horizon that does not formally include arh in the transition structure . ( we should clarify this statement by noting that we define transition structure as the purely molecular depiction and the transition state as the energetically complete analog that includes all the necessary fragments including those not yet actively participating . ) the structures affiliated with a2(arli)n and [ a4s4 ] higher aggregates have been discussed previously in the context of a2(arli)2 ladder structures . ( if we are forced to accept an a2(arli)3 mixed - pentamer - based transition structure , we have no ideas worthy of sharing . ) the role of a4s4 tetramers that we detected in the metalation rate studies was confirmed by nmr spectroscopic studies of lda showing significant tetramer - based subunit exchange . we provided a calculated reaction coordinate for a tetramer - based deaggregation of lda , dimer - to - tetramer - to - monomer , in scheme 4 . . we exploited large kies to detect or infer the existence of additional components of the reaction coordinate in scheme 1 . a higher [ a2s3 ] barrier 9 is inferred from direct detection of [ a2s2(arh)]and [ a2s3(arh ) ] transition structures 13 and 16 , which correspond to the ortholithiations ( proton / deuterium transfers ) as shown in scheme 5 . we include the computationally viable a2s2*[a2s3]a2s3 * ( 7912 ) transformation implicit in scheme 1 . the tetramer - based metalation of 15 could be inferred from the kinetically detectable [ a4s4 ] aggregation event , yet [ a4s4(arh)]-based lithiation was not kinetically visible and could be questioned in light of the previously noted tetramer - based deaggregation to monomers . all reactions of lda under nonequilibrium conditions studied to date have been accelerated by licl at ppm levels owing to the catalysis of dimer monomer exchange . the efficacy of licl relative to arli , in conjunction with saturation kinetics for both showing different rates at saturation , confirms that arli and licl catalyze different steps . indeed , in contrast to the arli - catalyzed dimer dimer equilibration , licl diverts the ortholithiation of the less reactive ard form through [ as3(ard)]-based transition structure 10 . open- and closed - monomer - based transition structures ( eq 31 ) are computationally viable . we have described nmr spectroscopic studies of the exchange of [ li]lda and [ li , n]lda . the most important finding is that lda subunits exchange slowly on laboratory time scales at 78 c consistent with the ortholithiation results . direct rate studies of the subunit exchanges as well as complementary lda coalescence studies implicated both dissociative ( dimer - based ) and associative ( tetramer - based ) exchange mechanisms , a satisfying result given that both are prominent in the lithiation rate studies . extensive computational studies of the dissociative pathways had been published . scheme 4 provides insight into the tetramer - based events , although the computational studies were difficult as noted above . overall , evidence that lda associates to tetramer en route to monomers is both convincing and provocative . this work calls out for a discussion of the basic principles underlying rate limitation . imagine the simplified scenario illustrated in scheme 6 in which an a2s2 partial deaggregation is followed by an a4s4 tetramer and subsequent post - rate - limiting lithiation of arh . we have chosen this particular sequence owing to its pedagogic value rather than its central importance . we further imposed the restriction that the relative barrier heights are similar and follow the order [ a2s2 ] > [ a4s4 ] > [ a4s4(arh ) ] , which is consistent with a subset of the experimental results . ( 1 ) how does the [ a4s4 ] barrier influence the reaction rate and rate law ? conventional wisdom suggests that [ a2s2 ] is the rate - limiting barrier , and [ a4s4 ] is irrelevant , but that is not altogether correct . the existence of seemingly post - rate - limiting intermediate a2s2 * imposes a barrier - weighted statistical factor on the rate . * has < 100% probability of proceeding to product . in the limit that [ a2s2 ] and [ a4s4 ] present barriers of equal height , that probability reduces to 50% the rate law would also reflect barrier - weighted contributions from [ a2s2 ] and [ a4s4 ] , including reaction orders that would be intermediate values rather than tidy integers . as the rate - limiting step shifts , there are isotopic labeling studies to probe post - rate - limiting steps through competition experiments ( see part 6 for example ) . more direct approaches either lower the obstructing barrier or elevate the subsequent barrier . recall that the energy diagrams are not static , but rather shift with changing concentrations of all participating species . ( 3 ) how does substrate concentration influence reaction rates and mechanism ? at high arh concentration , the barrier height for metalation is low as drawn in scheme 6 , and the metalation is post - rate - limiting . at lower concentrations , however , all minima and maxima drop relative to the [ a4s4 - -h -- ar ] barrier , rendering the metalation rate limiting . using eq 37 as an alternative perspective , post - rate - limiting metalation occurs when k1 k2[arh ] , and the intermediate denoted generically as amsn is efficiently converted to arli with high fidelity . at low arh concentrations , however , the trapping becomes inefficient , k1 k2[arh ] , and amsn is in a fully established equilibrium with a2s2 . a plot of rate versus [ arh ] over a wide concentration range would display saturation kinetics . although the rate - limiting metalations of arh were too fast to observe , we noted saturation behavior under several circumstances with ard ( figures 15 and 19 ) . deuteration , however , introduces enormous complexities ( see part 6).37 ( 4 ) how would changing the concentration of lda influence the rates ? elevated lda concentration would promote the higher - order step by lowering the [ a4s4 ] height relative to that of [ a2s2 ] , with the ironic effect of eliminating residual contributions of the tetramer - based step from the rate law . viewed from the alternative perspective in eq 38 , elevated lda concentration imposes k2[a2s2 ] k1 and renders the step corresponding to k1 rate - limiting . by contrast , lowering the lda concentration would raise [ a4s4 ] , causing [ a4s4 ] to come into parity and eventually dominate [ a2s2 ] . varying lda concentration over the full range would afford a rate law that reflects the shifting rate - limiting step by showing a shift from second - order lda dependence at low lda concentrations to a first - order dependence at elevated concentrations . although we detected dimer- and tetramer - based metalations in the experiments above , the concentration range is too narrow to observe such a shift . as a final note , the relative heights of the [ a4s4 ] and [ a4s4(arh ) ] barriers do not change with lda concentration . consequently , changing the lda concentration can not bring the tetramer - based metalation into view.38 ( 5 ) what are the consequences of catalyzing the dimer - to - dimer conversion , circumventing [ a2s2 ] in scheme 6 ? the short answer is that the rate - limiting step is shifted to transition structure [ a4s4 ] . to understand the role of catalysis let us consider the alternative perspective in eq 39 . when [ a2s2 ] is rate - limiting , catalysis of the forward step accelerates the formation of a2s2 * and the overall reaction . at elevated catalyst loading , catalysis of the back reaction makes k cat[cat ] k2[a2s2 ] with a consequent shift of the rate - limiting step to [ a4s4 ] . the kinetics would show saturation in catalyst ( as in figures 10 and 18 ) and an accompanying shift from first to second order in lda . here is the curious part : catalysis of the forward step is the source of acceleration whereas catalysis of the back reaction shifts the rate - limiting step.39 ( 6 ) how does isotopic substitution shift the rate - limiting step ? recall that the isotope effects are quite large , and the relative barrier heights for the different transition states cluster in a narrow energetic range . scheme 7 shows barriers for the highly simplified and generic reaction coordinate with barriers for ard superimposed on those for arh . of course , the core principle is that the rate depression through deuteration stems from a lower zero point energy ( zpe ) in the ground state that is eliminated at the transition state . ( although tunneling in the transition structure is certainly possible even probable it does not change the model . ) when applied to sequential barriers in scheme 7 , zpe also stabilizes the preceding transition state that includes both the [ amsn ] and ard components owing to net stabilization of ard with the consequent shift to rate - limiting metalation , [ amsn -- h(d)--ar ] . thus , the passive role of the arene zpe in the nonmetalation - based aggregation step shifts the rate - limiting step . ( 7 ) what are the origins of the competitive isotope effects and biphasic kinetics ? measurement of kinetic isotopes through competition of deuterated and protonated substrates necessarily leads to biphasic kinetics ( figures 14 and 20 ) if the metalation step is post rate limiting . we are unaware of other clean examples of such biphasic kinetics . a highly simplified model in scheme 3 biphasic kinetics ostensibly stem from efficient trapping by the more reactive protio form ( arh ) first and by the deuterio form ( ard ) only after the arh is consumed , but this outcome is misleading as written . imagine the competition of arh and ard represented in the idealized reaction coordinate diagram in scheme 8 . note that arh and ard are both included in a single thermochemical depiction of the ground state . h and c d stretches are lost in the transition state . the competitive isotope effect preferential proton versus deuterium transfer stems from the lower zpe of ard in the transition state . by using an arh ard mixture not only in the vessel but also in the thermochemical diagram , we have arithmetically shifted the isotopic contribution of zpe to the transition state . ( 8) how does isotopic labeling divert a reaction through an entirely different reaction coordinate ? using catalyzed conditions we observed a tetramer - based [ a4s4 ] rate - limiting step . anticipating that deuteration would suppress the metalation rate and bring a tetramer - based metalation ( [ a4sn(ard ) ] ) into view an assertion that should feel charged with intellectual risk at this point we were surprised to detect [ a2s2(ard ) ] and [ a2s3(ard ) ] ( dimer - based ) metalations . let us strip away the inordinate complexities of schemes 1 and 2 by gazing at just the relative barrier heights ( scheme 9 ) . in short , the relative energies of [ a2s2 ] and [ a4s4 ] aggregation events do not correlate with the relative energies of the [ a2s2(ard ) ] , [ a2s3(ard ) ] , and [ a4sn(ard ) ] metalations . in fact , there is no reason whatsoever to expect such a correlation of the two fundamentally different processes such as aggregation and metalation . this is easy to say in retrospect . by shifting the rate - limiting step , fleeting intermediates a2s2 * , a2s3 , and a4s4 are all formed at equilibrium with starting lda dimer a2s2 , causing the choice of pathway to derive exclusively from the relative facilities of the proton transfers . ( 9 ) can the reaction coordinate diagrams corresponding to reaction of arh ( scheme 1 ) and ard ( scheme 2 ) be presented as a single , self - consistent , coherent reaction coordinate diagram ? in short , they sure better be superimposable , and indeed such a depiction is self - consistent ( scheme 10 ) . as to whether the picture is coherent , we have our doubts , and adding labels to scheme 10 would likely not help . a unified depiction , however , is not impossible . the problem with superimposing schemes 1 and 2 to create scheme 10 is that there are six distinct minima and five maxima in which contributions from zpe create different energies corresponding to arh ( red ) and ard ( blue)12 minima and 14 maxima in total . taking a cue from the discussion of the competitive isotopic studies ( part 7 ) and from shifting the zpes to only four transition states , we offer a fully labeled variant as scheme 11 with no further comment except to marvel at the finished product . metalations of many substrates , including 1,4-difluorobenzene , using lda / thf at 78 c exhibit remarkable rate behaviors owing to the coincidence of barrier heights for aggregation events and metalations . trace impurities such as licl can accelerate metalations moderately or massively ( up to 100-fold ) , depending on the substrate . commercial lda and lda generated in situ are very different a difference that can be eliminated by adding traces of et3nhcl as a licl precursor to commercial lda . but the consequences are more subtle than that . given the hypersensitivity of the choice of substrate to the ensuing mechanism , nonequilibrating aggregates cause erratic regio- and stereoselectivities . such feedback loops cause regioselectivities to vary with percent conversion and with the number of equivalents of lda used . the metalation of arene 1 has provided the best view to date of how rate - limiting aggregation and solvation events involved in lda deaggregation can dictate rates and mechanisms . we found , for example , that lithium salts can catalyze different steps involved in the deaggregation . aryllithium 2 catalyzes lda closed - to - open dimer conversion , whereas licl catalyzes dimer - to - monomer conversion . this work offered a plethora of examples of saturation kinetics arising from shifting rate - limiting steps , often superimposing saturation behaviors . the plotline that emerged in many ways is more about understanding rate limitation and how to probe specific steps along a reaction coordinate than about organolithium chemistry per se . the job plots used to study autocatalysis represent exceedingly rare examples of job plots used to study reaction kinetics . although measured competitive kies reveal highly characteristic biphasic kinetics , we must confess to being unaware of others exploiting such diagnostic behavior . overall , the methodological developments required to study this remarkably complex organometallic problem are as poignant as the chemistry itself . thf , et2o , and hexanes were distilled from blue or purple solutions containing sodium benzophenone ketyl . literature procedures were modified to prepare lda as a licl- and ligand - free solid . ir spectra were recorded using an in situ ir spectrometer fitted with a 30-bounce , silicon - tipped probe . the spectra were acquired in 16 scans at a gain of 1 and a resolution of 4 cm . a representative reaction was carried out as follows : the ir probe was inserted through a nylon adapter and o - ring seal into an oven - dried , cylindrical flask fitted with a magnetic stir bar and a t - joint . the t - joint was capped by a septum for injections and a nitrogen line . after evacuation under full vacuum , heating , and flushing with nitrogen , the flask was charged with lda ( 108 mg , 1.01 mmol ) in thf and cooled in a dry ice licl was added via a thf stock solution prepared from et3nhcl and lda . after recording a background spectrum , we added arene 1 ( 0.76 mmol ) with stirring . for the most rapid reactions , ir spectra were recorded every 3 s with monitoring of the absorbance at 1510 cm over the course of the reaction . standard li , c , n , and f nmr spectra were recorded on a 500 mhz spectrometer at 73.57 , 125.79 , 50.66 , and 470.35 mhz , respectively . the li , c , and n resonances are referenced to 0.30 m [ li]licl / meoh at 90 c ( 0.0 ppm ) , the ch2o resonance of thf at 90 c ( 67.57 ppm ) , and neat me2net at 90 c ( 25.7 ppm ) , respectively . a 10.6 m solution of n - buli in hexanes ( 4.8 ml , 50.1 mmol ) was added via syringe pump to a solution of 1,4-difluorobenzene ( 1 , arh , 5.0 ml , 48.6 mmol ) in 150 ml of dry thf at 78 c under argon over 20 min . meod ( 2.05 ml , 50.1 mmol ) was added via syringe pump over 20 min . the mixture was allowed to stir for 30 min . without any intervening workups sequential additions of 1.1 equiv of n - buli and 1.1 equiv of meod a final aliquot of meod ( 10 ml , 5.0 equiv ) was added to quench the reaction fully . after the mixture was allowed to warm to room temperature , the ph was adjusted to 1.0 with 4.0 m aq hcl to dissolve all lithium salts . organic and aqueous layers were separated , and the organic layer was extracted with additional cold 0.020 m hcl to remove excess thf . the product was collected as a colorless liquid ( 1.75 g , 15.3 mmol ) via distillation at 88 c in 31.5% yield : c nmr 158.8 ( dqn , jc f = 13.2 hz ) ; lrms 118.1 m / z shows 98% 1-d4 . the time - dependent concentration plots obtained using ir spectroscopy were fit to mechanistic models expressed by a set of differential equations . the curve - fitting operation minimizes -square in searching for the coefficient values ( rate constants ) . marquardt algorithm was used for the -square minimization and is a form of nonlinear , least - squares fitting . the fitting procedure implements numeric integration based on the backward differentiation formula to solve the differential equations , yielding functions describing concentration versus time .

lithiation of 1,4-difluorobenzene with lithium diisopropylamide ( lda ) in thf at 78 c joins the ranks of a growing number of metalations that occur under conditions in which the rates of aggregate exchanges are comparable to the rates of metalation . as such , a substantial number of barriers vie for rate limitation . rate studies reveal that rate - limiting steps and even the choice of reaction coordinate depend on subtle variations in concentration . deuteration shifts the rate - limiting step and markedly alters the concentration dependencies and overall rate law . this narrative is less about ortholithiation per se and more about rate limitation and the dynamics of lda aggregate exchange .

Introduction Results Discussion Summary Conclusions Experimental Section

a survey of more than 500 total syntheses shows that lithium diisopropylamide ( lda ) is the most frequently used reagent in organic synthesis . for several practical reasons , we focused mechanistic studies on reactions that could be monitored at temperatures ranging from 55 to + 25. despite a large number of mechanistic variations arising from dozens of substrate solvent combinations , the aggregate equilibrations were rapid on the time scales of the rate - limiting substrate lithiation . there is necessarily a window of substrate reactivity a critical temperature range in which readily observed lithiations and lda aggregate exchanges occur at comparable rates . in an irony that will be lost on few organic chemists , this twilight zone for lda / tetrahydrofuran ( thf)-mediated lithiations is centered at 78 c : any lda / thf - mediated lithiation that proceeds at observable rates at 78c is occurring under nonlimiting conditions in which aggregation events and reactions with substrate vie to be rate limiting . under such nonlimiting conditions , the rules governing reactivity change markedly : aggregates are no longer in full equilibrium ; the rate - limiting step shifts unpredictably with subtle changes in reaction conditions ; catalysis by traces of extraneous lithium salts ( especially lithium chloride ) and autocatalysis by the developing product become acute ; and substituting deuterium for protium can change the rate , mechanism , and rate law . in short , lda / thf - mediated lithiations observed at 78 c are complex even by the standards of organolithium chemistry.1 we describe herein mechanistic studies of the lda / thf - mediated lithiation of 1,4-difluorobenzene ( 1 ) ( eq 1 ) . the mechanistic changes accompanying subtle changes in conditions are legion , but we have not peered beyond rate - limiting steps with such clarity as described below . the key to understanding the results is that changes in conditions essentially any changes shift the rate - limiting steps and consequentially alter the mathematical form of the accompanying rate law . f nmr spectroscopy shows the time dependence of a number of species : a downwardly curving decay of arh , a nearly linear formation of arli , and sigmoidal growth of an lda the mixed aggregate is much less relevant than one might think , and the other decays are much more complex than most could imagine . a c nmr spectrum shows the carbanionic carbon resonance of 2a as a triplet ( jli c = 6.0 hz ) further split by a large ( 130 hz ) two - bond f c coupling and a small ( 18.6 hz ) three - bond f f nmr spectrum of lda ( 0.10 m ) with arh ( 0.050 m ) and diisopropylamine ( 0.050 m ) in 3.5 m thf / hexanes at 78 c . hereafter , we draw arli 2 as trisolvate 2a.5 plot of y from eq 4 versus [ thf ] in hexanes cosolvent for the ortholithiation of arh ( 0.050 m ) with lda ( 0.10 m ) in the presence of added diisopropylamine ( 0.050 m ) measured by f nmr spectroscopy at 78 c . the distinction is not germane to the rate and mechanistic studies.678 plot of y ( eq 8) versus [ thf ] in hexanes cosolvent for the ortholithiation of arh ( 0.050 m ) with lda ( 0.10 m ) in the presence of diisopropylamine ( 0.050 m ) measured with f nmr spectroscopy at 78 c . under non - pseudo - first - order conditions in situations in which rate - limiting deaggregation dominates or under conditions in which autocatalysis caused deviation from a first - order decay , the initial rates were determined by following the reaction to 5% conversion and extracting the rate at t = 0 from a polynomial fit as described . rate studies reveal a rate law described by eq 9 that is consistent with rate - limiting deaggregation of dimer 4 ( eq 10 ) . the overall idealized rate law ( eq 9 ) is consistent with a dominant disolvated - dimer - based rate - limiting deaggregation ( eq 10 ) as noted in previous studies.910 representative plot showing linear decay for the ortholithiation of arh ( 0.0050 m ) with lda ( 0.10 m ) in 12.2 m thf monitored using ir spectroscopy at 78 c . inset shows a plot of initial rate versus [ arh ] ( initial arene concentration ) for the ortholithiation of arh with lda ( 0.10 m ) in thf ( 12.2 m ) measured with ir spectroscopy at 78 c . plot of initial rate versus [ lda ] in thf ( 12.2 m ) for the ortholithiation of arh ( 0.0050 m ) measured with ir spectroscopy at 78 c . we suspect that the disagreement is not about the mechanism per se but rather due to the sensitivity of such determinations.11 representative plot showing sigmoidal decay for the ortholithiation of arh ( 0.020 m ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . moreover , in contrast to normal job plots in which the curvature and position of the maximum provide insight into relative stoichiometries ( via parameters m and n in eq 12 ) , the shifting rate - limiting step precludes such a simple interpretation . instead , saturation corresponds to a shift in the rate - limiting step as described by eqs 13 and 14 . saturation occurs when rate - limiting deaggregation favored at zero or low arli concentration k1 + karli[arli ] k2 shifts to a new rate - limiting step at high arli concentration k1 + karli[arli ] k2. as the evidence shows , the new rate - limiting step still does not involve proton transfer.13 to ascertain the nature of the new rate - limiting step , we simply added sufficient arli at the outset of the reaction ( 0.020 m arli ) to establish full saturation ( plateau in figure 10 ) and determine a rate law . a zeroth - order in substrate , zeroth order in thf , and second order in lda ( figure 12 ) affords the idealized rate law in eq 15 and implicates a tetrasolvated tetramer - based lda aggregation as the rate - limiting step ( eq 16 ) . nonetheless , catalyzing the aggregate exchange of dimer 3 has revealed a rate - limiting tetramer pathway ( labeled 11 in scheme 1 ) lurking just beyond the first barrier . in practice , it is not that simple.1516 plot of initial rate versus [ lda ] in thf ( 12.2 m ) for the ortholithiation of arh ( 0.0050 m ) in the presence of 0.020 m arli monitored with ir spectroscopy at 78 c . licl ( 0.0010 m ) accelerates the lithiation of arh by lda / thf so much that rates can not be monitored at 78 c with technology available to us . representative plot showing the absorbance of arh versus time for the ortholithiation of arh ( 0.0050 m ) with lda ( 0.10 m ) in thf ( 12.2 m ) at 78 c ( curve a ) . as a reminder to the reader , the diagram is a static snapshot of a much more fluid picture in which the relative barriers vary markedly with changes in the concentrations of lda , thf , and ard . the biphasic kinetics and large isotope effects are highly characteristic of a dominantly post - rate - limiting lithiation in which the less reactive ard does not react until arh is consumed . competitive ortholithiation of arh ( 0.0050 m ) and ard ( 0.0050 m ) with lda ( 0.10 m ) in thf ( 12.2 m ) at 78 c . in contrast to arh in which the lithiation occurs in a post - rate - limiting step , rate limitation for ard depends on concentration ( figure 15 ) . the rate studies are consistent with the rate law described by eq 17 and mechanisms described by eqs 1820 . the evidence is presented in the limits of high and low ard concentration as follows.17181920high ard concentration limit:2122low ard concentration limit:232425 plot of initial rate versus [ ard ] for the ortholithiation of ard with lda ( 0.10 m ) in thf ( 12.2 m ) monitored with ir spectroscopy at 78 c . the rate law in eq 17 reduces to the much simpler rate law in eq 21 , which corresponds to the rate - limiting dimer fragmentation ( via 4 ) described by eq 10 . with stoichiometries of the rate - limiting transition structures in hand , we examined di- and trisolvated - dimer - based metalations computationally ( eq 26 ) . the energy difference is negligible.26 recall that metalations autocatalyzed by arli ( scheme 1 ) bypass the rate - limiting conversion of starting lda dimer 3 to putative open dimer a2s2 * 4 , revealing rate - limiting [ a4s4 ] transition structure 11 and a kinetically invisible post - rate - limiting metalation of arh . we treat the two limiting behaviors observed in the ard saturation kinetics separately.figure 19plot of initial rate versus [ ard ] for the ortholithiation of ard in the presence of 0.020 m arli ( 2-d3 ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . plot of initial rate versus [ ard ] for the ortholithiation of ard in the presence of 0.020 m arli ( 2-d3 ) with lda ( 0.10 m ) in 12.2 m thf monitored with ir spectroscopy at 78 c . ( 1 ) in the limit of low ard with added arli , the dependence on ard concentration attests to ard participation in the rate - limiting step . an arh / ard competition shows biphasic behavior ( figure 20 ) consistent with the trapping of a common intermediate in a post - rate - limiting step and a substantial kie ( kh / kd = 12 ) . competitive ortholithiation of arh ( 0.0050 m ) and ard ( 0.0050 m ) with lda ( 0.10 m ) in the presence of 0.020 m arli in 12.2 m thf at 78 c . the rate - limiting transition state is of stoichiometry [ a2s3]9 ( scheme 2 ) . the most unexpected aspect of the rate studies using arli as the catalyst is that the metalation of arh proceeds via a rate - limiting a4s4-tetramer - based aggregation event , whereas ard diverts to a2s2- and a2s3-dimer - based mechanisms . in previous studies of lda / thf - mediated lithiations at 78 c , the dramatic effects of licl on rates were traced to monomer - based lithiations without exception . a half - order lda dependence and second - order thf dependence ( figure 22 ) are consistent with the rate law in eq 27 and the generic trisolvated - monomer - based mechanism described by eqs 28 and 29.27282930computational studies probing the relative efficacies of the open and closed trisolvated - monomer - based transition structures support the closed form presumably owing to a strong li f interaction ( eq 31).31 plot of initial rate versus [ licl ] for the ortholithiation of ard ( 0.0020 m ) by 0.10 m lda in 12.2 m thf monitored with ir spectroscopy at 78 c . in the dissociative mechanism ( eq 32 ) , the rate - limiting step for subunit exchange is necessarily dimer - based , affording an overall first - order dependence ; once a dimer dissociates it is committed to exchange albeit statistically weighted based on the probability of reaggregating in a mixed isotopic form . the associative mechanism in eq 33 , by contrast , necessarily involves a tetramer - based rate - limiting step and would manifest an overall second - order dependence . this is satisfyingly consistent with the rate - limiting aggregation events detected in the ortholithiation rate studies . the rates are also independent of the thf concentration , implicating a2s2- and a4s4-based transition structures.34 li nmr spectra showing the li nuclear exchange of [ li]lda ( 0.10 m ) and doubly n - labeled [ li , n]lda ( 0.10 m ) in 12.2 m thf at 60 c ( eq 34 ) . ongoing studies of lda - mediated metalations under nonequilibrium conditions are , in essence , a study of lda deaggregation and rate limitation . paradoxical behaviors abound under these conditions in which aggregation exchanges and reactions with substrates battle to determine the rate - limiting step . changing concentrations of arh , lda , and thf alter the relative dominance of the barriers , resulting in wild swings in concentration dependencies and rate laws . autocatalysis by arli accelerates the metalation and shifts the rate - limiting steps , markedly changing the rate laws . saturation kinetics simultaneously superimposed saturation kinetics are legion owing to the relentlessly shifting rate - limiting steps . in short , the rules governing rates and mechanisms under conditions in which aggregates are in full equilibrium falter badly for nonequilibrium conditions . it represents a snapshot of a living , breathing reaction coordinate in which the relative barrier heights depend on many parameters . imagine the simplified scenario illustrated in scheme 6 in which an a2s2 partial deaggregation is followed by an a4s4 tetramer and subsequent post - rate - limiting lithiation of arh . the existence of seemingly post - rate - limiting intermediate a2s2 * imposes a barrier - weighted statistical factor on the rate . as the rate - limiting step shifts , there are isotopic labeling studies to probe post - rate - limiting steps through competition experiments ( see part 6 for example ) . at high arh concentration , the barrier height for metalation is low as drawn in scheme 6 , and the metalation is post - rate - limiting . using eq 37 as an alternative perspective , post - rate - limiting metalation occurs when k1 k2[arh ] , and the intermediate denoted generically as amsn is efficiently converted to arli with high fidelity . although the rate - limiting metalations of arh were too fast to observe , we noted saturation behavior under several circumstances with ard ( figures 15 and 19 ) . deuteration , however , introduces enormous complexities ( see part 6).37 ( 4 ) how would changing the concentration of lda influence the rates ? varying lda concentration over the full range would afford a rate law that reflects the shifting rate - limiting step by showing a shift from second - order lda dependence at low lda concentrations to a first - order dependence at elevated concentrations . the short answer is that the rate - limiting step is shifted to transition structure [ a4s4 ] . when [ a2s2 ] is rate - limiting , catalysis of the forward step accelerates the formation of a2s2 * and the overall reaction . at elevated catalyst loading , catalysis of the back reaction makes k cat[cat ] k2[a2s2 ] with a consequent shift of the rate - limiting step to [ a4s4 ] . here is the curious part : catalysis of the forward step is the source of acceleration whereas catalysis of the back reaction shifts the rate - limiting step.39 ( 6 ) how does isotopic substitution shift the rate - limiting step ? thus , the passive role of the arene zpe in the nonmetalation - based aggregation step shifts the rate - limiting step . using catalyzed conditions we observed a tetramer - based [ a4s4 ] rate - limiting step . by shifting the rate - limiting step , fleeting intermediates a2s2 * , a2s3 , and a4s4 are all formed at equilibrium with starting lda dimer a2s2 , causing the choice of pathway to derive exclusively from the relative facilities of the proton transfers . metalations of many substrates , including 1,4-difluorobenzene , using lda / thf at 78 c exhibit remarkable rate behaviors owing to the coincidence of barrier heights for aggregation events and metalations . given the hypersensitivity of the choice of substrate to the ensuing mechanism , nonequilibrating aggregates cause erratic regio- and stereoselectivities . this work offered a plethora of examples of saturation kinetics arising from shifting rate - limiting steps , often superimposing saturation behaviors . the plotline that emerged in many ways is more about understanding rate limitation and how to probe specific steps along a reaction coordinate than about organolithium chemistry per se . a 10.6 m solution of n - buli in hexanes ( 4.8 ml , 50.1 mmol ) was added via syringe pump to a solution of 1,4-difluorobenzene ( 1 , arh , 5.0 ml , 48.6 mmol ) in 150 ml of dry thf at 78 c under argon over 20 min .

macrophages are highly plastic monocyte - derived cells that acquire different molecular and functional phenotypes following exposure to different bioactive molecules and environments . the early studies on the interactions of macrophages and lymphocytes in battling bacterial infections revealed the t helper type 1 ( th1 ) secreted cytokine ifn to be involved in the classical activation of macrophages ( nathan et al . , 1983 ) . however , seminal studies by the groups of gordon and mantovani have extensively characterized additional macrophage subtypes activated in alternative manners ( reviewed in mantovani et al . , 2004 ; martinez et al . , 2009 ) . since the major polarizing cytokines initially found to be involved in classical and alternative activation were derived from th1 ( ifn ) and th2 ( il-4 and il-13 ) lymphocytes these activated macrophages were named m1 and m2 , respectively . later studies revealed that in addition to il-4 , alternative activation can also be induced by immune complexes and glucocorticoids ( martinez et al . , 2008 ) , and they are instrumental in immune responses against intracellular microbes and tumors ( mantovani et al . , m2 macrophages are more heterogeneous , but generally play a role in th2 responses , such as killing and encapsulation of extracellular parasites , resolving type 1 inflammation , and promoting tissue repair and remodeling . m2 macrophages are also playing a role in immune regulation and promote tumor progression ( mantovani et al . m1 and m2 macrophages are not only distinct in function , but also express different receptors and enzymes required for their activities . m1 macrophages express high levels of inflammatory cytokines ( il-12 , il-23 , tnf , il-1 , and il-6 ) and chemokines ( cxcl9 , 10 , and 11 , ccl2 , 3 , 4 , and 5 , and cxcl2 ) , as well as enzymes involved in the generation of reactive oxygen species ( ros ) and nitric oxide ( no ; mantovani et al . , 2005 ) . m2 macrophages express lower levels of inflammatory mediators , but high levels of il-10 , scavenger , mannose , and galactose receptors . importantly , in mice , m2 express the enzyme arginase-1 that intercepts the no generation pathway [ though inducible no synthase ( inos ) ] to generate ornithine and polyamines that are instrumental in tissue repair and fibrosis ( hesse et al . , 2001 ) . hence , the expression of inos and arginase-1 are major markers deciphering m1 and m2 macrophages . additional markers of m2 , such as ym1 and fizz1 , were later identified in mouse macrophages ( raes et al . , 2002 , 2005 ) . macrophages also undergo dramatic molecular and functional changes upon encounter , interaction with , and uptake of apoptotic cells ( efferocytosis ) during the resolution of inflammation . in this article we will highlight some of the similarities between m2 differentiation and transcriptional events activated by early efferocytosis . in addition , we will discuss recent results that support the notion that efferocytosis can eventually transform macrophages to another phenotype that is postulated to limit tissue repair / fibrosis and promote macrophage regulatory properties at remote sites . in this regard , it is important to note the early studies that indicated non - phlogistic activation of monocytes by the pro - resolving lipid mediators lipoxins . this bioactivity of lipoxins resulted in increased adhesion and migration of human monocytes ( maddox and serhan , 1996 ; maddox et al . , 1997 , 1998 ) hence prompting the notion that resolution - driven monocyte / macrophage activation promotes tissue repair and wound healing . the recognition , engulfment , and responsiveness to apoptotic cells are cardinal properties of resident and inflammatory macrophages and play a role in processes , such as tissue morphogenesis and homeostasis , embryonic development , hematopoiesis , immunity , and the resolution of inflammation ( savill et al . , 2002 ; erwig and henson , 2007 ; ravichandran and lorenz , 2007 ) . the recognition and uptake of apoptotic cells by macrophages through eat me signals ( and the absence of do not eat me signals ) expressed on their surface and their cognate receptors have been extensively studied and reviewed ( ravichandran , 2011 ) . however , apoptotic cells also transduce signals to the engulfing macrophages that result in significant molecular and functional adjustments that address physiological needs consequent to the identified cell death . during the resolution of inflammation , macrophages engulf apoptotic cells and consequently , apoptotic cell recognition evokes distinct signaling events ( patel et al . , 2006 ) that block the release of pro - inflammatory mediators from macrophages . this release is activated by bacterial moieties , and its blockage , which is termed immune - silencing ( voll et al . , 1997 ; 2004 ) , is accompanied by the production of tgf and il-10 ( byrne and reen , 2002 ; huynh et al . , 2002 ; the engulfment of apoptotic leukocytes by macrophages also leads to inhibition of inos expression and stimulates the expression of arginase-1 in the raw 264 macrophage cell line ( freire - de - lima et al . , in addition , the production of angiogenic growth factors ( golpon et al . , 2004 ) by macrophages is consequent to the uptake of apoptotic cells . elucidation of the signaling pathways activated by efferocytosis revealed significant roles for nuclear transcriptional regulators , such as peroxisome proliferator activated receptor ( ppar)- ( freire - de - lima et al . , 2006 ; johann et al . , 2006 ) and - ( mukundan et al . , 2009 ) as well as it is important to note that while macrophages engulf tissue - infiltrating apoptotic pmn during the resolution of inflammation , different experimental models used different sources of apoptotic cells , including jurkat t cells , mouse thymocytes , or human peripheral blood neutrophils . all types of apoptotic cells express phosphatidylserine on the outer leaflet of their cytoplasmic membrane , and this is apparently the major signaling module used by these cells to communicate their mortal status with phagocytic cells ( ravichandran , 2011 ) . are expressed on apoptotic cells of different sources to give a more detailed report as to the consequences of their demise . thus , the interpretation of the results obtained following incubations of macrophages with apoptotic cells of different sources should be evaluated carefully depending on the source of apoptotic cells used . the prototypic th2 cytokines il-4 , il-13 , and il-10 , as well as immune responses to parasites were found to promote many of the outcomes of efferocytosis in macrophages . these cytokines are well appreciated antagonists of the m1 response and macrophage pro - inflammatory properties ( martinez et al . , 2009 ) while il-4 and il-13 can also promote fibrosis through tgf production ( fichtner - feigl et al . , 2006 ; wynn , 2008 ) . il-13 was also found to promote vascular endothelial growth factor production during lung injury ( corne et al . , 2000 ) . , 1999 ; berry et al . , 2007 ) and ppar- ( kang et al . , 2008 ) to promote monocyte / macrophage alternative activation . lxr was recently found to synergize with il-4 in the induction of arginase-1 expression and promotion of an m2 phenotype in regressive atherosclerotic lesions ( pourcet et al . , 2011 ) . thus , efferocytosis induces phenotypic and molecular switches and activates signaling pathways in macrophages that resemble m2 polarization . moreover , m2 polarization promotes efferocytosis through induction of different molecular modules , whereas m1 macrophages exert reduced uptake of apoptotic cells . along these lines , recent studies also found that efferocytosis is a self - promoting process , and that m2 pathways play key roles in mediating this feature of macrophage function . ( 2011 ) in this research topic and will not be elaborated on here . nevertheless , while macrophages are paradoxically involved in both the generation of fibrosis and its resolution ( wynn and barron , 2010 ) and efferocytosis and m2 polarization generate a positive feedback loop during resolution of inflammation , it is much less clear what are the events and mediators that stop m2 differentiation and tissue repair / remodeling short of excessive , fibrotic outcomes . such events and mediators are inevitably required to complete the resolution of inflammation and restore homeostasis rather than end every infection with a debilitating scar . a major enzymatic pathway that mediates key events in the resolution of inflammation involves the expression and activation of 12/15-lipoxygenase ( lo ) in mice and 15-lo-1 in humans . 15-lo expression and activity are upregulated by il-4 and il-13 in murine and human monocytes , macrophages , and peripheral blood mononuclear cells ( levy et al . , 1993 ; nassar et al . , 1994 ; heydeck et al . this upregulation leads to the production of 15-lo products from eicosatetraenoic and docosahexaenoic acids ( eta and dha , respectively ) , such as 15-hydroxyeicosatetraenoic acid ( 15-hete ) , lipoxin ( lx ) a4 and b4 ( 5s,6s,15s - trihydroxy-7e,9e,11z,13e - epa , and 5s,14r,15s - trihydroxy-6e,8z,10e,12e - epa , respectively ) , 17s - hydroxy - dha ( 17s - hydroxy-4z,7z,10z,13z,15e,19z - dha ) , and protectin d1 ( 10r,17s - dihydroxy-4z,7z,11e,13e,15z,19z - dha ) . macrophage expression of 12/15-lo was found to promote the production of resolvin ( rv ) d1 ( 7s,8r,17s - trihydroxy-4z,9e,11e,13z,15e,19z - dha ) and maresin 1 ( 7,14-dihydroxy-4z,8,10,12,16z,19z - dha ) , in addition to lxa4 and pd1 ( merched et al . the expression of 12/15-lo was also found to be upregulated in mouse macrophages following their incubation with apoptotic cells ( freire - de - lima et al . , 2006 ; schif - zuck et al . , 2011 ) and resulted in the production of 15-hete and lxa4 ( freire - de - lima et al . , 2006 ) . macrophages from chronic granulomatous disease ( cgd ) mice display impaired efferocytosis that could be repaired by il-4 through the expression of 12/15-lo and activation of ppar- ( fernandez - boyanapalli et al . , 2009 ) . hence , 15-lo - mediated signaling seems to be a major convergence point for efferocytosis and m2 polarization , and its down - stream signaling pathways could play a paramount role in deciphering whether macrophages will become pro - fibrotic or will finalize the resolution sequel to restore tissue homeostasis . along these reasoning , 12/15-lo products have been shown to be anti - inflammatory and to promote tissue repair , while playing an anti - fibrotic and immune - regulatory role ( serhan , 2010 ) . the major bioactive 12/15-lo products could be produced from arachidonic acid to yield 15-hete or lipoxins , or from dha to generate protectin d ( pd)1 , resolvins of the d series , and the recently identified macrophage product maresin 1 ( serhan , 2010 ) . while 15-hete binds ppar to mediate its anti - inflammatory actions ( huang et al . , 1999 ) , lxa4 , pd1 , and resolvin d1 seem to act through binding to cell surface gpcrs ( serhan et al . , 2011 ) , as well as the aryl hydrocarbon receptor ( that binds lxa4 ; machado et al . , 2006 all these 12/15-lo products induce a broad spectrum of anti - inflammatory actions on neutrophils and macrophages , as well as other cell types ( wittwer and hersberger , 2007 ; serhan et al . , 2011 ) . lipoxins and pd1 are produced during epithelial injury in the cornea and mediate wound repair in addition to counteracting inflammation ( gronert et al . , 2005 ) . on the other hand , 12/15-lo products also induce unique pro - resolving properties of macrophages and promote regulatory pathways in lymphocytes . lxa4 , pd1 , rvd1 , and ppar agonists were all found to promote efferocytosis and enhance pmn clearance during resolution ( godson et al . , 2000 ; schwab et al . , 2007 ; fernandez - boyanapalli et al . , 2009 ; krishnamoorthy et al . , 2010 ) in addition , pd1 and rvd1 were found to promote macrophage departure of resolving inflammation sites ( schwab et al . , 2003 , 2005 ) and enhanced ccr5 expression on apoptotic pmn to promote clearance of its pro - inflammatory ligands ( ariel et al . , 2006 ) . moreover , lxa4 was recently found to play a role in the generation of myeloid - derived suppressor cells ( zhang et al . , 2010 ) . of note , lxa4 , pd1 , and rvd1 are potent inhibitors of fibrosis in the lung and kidney ( duffield et al . , 2006 ; martins et al . therefore , 15-lo products can be generated by macrophages following their interaction with apoptotic cells and/or polarization to the m2 phenotype . in turn , these products not only block inflammation but can also shift the macrophage healing balance from tissue repair / fibrosis to pro - resolution , anti - fibrotic , and regulatory functions . the exact mode of production and action for the different 15-lo products is probably dependent on substrate availability , concentration formed in the healing tissue and additional cues from the resolving environment . nevertheless , they seem to act in concert to promote post - inflammation tissue healing and return to homeostasis . a major enzymatic pathway that mediates key events in the resolution of inflammation involves the expression and activation of 12/15-lipoxygenase ( lo ) in mice and 15-lo-1 in humans . 15-lo expression and activity are upregulated by il-4 and il-13 in murine and human monocytes , macrophages , and peripheral blood mononuclear cells ( levy et al . , 1993 ; nassar et al . , 1994 ; heydeck et al . , 1998 ; huang et al . , 1999 ; this upregulation leads to the production of 15-lo products from eicosatetraenoic and docosahexaenoic acids ( eta and dha , respectively ) , such as 15-hydroxyeicosatetraenoic acid ( 15-hete ) , lipoxin ( lx ) a4 and b4 ( 5s,6s,15s - trihydroxy-7e,9e,11z,13e - epa , and 5s,14r,15s - trihydroxy-6e,8z,10e,12e - epa , respectively ) , 17s - hydroxy - dha ( 17s - hydroxy-4z,7z,10z,13z,15e,19z - dha ) , and protectin d1 ( 10r,17s - dihydroxy-4z,7z,11e,13e,15z,19z - dha ) . macrophage expression of 12/15-lo was found to promote the production of resolvin ( rv ) d1 ( 7s,8r,17s - trihydroxy-4z,9e,11e,13z,15e,19z - dha ) and maresin 1 ( 7,14-dihydroxy-4z,8,10,12,16z,19z - dha ) , in addition to lxa4 and pd1 ( merched et al . , 2008 ; serhan et al . , the expression of 12/15-lo was also found to be upregulated in mouse macrophages following their incubation with apoptotic cells ( freire - de - lima et al . , 2006 ; schif - zuck et al . , 2011 ) and resulted in the production of 15-hete and lxa4 ( freire - de - lima et al . , 2006 ) . macrophages from chronic granulomatous disease ( cgd ) mice display impaired efferocytosis that could be repaired by il-4 through the expression of 12/15-lo and activation of ppar- ( fernandez - boyanapalli et al . , 2009 ) . hence , 15-lo - mediated signaling seems to be a major convergence point for efferocytosis and m2 polarization , and its down - stream signaling pathways could play a paramount role in deciphering whether macrophages will become pro - fibrotic or will finalize the resolution sequel to restore tissue homeostasis . along these reasoning , 12/15-lo products have been shown to be anti - inflammatory and to promote tissue repair , while playing an anti - fibrotic and immune - regulatory role ( serhan , 2010 ) . the major bioactive 12/15-lo products could be produced from arachidonic acid to yield 15-hete or lipoxins , or from dha to generate protectin d ( pd)1 , resolvins of the d series , and the recently identified macrophage product maresin 1 ( serhan , 2010 ) . while 15-hete binds ppar to mediate its anti - inflammatory actions ( huang et al . , 1999 ) , lxa4 , pd1 , and resolvin d1 seem to act through binding to cell surface gpcrs ( serhan et al . , 2011 ) , as well as the aryl hydrocarbon receptor ( that binds lxa4 ; machado et al . , 2006 ) all these 12/15-lo products induce a broad spectrum of anti - inflammatory actions on neutrophils and macrophages , as well as other cell types ( wittwer and hersberger , 2007 ; serhan et al . , 2011 ) . lipoxins and pd1 are produced during epithelial injury in the cornea and mediate wound repair in addition to counteracting inflammation ( gronert et al . , 2005 ) . on the other hand , 12/15-lo products also induce unique pro - resolving properties of macrophages and promote regulatory pathways in lymphocytes . lxa4 , pd1 , rvd1 , and ppar agonists were all found to promote efferocytosis and enhance pmn clearance during resolution ( godson et al . , 2000 ; schwab et al . , 2007 ; fernandez - boyanapalli et al . , 2009 ; krishnamoorthy et al . , 2010 ) . in addition , pd1 and rvd1 were found to promote macrophage departure of resolving inflammation sites ( schwab et al . , 2003 , 2005 ) and enhanced ccr5 expression on apoptotic pmn to promote clearance of its pro - inflammatory ligands ( ariel et al . , 2006 ) . moreover , lxa4 was recently found to play a role in the generation of myeloid - derived suppressor cells ( zhang et al . , 2010 ) . of note , lxa4 , pd1 , and rvd1 are potent inhibitors of fibrosis in the lung and kidney ( duffield et al . , 2006 ; martins et al . , 2009 ; borgeson et al . therefore , 15-lo products can be generated by macrophages following their interaction with apoptotic cells and/or polarization to the m2 phenotype . in turn , these products not only block inflammation but can also shift the macrophage healing balance from tissue repair / fibrosis to pro - resolution , anti - fibrotic , and regulatory functions . the exact mode of production and action for the different 15-lo products is probably dependent on substrate availability , concentration formed in the healing tissue and additional cues from the resolving environment . nevertheless , they seem to act in concert to promote post - inflammation tissue healing and return to homeostasis . recent reports have indicated the co - existence of various macrophage phenotypes in resolving peritoneal cavities ( bystrom et al . , 2008 ; schif - zuck et al . , 2011 ) . macrophages from resolving murine peritonitis expressed an alternatively activated phenotype albeit with increase expression of m1 markers , such as cyclooxygenase 2 ( cox 2 ) and inos ( bystrom et al . , 2008 ) . thus , these macrophages were termed resolution - phase macrophages ( rms ) and were postulated to have a hybrid phenotype of classically and alternatively activated macrophages ( bystrom et al . , 2008 ) . a recent report from the same group has indicated that rms could be divided to at least three distinct populations based on f4/80 and ly-6c expression , with varying expression of additional pro - inflammatory and anti - inflammatory markers as well as cd11b ( stables et al . , 2011 ) . along these lines , we have recently characterized f4/80 macrophages from resolving peritoneal exudates into two distinct macrophage subtypes : cd11b and cd11b ( schif - zuck et al . , 2011 ) . cd11b macrophages were found to express low to intermediate levels of the m1 markers inos , cox 2 , and matrix metalloproteinase ( mmp)-9 and high levels of the m2 marker arginase-1 . in addition , these macrophages secret medium levels of inflammatory cytokines and chemokines , as well as il-10 , in response to tlr ligands , are highly phagocytic , and do not migrate to lymphoid tissues . cd11b macrophages express even lower levels of inos , cox 2 , and mmp-9 than cd11b ones , but they also do not express arginase-1 . in addition , these macrophages secrete very low levels of inflammatory cytokines and chemokines , and il-10 , but higher amounts of tgf. moreover , cd11b macrophages , despite containing higher numbers of apoptotic pmn , are no longer phagocytic and are prone to emigrate to remote sites . hence , cd11b macrophages were termed satiated ( schif - zuck et al . , 2011 ) . a seminal report from ravichandran and colleagues ( park et al . , 2011 ) has recently revealed that the mitochondrial membrane protein ucp2 controls satiation vs. continued clearance of apoptotic cells , and it would be interesting to examine its role in the generation of cd11b macrophages . the integration of the results from schif - zuck et al . , bystrom et al . , and stables et al . suggests rm / cd11b macrophages are a mixed macrophage population with dominant m2-like characteristics , and some low - grade m1 activity and that early efferocytosis promotes the conversion of the m1-like population to an m2-like phenotype ( fadok et al . , 1998 ; freire - de - lima et al . , 2006 ; korns et al . , 2011 ) as well as enhanced phagocytosis / efferocytosis . however , the cd11b subset of macrophages , although converting from the cd11b subset ex vivo and in vivo ( following late , threshold - meeting , efferocytosis ; schif - zuck et al . , 2011 ) , are not m2-like , but rather display a distinct phenotype with its own molecular and functional characteristic ( figure 1 ) . of interest , a similar series of macrophage phenotype switches was found to take place during muscle injury and repair . these switches were induced by the engulfment of muscle debris that promoted tgf production and muscle regeneration ( arnold et al . , 2007 ; importantly , the macrophage phenotype switch was mediated by a signaling cascade involving mapk ( perdiguero et al . , 2011 ) an essential module in macrophage inflammatory signaling ( kim et al . , 2008 ) . a monocyte that infiltrated an inflamed tissue differentiates to a macrophage and adopts an m1-like phenotype previous to encounter with apoptotic pmns ( a ) . once it encounters apoptotic pmn and starts to engulf them ( early efferocytosis ) , the macrophage switches to an m2-like phenotype that is anti - inflammatory , highly efferocytic , and involved in tissue repair and return to homeostasis , but can also promote fibrosis and scar formation ( b ) . as the engulfment of apoptotic pmn by the macrophage continues and reaches a threshold level determined by the resolving milieu ( satiating - efferocytosis ) the macrophage undergoes another switch to the mres phenotype ( c ) . these macrophages reduce the expression of pro - fibrotic arginase-1 and display reduced phagocytosis of extracellular particle including apoptotic cells . consequently , rapid mres departure of the resolving tissue and emigration to remote sites takes place . at these target organs mres macrophages presumably produce 12/15-lo - derived pro - resolving lipid mediators , and deliver homeostatic signals to antigen presenting cells and lymphocytes . moreover , mres that stay in the resolving tissue might express higher levels of anti - inflammatory , anti - fibrotic , and anti - oxidant proteins to limit tissue damage and fibrosis . 12/15-lo - derived lipid mediators probably also contribute to the anti - inflammatory and anti - fibrotic properties of mres in the resolving tissue . early and satiating - efferocytosis can be modulated by pro - resolving and anti - inflammatory mediators , such as lipoxins , resolvins , protectins , maresin , gc , il-4 , tgf , il-10 , and ppar ligands ( d ) . this modulation can enhance the immune - silencing and departure of mres to the lymphatics , where they can contribute to the termination of acquired immune responses . macrophages are important in limiting inflammation , excessive tissue repair , and fibrosis ( wynn and barron , 2010 ) . they also act at remote sites , such as lymphoid organs and adipose tissue ( schwab et al . , 2007 ; , 2009 ; odegaard et al . , 2007 ; titos et al . , 2011 ) to regulate acquired immune responses and metabolism . since cd11b macrophages are distinct from either m1 or m2 , do not express the pro - fibrotic enzyme arginase-1 , stop phagocytosing foreign particles and can be found at lymphoid organs and adipose tissue ( schif - zuck et al . , 2011 ; titos et al . , 2011 ) , we suggest these macrophages display a new phenotype , now termed resolution - promoting macrophages ( mres ) , which might be involved in anti - fibrotic , immune - regulatory , and metabolic processes , and hence is critical for the local and systemic termination of inflammatory episodes . the decision - making of macrophages on which phenotype will be expressed at a given time and setting is probably controlled by multiple variants in their milieu , including the number of apoptotic pmn they acquired and local concentrations of pro - resolving lipid mediators ( from 15-lo and other pathways ) and glucocorticoids ( schif - zuck et al . other macrophage - inactivating and resolution - promoting cytokines , growth factors and lipid mediators , such as il-10 , tgf , and ppar ligands are likely to also be important in regulating the fate of macrophages during the resolution of inflammation and the return of tissues to homeostasis . charles n. serhan is an inventor on patents ( resolvins ) assigned to bwh and licensed to resolvyx pharmaceuticals . charles n. serhan is a scientific founder of resolvyx pharmaceuticals and owns equity in the company . charles n. serhan interests were reviewed and are managed by the brigham and women 's hospital and partners healthcare in accordance with their conflict of interest policies .

monocytes that migrate into tissues during inflammatory episodes and differentiate to macrophages were previously classified as classically ( m1 ) or alternatively ( m2 ) activated macrophages , based on their exposure to different fate - determining mediators . these macrophage subsets display distinct molecular markers and differential functions . at the same time , studies from recent years found that the encounter of apoptotic leukocytes with macrophages leads to the clearance of this cellular debris by the macrophages , while concomitantly reprogramming / immune - silencing the macrophages . while some of the features of m2 differentiation , such as arginase-1 ( murine ) and 15-lipoxygenases ( human and murine ) expression , were also displayed by macrophages following the engulfment of apoptotic cells , it was not clear whether apoptotic cells can be regarded as an m2-like differentiating signal . in this manuscript we review the recent information regarding the impact of apoptotic cells on macrophage phenotype changes in molecular terms . we will focus on recent evidence for the in vivo existence of distinct pro - resolving macrophages and the role of apoptotic cells , specialized lipid mediators , and glucocorticoids in their generation . consequently , we will suggest that these pro - resolving cd11blow macrophages have metamorphed from m2-like macrophages , and modulated their protein profile to accommodate the changes in their function .

Introduction Efferocytosis as an Alternative Mode of Macrophage Activation 15-lipoxygenase and its products CD11B Conflict of Interest Statement

macrophages are highly plastic monocyte - derived cells that acquire different molecular and functional phenotypes following exposure to different bioactive molecules and environments . since the major polarizing cytokines initially found to be involved in classical and alternative activation were derived from th1 ( ifn ) and th2 ( il-4 and il-13 ) lymphocytes these activated macrophages were named m1 and m2 , respectively . , m2 macrophages are more heterogeneous , but generally play a role in th2 responses , such as killing and encapsulation of extracellular parasites , resolving type 1 inflammation , and promoting tissue repair and remodeling . m1 macrophages express high levels of inflammatory cytokines ( il-12 , il-23 , tnf , il-1 , and il-6 ) and chemokines ( cxcl9 , 10 , and 11 , ccl2 , 3 , 4 , and 5 , and cxcl2 ) , as well as enzymes involved in the generation of reactive oxygen species ( ros ) and nitric oxide ( no ; mantovani et al . m2 macrophages express lower levels of inflammatory mediators , but high levels of il-10 , scavenger , mannose , and galactose receptors . additional markers of m2 , such as ym1 and fizz1 , were later identified in mouse macrophages ( raes et al . macrophages also undergo dramatic molecular and functional changes upon encounter , interaction with , and uptake of apoptotic cells ( efferocytosis ) during the resolution of inflammation . in this article we will highlight some of the similarities between m2 differentiation and transcriptional events activated by early efferocytosis . in addition , we will discuss recent results that support the notion that efferocytosis can eventually transform macrophages to another phenotype that is postulated to limit tissue repair / fibrosis and promote macrophage regulatory properties at remote sites . in this regard , it is important to note the early studies that indicated non - phlogistic activation of monocytes by the pro - resolving lipid mediators lipoxins . the recognition , engulfment , and responsiveness to apoptotic cells are cardinal properties of resident and inflammatory macrophages and play a role in processes , such as tissue morphogenesis and homeostasis , embryonic development , hematopoiesis , immunity , and the resolution of inflammation ( savill et al . the recognition and uptake of apoptotic cells by macrophages through eat me signals ( and the absence of do not eat me signals ) expressed on their surface and their cognate receptors have been extensively studied and reviewed ( ravichandran , 2011 ) . however , apoptotic cells also transduce signals to the engulfing macrophages that result in significant molecular and functional adjustments that address physiological needs consequent to the identified cell death . during the resolution of inflammation , macrophages engulf apoptotic cells and consequently , apoptotic cell recognition evokes distinct signaling events ( patel et al . , 2006 ) that block the release of pro - inflammatory mediators from macrophages . this release is activated by bacterial moieties , and its blockage , which is termed immune - silencing ( voll et al . , 1997 ; 2004 ) , is accompanied by the production of tgf and il-10 ( byrne and reen , 2002 ; huynh et al . , 2002 ; the engulfment of apoptotic leukocytes by macrophages also leads to inhibition of inos expression and stimulates the expression of arginase-1 in the raw 264 macrophage cell line ( freire - de - lima et al . , 2004 ) by macrophages is consequent to the uptake of apoptotic cells . elucidation of the signaling pathways activated by efferocytosis revealed significant roles for nuclear transcriptional regulators , such as peroxisome proliferator activated receptor ( ppar)- ( freire - de - lima et al . , 2009 ) as well as it is important to note that while macrophages engulf tissue - infiltrating apoptotic pmn during the resolution of inflammation , different experimental models used different sources of apoptotic cells , including jurkat t cells , mouse thymocytes , or human peripheral blood neutrophils . all types of apoptotic cells express phosphatidylserine on the outer leaflet of their cytoplasmic membrane , and this is apparently the major signaling module used by these cells to communicate their mortal status with phagocytic cells ( ravichandran , 2011 ) . are expressed on apoptotic cells of different sources to give a more detailed report as to the consequences of their demise . thus , the interpretation of the results obtained following incubations of macrophages with apoptotic cells of different sources should be evaluated carefully depending on the source of apoptotic cells used . the prototypic th2 cytokines il-4 , il-13 , and il-10 , as well as immune responses to parasites were found to promote many of the outcomes of efferocytosis in macrophages . these cytokines are well appreciated antagonists of the m1 response and macrophage pro - inflammatory properties ( martinez et al . moreover , m2 polarization promotes efferocytosis through induction of different molecular modules , whereas m1 macrophages exert reduced uptake of apoptotic cells . along these lines , recent studies also found that efferocytosis is a self - promoting process , and that m2 pathways play key roles in mediating this feature of macrophage function . ( 2011 ) in this research topic and will not be elaborated on here . nevertheless , while macrophages are paradoxically involved in both the generation of fibrosis and its resolution ( wynn and barron , 2010 ) and efferocytosis and m2 polarization generate a positive feedback loop during resolution of inflammation , it is much less clear what are the events and mediators that stop m2 differentiation and tissue repair / remodeling short of excessive , fibrotic outcomes . 15-lo expression and activity are upregulated by il-4 and il-13 in murine and human monocytes , macrophages , and peripheral blood mononuclear cells ( levy et al . this upregulation leads to the production of 15-lo products from eicosatetraenoic and docosahexaenoic acids ( eta and dha , respectively ) , such as 15-hydroxyeicosatetraenoic acid ( 15-hete ) , lipoxin ( lx ) a4 and b4 ( 5s,6s,15s - trihydroxy-7e,9e,11z,13e - epa , and 5s,14r,15s - trihydroxy-6e,8z,10e,12e - epa , respectively ) , 17s - hydroxy - dha ( 17s - hydroxy-4z,7z,10z,13z,15e,19z - dha ) , and protectin d1 ( 10r,17s - dihydroxy-4z,7z,11e,13e,15z,19z - dha ) . macrophage expression of 12/15-lo was found to promote the production of resolvin ( rv ) d1 ( 7s,8r,17s - trihydroxy-4z,9e,11e,13z,15e,19z - dha ) and maresin 1 ( 7,14-dihydroxy-4z,8,10,12,16z,19z - dha ) , in addition to lxa4 and pd1 ( merched et al . the expression of 12/15-lo was also found to be upregulated in mouse macrophages following their incubation with apoptotic cells ( freire - de - lima et al . , 2011 ) and resulted in the production of 15-hete and lxa4 ( freire - de - lima et al . hence , 15-lo - mediated signaling seems to be a major convergence point for efferocytosis and m2 polarization , and its down - stream signaling pathways could play a paramount role in deciphering whether macrophages will become pro - fibrotic or will finalize the resolution sequel to restore tissue homeostasis . along these reasoning , 12/15-lo products have been shown to be anti - inflammatory and to promote tissue repair , while playing an anti - fibrotic and immune - regulatory role ( serhan , 2010 ) . the major bioactive 12/15-lo products could be produced from arachidonic acid to yield 15-hete or lipoxins , or from dha to generate protectin d ( pd)1 , resolvins of the d series , and the recently identified macrophage product maresin 1 ( serhan , 2010 ) . on the other hand , 12/15-lo products also induce unique pro - resolving properties of macrophages and promote regulatory pathways in lymphocytes . lxa4 , pd1 , rvd1 , and ppar agonists were all found to promote efferocytosis and enhance pmn clearance during resolution ( godson et al . , 2003 , 2005 ) and enhanced ccr5 expression on apoptotic pmn to promote clearance of its pro - inflammatory ligands ( ariel et al . of note , lxa4 , pd1 , and rvd1 are potent inhibitors of fibrosis in the lung and kidney ( duffield et al . therefore , 15-lo products can be generated by macrophages following their interaction with apoptotic cells and/or polarization to the m2 phenotype . in turn , these products not only block inflammation but can also shift the macrophage healing balance from tissue repair / fibrosis to pro - resolution , anti - fibrotic , and regulatory functions . 15-lo expression and activity are upregulated by il-4 and il-13 in murine and human monocytes , macrophages , and peripheral blood mononuclear cells ( levy et al . , 1999 ; this upregulation leads to the production of 15-lo products from eicosatetraenoic and docosahexaenoic acids ( eta and dha , respectively ) , such as 15-hydroxyeicosatetraenoic acid ( 15-hete ) , lipoxin ( lx ) a4 and b4 ( 5s,6s,15s - trihydroxy-7e,9e,11z,13e - epa , and 5s,14r,15s - trihydroxy-6e,8z,10e,12e - epa , respectively ) , 17s - hydroxy - dha ( 17s - hydroxy-4z,7z,10z,13z,15e,19z - dha ) , and protectin d1 ( 10r,17s - dihydroxy-4z,7z,11e,13e,15z,19z - dha ) . , the expression of 12/15-lo was also found to be upregulated in mouse macrophages following their incubation with apoptotic cells ( freire - de - lima et al . , 2011 ) and resulted in the production of 15-hete and lxa4 ( freire - de - lima et al . hence , 15-lo - mediated signaling seems to be a major convergence point for efferocytosis and m2 polarization , and its down - stream signaling pathways could play a paramount role in deciphering whether macrophages will become pro - fibrotic or will finalize the resolution sequel to restore tissue homeostasis . along these reasoning , 12/15-lo products have been shown to be anti - inflammatory and to promote tissue repair , while playing an anti - fibrotic and immune - regulatory role ( serhan , 2010 ) . the major bioactive 12/15-lo products could be produced from arachidonic acid to yield 15-hete or lipoxins , or from dha to generate protectin d ( pd)1 , resolvins of the d series , and the recently identified macrophage product maresin 1 ( serhan , 2010 ) . , 1999 ) , lxa4 , pd1 , and resolvin d1 seem to act through binding to cell surface gpcrs ( serhan et al . on the other hand , 12/15-lo products also induce unique pro - resolving properties of macrophages and promote regulatory pathways in lymphocytes . , 2003 , 2005 ) and enhanced ccr5 expression on apoptotic pmn to promote clearance of its pro - inflammatory ligands ( ariel et al . therefore , 15-lo products can be generated by macrophages following their interaction with apoptotic cells and/or polarization to the m2 phenotype . in turn , these products not only block inflammation but can also shift the macrophage healing balance from tissue repair / fibrosis to pro - resolution , anti - fibrotic , and regulatory functions . the exact mode of production and action for the different 15-lo products is probably dependent on substrate availability , concentration formed in the healing tissue and additional cues from the resolving environment . recent reports have indicated the co - existence of various macrophage phenotypes in resolving peritoneal cavities ( bystrom et al . macrophages from resolving murine peritonitis expressed an alternatively activated phenotype albeit with increase expression of m1 markers , such as cyclooxygenase 2 ( cox 2 ) and inos ( bystrom et al . thus , these macrophages were termed resolution - phase macrophages ( rms ) and were postulated to have a hybrid phenotype of classically and alternatively activated macrophages ( bystrom et al . a recent report from the same group has indicated that rms could be divided to at least three distinct populations based on f4/80 and ly-6c expression , with varying expression of additional pro - inflammatory and anti - inflammatory markers as well as cd11b ( stables et al . along these lines , we have recently characterized f4/80 macrophages from resolving peritoneal exudates into two distinct macrophage subtypes : cd11b and cd11b ( schif - zuck et al . cd11b macrophages were found to express low to intermediate levels of the m1 markers inos , cox 2 , and matrix metalloproteinase ( mmp)-9 and high levels of the m2 marker arginase-1 . cd11b macrophages express even lower levels of inos , cox 2 , and mmp-9 than cd11b ones , but they also do not express arginase-1 . in addition , these macrophages secrete very low levels of inflammatory cytokines and chemokines , and il-10 , but higher amounts of tgf. moreover , cd11b macrophages , despite containing higher numbers of apoptotic pmn , are no longer phagocytic and are prone to emigrate to remote sites . , 2011 ) has recently revealed that the mitochondrial membrane protein ucp2 controls satiation vs. continued clearance of apoptotic cells , and it would be interesting to examine its role in the generation of cd11b macrophages . suggests rm / cd11b macrophages are a mixed macrophage population with dominant m2-like characteristics , and some low - grade m1 activity and that early efferocytosis promotes the conversion of the m1-like population to an m2-like phenotype ( fadok et al . however , the cd11b subset of macrophages , although converting from the cd11b subset ex vivo and in vivo ( following late , threshold - meeting , efferocytosis ; schif - zuck et al . these switches were induced by the engulfment of muscle debris that promoted tgf production and muscle regeneration ( arnold et al . once it encounters apoptotic pmn and starts to engulf them ( early efferocytosis ) , the macrophage switches to an m2-like phenotype that is anti - inflammatory , highly efferocytic , and involved in tissue repair and return to homeostasis , but can also promote fibrosis and scar formation ( b ) . as the engulfment of apoptotic pmn by the macrophage continues and reaches a threshold level determined by the resolving milieu ( satiating - efferocytosis ) the macrophage undergoes another switch to the mres phenotype ( c ) . these macrophages reduce the expression of pro - fibrotic arginase-1 and display reduced phagocytosis of extracellular particle including apoptotic cells . consequently , rapid mres departure of the resolving tissue and emigration to remote sites takes place . at these target organs mres macrophages presumably produce 12/15-lo - derived pro - resolving lipid mediators , and deliver homeostatic signals to antigen presenting cells and lymphocytes . moreover , mres that stay in the resolving tissue might express higher levels of anti - inflammatory , anti - fibrotic , and anti - oxidant proteins to limit tissue damage and fibrosis . 12/15-lo - derived lipid mediators probably also contribute to the anti - inflammatory and anti - fibrotic properties of mres in the resolving tissue . early and satiating - efferocytosis can be modulated by pro - resolving and anti - inflammatory mediators , such as lipoxins , resolvins , protectins , maresin , gc , il-4 , tgf , il-10 , and ppar ligands ( d ) . this modulation can enhance the immune - silencing and departure of mres to the lymphatics , where they can contribute to the termination of acquired immune responses . macrophages are important in limiting inflammation , excessive tissue repair , and fibrosis ( wynn and barron , 2010 ) . they also act at remote sites , such as lymphoid organs and adipose tissue ( schwab et al . since cd11b macrophages are distinct from either m1 or m2 , do not express the pro - fibrotic enzyme arginase-1 , stop phagocytosing foreign particles and can be found at lymphoid organs and adipose tissue ( schif - zuck et al . , 2011 ) , we suggest these macrophages display a new phenotype , now termed resolution - promoting macrophages ( mres ) , which might be involved in anti - fibrotic , immune - regulatory , and metabolic processes , and hence is critical for the local and systemic termination of inflammatory episodes . the decision - making of macrophages on which phenotype will be expressed at a given time and setting is probably controlled by multiple variants in their milieu , including the number of apoptotic pmn they acquired and local concentrations of pro - resolving lipid mediators ( from 15-lo and other pathways ) and glucocorticoids ( schif - zuck et al . other macrophage - inactivating and resolution - promoting cytokines , growth factors and lipid mediators , such as il-10 , tgf , and ppar ligands are likely to also be important in regulating the fate of macrophages during the resolution of inflammation and the return of tissues to homeostasis . charles n. serhan interests were reviewed and are managed by the brigham and women 's hospital and partners healthcare in accordance with their conflict of interest policies .

recently , acupuncture treatment as a complementary and alternative medicine ( cam ) has been the focus of studies from advanced basic research ( 1 ) to clinical reports ( 2 ) on pain . although much more clinical work has centered on the effects of acupuncture on the digestive tract than for other organs ( 35 ) , far less work has been done on the mechanism of these effects . japan has a long history of research on acupuncture for the digestive function . in 1912 , the japanese government asked miura , a professor at tokyo university , to study acupuncture treatment . he observed and reported changes in the abdominal wall for the gastrointestinal motility of rabbits caused by acupuncture ( 6 ) . his work , however , only encompassed physical responses to acupuncture , and until recently there have been no studies on the mechanisms by which these responses are produced . basic studies on somato - visceral reflexes found a high association with acupuncture mechanisms , showing that somato - sensory stimulations to the skin or muscles changed the gastrointestinal reflex function in anesthetized animals ( 7,8 ) . recent studies have explored changes in gastrointestinal motilities of the gastropyloric region and duodenum via somato - autonomic reflexes . in 1975 , sato et al . observed that pinching the abdomen of anesthetized rats decreased intragastric pressure . because the decrease of intragastric pressure occurred on spinalized rats , but disappeared when the splanchnic nerves on both sides of the rats were severed , they concluded that the decrease was a reflex response that originated in the spine and was expressed via gastric sympathetic nerves ( 9 ) . in more detailed investigations of intragastric pressure decrease , pinched anesthetized rats in various regions of the body and observed that stimulating the body trunk inhibited gastric motilities , while stimulating the limbs excited responses in the motilities . in the inhibitory response , the gastric sympathetic nerves were more active than normal while with an excitatory response , the gastric vagus nerves were more active than normal . since only the inhibitory response was observed in spinalized animals , they discovered that the excitatory response to acupuncture of the limbs occurred through the excitation of vagus nerves via supraspinal reflexes ( 10 ) . ( a ) a frame format showing gastric motilities caused by acupuncture in each dermatome . open or closed circles showed regions of the excitatory or inhibitory gastric motilities . ( b , c ) effects of acupuncture stimulation of the abdomen ( b ) and hind paw ( c ) . gastric motilities ( upper traces ) , efferent gastric sympathetic nerve activity ( middle trace ) and efferent vagus nerve activity ( lowest trace ) after acupuncture . activity of each nerve was continuously measured and level of acupuncture stimulation / minute is expressed with horizontal bars . ( a ) a frame format showing gastric motilities caused by acupuncture in each dermatome . open or closed circles showed regions of the excitatory or inhibitory gastric motilities . ( b , c ) effects of acupuncture stimulation of the abdomen ( b ) and hind paw ( c ) . gastric motilities ( upper traces ) , efferent gastric sympathetic nerve activity ( middle trace ) and efferent vagus nerve activity ( lowest trace ) after acupuncture . activity of each nerve was continuously measured and level of acupuncture stimulation / minute is expressed with horizontal bars . we present recent basic studies mainly by our group on the mechanism of acupuncture by somato - autonomic reflexes and on the acupuncture mechanism of reflex regulation of the gastroduodenal function in anesthetized rats . when mori et al . observed gastric motility in anesthetized rabbits by the balloon method , when they were stimulated by tapping on acupuncture point st36 in 1978 , they discovered that intragastric pressure increased by acupuncture stimulation and that the response disappeared when the central side of the sciatic nerves , which govern the nerves at point st36 , were severed ( 11 ) . in 1991 , kudo et al . observed by electrogastrogram that electro - acupuncture to anesthetized dogs on acupuncture point bl19 , which is on the back , delayed kinetic rhythms of the stomach and inhibited electric activity of the stomach wall ( 12 ) . these reports suggest that acupuncture changes gastric motilities in anesthetized animals , as somato - sensory stimulation does with pinching . in 1993 , sato et al . observed the effect of manual acupuncture stimulation to anesthetized rats through continuous measurement of the intragastric pressure by means of a balloon inserted into the gastropyrolic region . they discovered that when they inserted a needle ( with a diameter of 340 m ) about 45 mm deep into the subcutaneous muscular layer at many points on the whole body and gave the tested rats acupuncture by twisting the needle for 60 seconds , gastric motilities either decreased or increased . acupuncture inhibited gastric motilities when the abdomen was stimulated as a result of increased activity of the efferent fibers of the gastric sympathetic nerves . increase of gastric motilities in the case of acupuncture stimulations to limbs resulted from increased activity of the efferent fibers of the gastric vagus nerves . these inhibitory or excitatory responses were induced by acupuncture to both the skin and the muscle , the skin only and the subcutaneous muscles only . acupuncture to the abdomen increased the activity of afferent fibers of the spinal nerves in the lower chest and , when the spinal nerves in the chest were severed , the inhibition of gastric motilities disappeared even when the abdomen was stimulated . similarly , acupuncture to the hind paw increased the activity of the thigh and the afferent fibers of the sciatic nerves and , when the thigh and sciatic nerves were severed , the response to increase gastric motilities disappeared even when the hind paw was stimulated . ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . significant inhibitory changes were induced by intense stimulation above the threshold of group c fibers . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . * p < 0.05 ( * * p < 0.01 ) indicates significant differences in reference to prestimulatory values with paired t - test analysis . [ ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . significant inhibitory changes were induced by intense stimulation above the threshold of group c fibers . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . p < 0.05 ( * * p < 0.01 ) indicates significant differences in reference to prestimulatory values with paired t - test analysis . gastric motilities were inhibited by acupuncture to the abdomen in spinalized rats but acupuncture to the hind paw did not excite gastric motilities proving that inhibitory or excitatory responses to gastric motilities by acupuncture to the abdomen or the hind paw were spinal or supraspinal reflex responses . furthermore the responses did not disappear when naloxone was given , suggesting that responses occur through a different mechanism from acupuncture analgesia ( 13 ) . in the same study group , confirmed that electro - acupuncture of various intensities to the abdomen or hind limbs caused excitatory or inhibitory responses in gastric motilities . they further recorded the activity of afferent fibers from intercostal nerves or tibial nerves when the abdomen or hind paw were stimulated and examined the relationship of the intensity of the stimulation to response in gastric motilities . they identified the nerves through which the electro - acupuncture stimulations worked , observing that the responses occurred above the threshold level of electro - acupuncture stimulation intensity at which c fibers are excited in the abdomen , but above the threshold level at which a and c fibers are excited in the hind paw . accordingly , the stimulation of body trunk and hind limbs were transmitted through different nerve fibers ( 14 ) . for duodenal motilities , sato et al . measured motilities caused by pinching stimulation and reported that noxious stimulation to the abdomen produced an inhibitory response by spinal reflexes . in 2003 , noguchi et al . measured duodenal motilities by a method similar to that used to measure gastric motilities , and examined the relationship between intensities of electro - acupuncture to changes in duodenal motilities . their results revealed that to decrease duodenal motilities , electro - acupuncture stimulation to the abdomen needed to be strong enough to excite group iv fibers of intercostal nerves . to increase motilities , electro - acupuncture stimulation to the abdomen needs to be strong enough to excite the higher - threshold group iii fibers of tibial nerves . they also proved that the responses occur through a path similar to that of gastric motilities ( 15 ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity that excited group iv fibers . ( a-2 ) the threshold of afferent tibial nerve activity innervating the stimulated hind paw region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity threshold of group iii or iv fibers with high thresholds . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity that excited group iv fibers . ( a-2 ) the threshold of afferent tibial nerve activity innervating the stimulated hind paw region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity threshold of group iii or iv fibers with high thresholds . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . when mori et al . observed gastric motility in anesthetized rabbits by the balloon method , when they were stimulated by tapping on acupuncture point st36 in 1978 , they discovered that intragastric pressure increased by acupuncture stimulation and that the response disappeared when the central side of the sciatic nerves , which govern the nerves at point st36 , were severed ( 11 ) . in 1991 , kudo et al . observed by electrogastrogram that electro - acupuncture to anesthetized dogs on acupuncture point bl19 , which is on the back , delayed kinetic rhythms of the stomach and inhibited electric activity of the stomach wall ( 12 ) . these reports suggest that acupuncture changes gastric motilities in anesthetized animals , as somato - sensory stimulation does with pinching . in 1993 , sato et al . observed the effect of manual acupuncture stimulation to anesthetized rats through continuous measurement of the intragastric pressure by means of a balloon inserted into the gastropyrolic region . they discovered that when they inserted a needle ( with a diameter of 340 m ) about 45 mm deep into the subcutaneous muscular layer at many points on the whole body and gave the tested rats acupuncture by twisting the needle for 60 seconds , gastric motilities either decreased or increased . acupuncture inhibited gastric motilities when the abdomen was stimulated as a result of increased activity of the efferent fibers of the gastric sympathetic nerves . increase of gastric motilities in the case of acupuncture stimulations to limbs resulted from increased activity of the efferent fibers of the gastric vagus nerves . these inhibitory or excitatory responses were induced by acupuncture to both the skin and the muscle , the skin only and the subcutaneous muscles only . acupuncture to the abdomen increased the activity of afferent fibers of the spinal nerves in the lower chest and , when the spinal nerves in the chest were severed , the inhibition of gastric motilities disappeared even when the abdomen was stimulated . similarly , acupuncture to the hind paw increased the activity of the thigh and the afferent fibers of the sciatic nerves and , when the thigh and sciatic nerves were severed , the response to increase gastric motilities disappeared even when the hind paw was stimulated . ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . significant inhibitory changes were induced by intense stimulation above the threshold of group c fibers . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . * p < 0.05 ( * * p < 0.01 ) indicates significant differences in reference to prestimulatory values with paired t - test analysis . [ ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . significant inhibitory changes were induced by intense stimulation above the threshold of group c fibers . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . p < 0.05 ( * * p < 0.01 ) indicates significant differences in reference to prestimulatory values with paired t - test analysis . gastric motilities were inhibited by acupuncture to the abdomen in spinalized rats but acupuncture to the hind paw did not excite gastric motilities proving that inhibitory or excitatory responses to gastric motilities by acupuncture to the abdomen or the hind paw were spinal or supraspinal reflex responses . furthermore the responses did not disappear when naloxone was given , suggesting that responses occur through a different mechanism from acupuncture analgesia ( 13 ) . yamaguchi et al . , in the same study group , confirmed that electro - acupuncture of various intensities to the abdomen or hind limbs caused excitatory or inhibitory responses in gastric motilities . they further recorded the activity of afferent fibers from intercostal nerves or tibial nerves when the abdomen or hind paw were stimulated and examined the relationship of the intensity of the stimulation to response in gastric motilities . they identified the nerves through which the electro - acupuncture stimulations worked , observing that the responses occurred above the threshold level of electro - acupuncture stimulation intensity at which c fibers are excited in the abdomen , but above the threshold level at which a and c fibers are excited in the hind paw . accordingly , the stimulation of body trunk and hind limbs were transmitted through different nerve fibers ( 14 ) . for duodenal motilities , sato et al . measured motilities caused by pinching stimulation and reported that noxious stimulation to the abdomen produced an inhibitory response by spinal reflexes . in 2003 , noguchi et al . measured duodenal motilities by a method similar to that used to measure gastric motilities , and examined the relationship between intensities of electro - acupuncture to changes in duodenal motilities . their results revealed that to decrease duodenal motilities , electro - acupuncture stimulation to the abdomen needed to be strong enough to excite group iv fibers of intercostal nerves . to increase motilities , electro - acupuncture stimulation to the abdomen needs to be strong enough to excite the higher - threshold group iii fibers of tibial nerves . they also proved that the responses occur through a path similar to that of gastric motilities ( 15 ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity that excited group iv fibers . ( a-2 ) the threshold of afferent tibial nerve activity innervating the stimulated hind paw region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity threshold of group iii or iv fibers with high thresholds . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity that excited group iv fibers . ( a-2 ) the threshold of afferent tibial nerve activity innervating the stimulated hind paw region , and the inhibitory change rates of duodenal motilities . a significant inhibitory response appeared with stimulation above the intensity threshold of group iii or iv fibers with high thresholds . b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . the first paper given at a conference in the west to relate acupuncture treatment with gastric - acid secretion was given by sodipo et al . at the university of lagos in nigeria in 1979 they treated a group of duodenal ulcer ( du ) cases and a group of non - ulcer dyspepsia ( nud ) cases by various acupuncture regimens for 6 weeks . they reported that treatment relieved stomach pain , and that stimulated maximal acid output ( mao ) decreased in the du group . ( a ) effects of severing the splanchnic nerve and vagus nerve on excitatory response of gastric acid secretion with electro - acupuncture of the hind paw of the anesthetized rats at acupuncture point s-36 excitatory response of gastric acid secretion with electro - acupuncture disappeared after vagotomy ( open squares ) and ischiadic nerve section ( closed squares ) , while that after the splanchnic nerve section did not disappear . ( b ) effects of electro - acupuncture stimulation on gastric acid secretion after amino acid intake in conscious dogs . no ap : control without stimulation , eap : electro - acupuncture , eap + naloxone : acupuncture stimulation + naloxone ( 40 g / kg / h ) , aameal : amino acid intake ( given in all experiments ) . electro - acupuncture inhibited gastric acid secretion after amino acid intake ( open squares ) . however , ( a ) effects of severing the splanchnic nerve and vagus nerve on excitatory response of gastric acid secretion with electro - acupuncture of the hind paw of the anesthetized rats at acupuncture point s-36 excitatory response of gastric acid secretion with electro - acupuncture disappeared after vagotomy ( open squares ) and ischiadic nerve section ( closed squares ) , while that after the splanchnic nerve section did not disappear . horizontal lines ( eas ) indicate the period of acupuncture stimulation . ( b ) effects of electro - acupuncture stimulation on gastric acid secretion after amino acid intake in conscious dogs . no ap : control without stimulation , eap : electro - acupuncture , eap + naloxone : acupuncture stimulation + naloxone ( 40 g / kg / h ) , aameal : amino acid intake ( given in all experiments ) . electro - acupuncture inhibited gastric acid secretion after amino acid intake ( open squares ) . [ cited from jin et al . with modification ( 18 ) ] . in the field of basic acupuncture studies , zhou et al . observed in 1984 that gastric - acid secretion was inhibited in conscious dogs with a pavlov pouch when they gave them electro - acupuncture to acupuncture points st36 , pc6 and bl20 for 2 hours . this response disappeared when they gave the dogs a vagus nerve blocker ( atropine ) or local anesthetic ( procaine ) to the regions that received acupuncture . they then concluded that gastric - acid secretion was inhibited by somato - autonomoic reflexes ( 17 ) . in 1996 , jin et al . observed that electro - acupuncture of conscious dogs inhibited rises in gastric - acid secretion that should have occurred when they ingested amino acid foods they proposed that the mechanism involved endogenous opioids such as occurs in acupuncture analgesia ( 18 ) . hang et al . in shanghai , observed gastric - acid secretion through changes in the ph value of physiologic saline circulated in the stomach of anesthetized rats ( ghosh & schild method ) , and observed an increase in secretion by electro - acupuncture to acupuncture point st36 with no effect from severing the ischiadic and vagus nerves or the administration of naloxone ( 19 ) . when noguchi et al . observed gastric - acid secretion in anesthetized rats , using a similar gastric circulation , they discovered that the response to increasing gastric - acid secretion occurred by electro - acupuncture stimulations to acupuncture point st36 , and that it disappeared when the ischiadic nerves or vagus nerves were severed , but did not disappear when the splanchnic nerves were severed . they concluded that the excitatory response occurred through somato - autonomic nervous reflexes ( 20 ) . although many scientists have reported on the responses of gastric - acid secretion to acupuncture stimulations , the results were not definitive and the underlying mechanism remains unknown . pomeranz pointed out in his paper ( 21 ) , that the greatest difference between noguchi 's ( 1996 ) and jin 's ( 1996 ) reports was whether the animals were anesthetized or not . in order to observe the response to acupuncture in conscious animals , the problem of stress caused by acupuncture needed to be solved . in the study of gastric - acid secretion accordingly many concerns must be solved to clarify the effect of acupuncture stimulations on gastric - acid secretion . the following mechanisms for acupuncture stimulation to anesthetized rats have been proven to exert effects on gastric motilities . acupuncture to the abdomen of anesthetized rats increases gastric motilities by exciting sympathetic nerves via spinal reflexes . stimulating the limbs of rats caused an increase in gastric motilities by exciting vagus nerves via supraspinal reflexes . in addition , acupuncture simulation of the abdomen must be strong enough to excite group vi fibers of the afferent fibers of intercostal nerves in order to produce an inhibitory response . stimulation of hind limbs should be strong enough to excite the high - threshold group iii fibers of the tibial nerves in order to produce the excitatory response . as for duodenal motilities , acupuncture works through a neural mechanism similar to that of gastric motilities in terms of stimulated regions and intensity of stimulation . reports on inhibitory and excitatory effects of acupuncture on gastric - acid secretion are mixed . for the neural mechanism , somato - autonomic reflexes and responses of intervening endogenous opioids

many clinical studies focus on the effects of acupuncture on digestive disorders . however , few studies describe the mechanism by which these effects are produced . we present some recent experimental work on the mechanism of acupuncture for reflex regulation of gastroduodenal function in anesthetized rats . in anesthetized rats , it has been proven that acupuncture to the abdomen excites sympathetic nerves via spinal reflexes causing inhibition of motilities while acupuncture of limbs excites vagus nerves via supraspinal reflexes causing an increase in the motilities . it has also been shown that in order to inhibit gastric motilities , acupuncture stimulation of the abdomen must be strong enough to excite group vi fibers of the afferent intercostal nerves . to increase gastric motilities , acupuncture stimulation to hind limbs must be strong enough to excite the high - threshold group iii fibers of tibial nerves . it has also been shown that the neural mechanism of duodenal motility stimulation by acupuncture involves the same body regions and intensity of stimulation as that of gastric motilities . theories regarding the underlying mechanism have proposed somato - autonomic reflexes and responses via endogenous opioids , etc . , but without definitive conclusions .

Introduction Gastroduodenal Motility Acupuncture Stimulation Electro-acupuncture Stimulation Gastric-acid Secretion Conclusion

recently , acupuncture treatment as a complementary and alternative medicine ( cam ) has been the focus of studies from advanced basic research ( 1 ) to clinical reports ( 2 ) on pain . although much more clinical work has centered on the effects of acupuncture on the digestive tract than for other organs ( 35 ) , far less work has been done on the mechanism of these effects . he observed and reported changes in the abdominal wall for the gastrointestinal motility of rabbits caused by acupuncture ( 6 ) . his work , however , only encompassed physical responses to acupuncture , and until recently there have been no studies on the mechanisms by which these responses are produced . basic studies on somato - visceral reflexes found a high association with acupuncture mechanisms , showing that somato - sensory stimulations to the skin or muscles changed the gastrointestinal reflex function in anesthetized animals ( 7,8 ) . recent studies have explored changes in gastrointestinal motilities of the gastropyloric region and duodenum via somato - autonomic reflexes . observed that pinching the abdomen of anesthetized rats decreased intragastric pressure . because the decrease of intragastric pressure occurred on spinalized rats , but disappeared when the splanchnic nerves on both sides of the rats were severed , they concluded that the decrease was a reflex response that originated in the spine and was expressed via gastric sympathetic nerves ( 9 ) . in more detailed investigations of intragastric pressure decrease , pinched anesthetized rats in various regions of the body and observed that stimulating the body trunk inhibited gastric motilities , while stimulating the limbs excited responses in the motilities . in the inhibitory response , the gastric sympathetic nerves were more active than normal while with an excitatory response , the gastric vagus nerves were more active than normal . since only the inhibitory response was observed in spinalized animals , they discovered that the excitatory response to acupuncture of the limbs occurred through the excitation of vagus nerves via supraspinal reflexes ( 10 ) . ( a ) a frame format showing gastric motilities caused by acupuncture in each dermatome . open or closed circles showed regions of the excitatory or inhibitory gastric motilities . ( b , c ) effects of acupuncture stimulation of the abdomen ( b ) and hind paw ( c ) . activity of each nerve was continuously measured and level of acupuncture stimulation / minute is expressed with horizontal bars . ( a ) a frame format showing gastric motilities caused by acupuncture in each dermatome . ( b , c ) effects of acupuncture stimulation of the abdomen ( b ) and hind paw ( c ) . activity of each nerve was continuously measured and level of acupuncture stimulation / minute is expressed with horizontal bars . we present recent basic studies mainly by our group on the mechanism of acupuncture by somato - autonomic reflexes and on the acupuncture mechanism of reflex regulation of the gastroduodenal function in anesthetized rats . observed gastric motility in anesthetized rabbits by the balloon method , when they were stimulated by tapping on acupuncture point st36 in 1978 , they discovered that intragastric pressure increased by acupuncture stimulation and that the response disappeared when the central side of the sciatic nerves , which govern the nerves at point st36 , were severed ( 11 ) . observed by electrogastrogram that electro - acupuncture to anesthetized dogs on acupuncture point bl19 , which is on the back , delayed kinetic rhythms of the stomach and inhibited electric activity of the stomach wall ( 12 ) . these reports suggest that acupuncture changes gastric motilities in anesthetized animals , as somato - sensory stimulation does with pinching . observed the effect of manual acupuncture stimulation to anesthetized rats through continuous measurement of the intragastric pressure by means of a balloon inserted into the gastropyrolic region . they discovered that when they inserted a needle ( with a diameter of 340 m ) about 45 mm deep into the subcutaneous muscular layer at many points on the whole body and gave the tested rats acupuncture by twisting the needle for 60 seconds , gastric motilities either decreased or increased . acupuncture inhibited gastric motilities when the abdomen was stimulated as a result of increased activity of the efferent fibers of the gastric sympathetic nerves . increase of gastric motilities in the case of acupuncture stimulations to limbs resulted from increased activity of the efferent fibers of the gastric vagus nerves . these inhibitory or excitatory responses were induced by acupuncture to both the skin and the muscle , the skin only and the subcutaneous muscles only . acupuncture to the abdomen increased the activity of afferent fibers of the spinal nerves in the lower chest and , when the spinal nerves in the chest were severed , the inhibition of gastric motilities disappeared even when the abdomen was stimulated . similarly , acupuncture to the hind paw increased the activity of the thigh and the afferent fibers of the sciatic nerves and , when the thigh and sciatic nerves were severed , the response to increase gastric motilities disappeared even when the hind paw was stimulated . ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . [ ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . gastric motilities were inhibited by acupuncture to the abdomen in spinalized rats but acupuncture to the hind paw did not excite gastric motilities proving that inhibitory or excitatory responses to gastric motilities by acupuncture to the abdomen or the hind paw were spinal or supraspinal reflex responses . in the same study group , confirmed that electro - acupuncture of various intensities to the abdomen or hind limbs caused excitatory or inhibitory responses in gastric motilities . they further recorded the activity of afferent fibers from intercostal nerves or tibial nerves when the abdomen or hind paw were stimulated and examined the relationship of the intensity of the stimulation to response in gastric motilities . they identified the nerves through which the electro - acupuncture stimulations worked , observing that the responses occurred above the threshold level of electro - acupuncture stimulation intensity at which c fibers are excited in the abdomen , but above the threshold level at which a and c fibers are excited in the hind paw . accordingly , the stimulation of body trunk and hind limbs were transmitted through different nerve fibers ( 14 ) . measured motilities caused by pinching stimulation and reported that noxious stimulation to the abdomen produced an inhibitory response by spinal reflexes . measured duodenal motilities by a method similar to that used to measure gastric motilities , and examined the relationship between intensities of electro - acupuncture to changes in duodenal motilities . their results revealed that to decrease duodenal motilities , electro - acupuncture stimulation to the abdomen needed to be strong enough to excite group iv fibers of intercostal nerves . to increase motilities , electro - acupuncture stimulation to the abdomen needs to be strong enough to excite the higher - threshold group iii fibers of tibial nerves . they also proved that the responses occur through a path similar to that of gastric motilities ( 15 ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . observed gastric motility in anesthetized rabbits by the balloon method , when they were stimulated by tapping on acupuncture point st36 in 1978 , they discovered that intragastric pressure increased by acupuncture stimulation and that the response disappeared when the central side of the sciatic nerves , which govern the nerves at point st36 , were severed ( 11 ) . observed by electrogastrogram that electro - acupuncture to anesthetized dogs on acupuncture point bl19 , which is on the back , delayed kinetic rhythms of the stomach and inhibited electric activity of the stomach wall ( 12 ) . these reports suggest that acupuncture changes gastric motilities in anesthetized animals , as somato - sensory stimulation does with pinching . observed the effect of manual acupuncture stimulation to anesthetized rats through continuous measurement of the intragastric pressure by means of a balloon inserted into the gastropyrolic region . they discovered that when they inserted a needle ( with a diameter of 340 m ) about 45 mm deep into the subcutaneous muscular layer at many points on the whole body and gave the tested rats acupuncture by twisting the needle for 60 seconds , gastric motilities either decreased or increased . acupuncture inhibited gastric motilities when the abdomen was stimulated as a result of increased activity of the efferent fibers of the gastric sympathetic nerves . increase of gastric motilities in the case of acupuncture stimulations to limbs resulted from increased activity of the efferent fibers of the gastric vagus nerves . these inhibitory or excitatory responses were induced by acupuncture to both the skin and the muscle , the skin only and the subcutaneous muscles only . acupuncture to the abdomen increased the activity of afferent fibers of the spinal nerves in the lower chest and , when the spinal nerves in the chest were severed , the inhibition of gastric motilities disappeared even when the abdomen was stimulated . similarly , acupuncture to the hind paw increased the activity of the thigh and the afferent fibers of the sciatic nerves and , when the thigh and sciatic nerves were severed , the response to increase gastric motilities disappeared even when the hind paw was stimulated . ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . [ ( b ) excitatory changes of gastric motilities by stimulation of hind limb with electro - acupuncture . ( c ) comparison between the intensity of abdominal stimulation with electro - acupuncture and the rates of gastric motility changes . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers in the afferent intercostal nerve activity . ( d ) comparison between the intensity of hind paw stimulation with electro - acupuncture and the rates of gastric motility change . as shown with arrows , ta and tc indicate the average intensity of threshold of group a and b fibers of the tibial nerves , respectively . gastric motilities were inhibited by acupuncture to the abdomen in spinalized rats but acupuncture to the hind paw did not excite gastric motilities proving that inhibitory or excitatory responses to gastric motilities by acupuncture to the abdomen or the hind paw were spinal or supraspinal reflex responses . , in the same study group , confirmed that electro - acupuncture of various intensities to the abdomen or hind limbs caused excitatory or inhibitory responses in gastric motilities . they further recorded the activity of afferent fibers from intercostal nerves or tibial nerves when the abdomen or hind paw were stimulated and examined the relationship of the intensity of the stimulation to response in gastric motilities . they identified the nerves through which the electro - acupuncture stimulations worked , observing that the responses occurred above the threshold level of electro - acupuncture stimulation intensity at which c fibers are excited in the abdomen , but above the threshold level at which a and c fibers are excited in the hind paw . measured motilities caused by pinching stimulation and reported that noxious stimulation to the abdomen produced an inhibitory response by spinal reflexes . measured duodenal motilities by a method similar to that used to measure gastric motilities , and examined the relationship between intensities of electro - acupuncture to changes in duodenal motilities . their results revealed that to decrease duodenal motilities , electro - acupuncture stimulation to the abdomen needed to be strong enough to excite group iv fibers of intercostal nerves . to increase motilities , electro - acupuncture stimulation to the abdomen needs to be strong enough to excite the higher - threshold group iii fibers of tibial nerves . they also proved that the responses occur through a path similar to that of gastric motilities ( 15 ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . ( a ) intensity of electro - acupuncture to the abdomen and hind paw and the rate of changes in duodenal motilities . ( a-1 ) the threshold of afferent intercostal nerve activity innervating the stimulated abdominal region , and the inhibitory change rates of duodenal motilities . b-1 ) no inhibitory response of the duodenal motilities , ( b-2 ) disappearance of excitatory response . ( c-1 ) disappearance of inhibitory response of duodenal motilities , ( c-2 ) : no effect on excitatory response . ( d-1 ) no effect on inhibitory response of duodenal motilities , ( d-2 ) disappearance of excitatory response ( cited from noguchi with modification ) . ( a ) effects of severing the splanchnic nerve and vagus nerve on excitatory response of gastric acid secretion with electro - acupuncture of the hind paw of the anesthetized rats at acupuncture point s-36 excitatory response of gastric acid secretion with electro - acupuncture disappeared after vagotomy ( open squares ) and ischiadic nerve section ( closed squares ) , while that after the splanchnic nerve section did not disappear . ( b ) effects of electro - acupuncture stimulation on gastric acid secretion after amino acid intake in conscious dogs . however , ( a ) effects of severing the splanchnic nerve and vagus nerve on excitatory response of gastric acid secretion with electro - acupuncture of the hind paw of the anesthetized rats at acupuncture point s-36 excitatory response of gastric acid secretion with electro - acupuncture disappeared after vagotomy ( open squares ) and ischiadic nerve section ( closed squares ) , while that after the splanchnic nerve section did not disappear . horizontal lines ( eas ) indicate the period of acupuncture stimulation . ( b ) effects of electro - acupuncture stimulation on gastric acid secretion after amino acid intake in conscious dogs . observed that electro - acupuncture of conscious dogs inhibited rises in gastric - acid secretion that should have occurred when they ingested amino acid foods they proposed that the mechanism involved endogenous opioids such as occurs in acupuncture analgesia ( 18 ) . in shanghai , observed gastric - acid secretion through changes in the ph value of physiologic saline circulated in the stomach of anesthetized rats ( ghosh & schild method ) , and observed an increase in secretion by electro - acupuncture to acupuncture point st36 with no effect from severing the ischiadic and vagus nerves or the administration of naloxone ( 19 ) . observed gastric - acid secretion in anesthetized rats , using a similar gastric circulation , they discovered that the response to increasing gastric - acid secretion occurred by electro - acupuncture stimulations to acupuncture point st36 , and that it disappeared when the ischiadic nerves or vagus nerves were severed , but did not disappear when the splanchnic nerves were severed . they concluded that the excitatory response occurred through somato - autonomic nervous reflexes ( 20 ) . although many scientists have reported on the responses of gastric - acid secretion to acupuncture stimulations , the results were not definitive and the underlying mechanism remains unknown . in order to observe the response to acupuncture in conscious animals , the problem of stress caused by acupuncture needed to be solved . in the study of gastric - acid secretion accordingly many concerns must be solved to clarify the effect of acupuncture stimulations on gastric - acid secretion . the following mechanisms for acupuncture stimulation to anesthetized rats have been proven to exert effects on gastric motilities . acupuncture to the abdomen of anesthetized rats increases gastric motilities by exciting sympathetic nerves via spinal reflexes . stimulating the limbs of rats caused an increase in gastric motilities by exciting vagus nerves via supraspinal reflexes . in addition , acupuncture simulation of the abdomen must be strong enough to excite group vi fibers of the afferent fibers of intercostal nerves in order to produce an inhibitory response . stimulation of hind limbs should be strong enough to excite the high - threshold group iii fibers of the tibial nerves in order to produce the excitatory response . as for duodenal motilities , acupuncture works through a neural mechanism similar to that of gastric motilities in terms of stimulated regions and intensity of stimulation . reports on inhibitory and excitatory effects of acupuncture on gastric - acid secretion are mixed . for the neural mechanism , somato - autonomic reflexes and responses of intervening endogenous opioids

in highly developed countries , in order to enhance the efficiency of health care systems , the search for better practices and new organizational models is an issue . because of economic pressures and a succession of failed attempts to improve services coordination , the concept of integrated service networks has recently emerged as a way of structuring health care , particularly for chronic and complex problems . coordination and continuity of services is an old preoccupation , but what emerged mostly in the 1990s is the idea of creating a network of services linking autonomous health care and social service providers in a given district to treat specific health problems or clienteles such as serious mental health disorders or the frail elderly . since then , a wealth of literature has been published defining what integrated service networks , also known as organized delivery systems , integrated delivery systems or disease management , are or should be . despite the abundance of writings on integrated service networks , few empirical studies have been published , and still fewer have explored models to suit the various implementation contexts [ 25 ] . leutz 's questions such as who should be in charge of integration , the degree of financial and administrative integration needed to achieve clinical integration , and what support structures and processes are needed for integrated service networks require further investigation . there have been significant advances in knowledge concerning factors that facilitate or hinder the development of integrated service networks , but more is required to map out their evolution [ 79 ] . this article explores the question of the organization of integrated service networks for the serious mental health disorders , based on the quebec ( canada ) context and on three recent research projects conducted in that province . first , the concept of integrated service network will be examined , focusing on fundamental dimensions relevant to its organization . after having outlined our methodology , the presentation of four mental health models of integrated service networks in rural , urban or semi - urban , and metropolitan areas will follow . we will then elaborate on key conditions of , and main obstacles to , the application of those models , referring to the three questions raised by leutz , cited in the preceding paragraph . to conclude , we will look at how these models of integrated service networks in mental health can be applied to other health care sectors and even generalized . as mentioned in the introduction , numerous works have been published on services integration and integrated service networks [ 5 , 1014 ] . services integration has three components : functional / administrative , clinical , and physician - system . the functional / administrative part deals with systems of governance , management , information , resources allocation and evaluation . it implies care at the vertical and horizontal levels , which means taking into account service needs between different periods of care ( i.e. pre- and post- hospitalization ) as well as services provided within a period of care ( i.e. psychosocial community support , housing and employment ) . the physician - system component stems from the importance of the physicians participation in the network , for they are ultimately clinically responsible for the care given [ 5 , 6 , 16 ] . it involves restructuring clinical practices for the clientele and the relationships between health care professionals , service lines and organizations at the different levels of governance : strategic ( upper management ) , tactical ( middle management ) and operational ( staff ) . the concept of network brings a territorial dimension to the idea of integration , and pertains to the organization of services for a specific health sector . all organizations involved in a given area and with a targeted clientele will thus be mobilized to coordinate their action . depending on client and resources volume integrated service networks are based on an acknowledgement of considerable interdependence among the actors and organizations in a given district and sector of intervention . on a continuum of intensity of inter - organizational relationships ( e.g. partnership , mutual adjustment ) , integrated service networks constitute one of the most advanced and formalized forms of coordination between independent organizations . they involve rationalization in order to provide a diversified range of services to meet client needs . in the literature , they correspond to the virtual integration usually viewed as more effective for complex systems that present a high degree of diversification . the situation is all the more evident in the health care sector , where organizations are viewed as professional bureaucracies with unclear goals and fluid and ambiguous authority . to implement functional / administrative , clinical and physician - system integration , integration strategies are mechanisms or processes to promote more coherent , coordinated and efficient services in a network [ 13 , 14 , 18 , 2123 ] . functional / administrative strategies aim at , for example , reducing services duplication , increasing flexibility in resources allocation so as to finance organizations in a network that offer more cost - effective interventions , improving and relating clinical information between organizations and better controlling the system . in functional / administrative integration , we find strategies such as electronic client information and management systems as well as methods for allocating and managing material , financial and personnel resources . planning , inter - organizational protocols ( memorandum of understanding ) , grouping of institutions and a single point of entry are featured as well . the way governance is structured is a key element for good network functioning [ 24 , 25 ] . at the clinical level , the importance of creating a network is based on the actors interest in : ( a ) sharing skills and knowledge regarding a clientele difficult to treat and involving various types of clinical staff and organizations ; ( b ) building peer support that may encompass diversified skills ; ( c ) reinforcing consultation or sharing expertise and support among various lines of services ; ( d ) becoming more familiar with a district 's resources in order to better refer clientele ; ( e ) clarifying organizations missions and their referral processes , particularly appointing liaison officers to facilitate continuity ; ( f ) identifying key staff members to whom clients can be referred according to their needs ; ( g ) developing common tools for the network ; and ( h ) for more complex cases , facilitating joint interventions among professionals from different organizations . they include community follow - up , shared care , continuing education , clinical coaching , inter - organizational internships and programs , shared staff , and individualized service plans . additional provision includes : follow - up protocols that help standardize and rationalize practices , common electronic needs assessment grids and confidentiality protocols that allow for inter - organization exchange of information on clients , and so on . community follow - up may vary in intensity and imply staff workers from one or more organizations . shared care consists in support provided by psychiatrists to general practitioners treating clients with mental health problems . an example of inter - organizational program would be crisis services provided jointly by a local community centre and a community organization . those integration strategies are crucial considering that integrated service networks involve sweeping changes in the organization of the health care system , in inter - organizational practices and in the delivery of services . the canadian health care system is under provincial jurisdiction . it is mainly public , with extensive private sector collaboration ( e.g. residential / nursing - home services , dental and optometric services , pharmacists ) . in the province of quebec , control over that system is the responsibility of two regulatory bodies : the ministry of health and social services and the regional agencies . the ministry defines the strategic functions of the system , establishes the main rules governing its operations and distributes resources equitably among regions . it also retains some important functions in managing the health care system , such as developing guidelines for human , material and financial resources , enacting labour force adjustment policies and programs , and organizing training and research . the regional agencies , numbering eighteen for nineteen health and social services regions , are responsible for planning , organizing , coordinating , budgeting and evaluating health care and social services in their respective regions . they manage the health care system from local districts and on the basis of nine programs or clienteles ( e.g. physical , mental and public health , physical and mental disabilities , the frail elderly ) . thus , the system is relatively decentralized at the regional level , despite the fact that on one side , the ministry holds strategic responsibilities and that on the other side , the local health care organizations retain a high degree of autonomy by handling such responsibilities as overall budget management . the idea of ensuring greater coherence and coordination among organizations that deliver health services based on programs for specific clienteles in a given district emerged in quebec around 1990 . , strategies for integrating services were developed through regional planning and regional and local coordination committees [ 27 , 28 ] . the mid-1990s witnessed extensive mergers of institutions , which became the main approach in achieving better integration of the health and welfare system . through mergers , the 917 quebec health care establishments that made up the system in 1990 implementation of integrated service networks went into full swing between 1997 and 2001 , owing to non - recurrent funding granted by the federal government to encourage innovation and system efficiency . owing to those funds , a number of integrated networks emerged for complex or chronic health problems , notably for the frail elderly , for type 2 diabetics and for cancer patients . in parallel to the federal initiative , provincial policies were elaborated to promote integrated service networks for clienteles needing multiple services on an intensive basis for a substantive period of time . in the policy - makers views , such an organization of services allows for better client follow - up and response to people 's needs , ensures efficiency in the system by reducing duplication of services and encourages providers to work in coordination . in that context , the development of integrated service networks for people with serious mental health disorders was emphasized . several ministerial documents testify to that orientation [ 3133 ] . beside some broad guidelines such as implementing a basic range of diversified services , the way networks no timescale , incentive or follow - up of the policy implementation was specifically given by the government to promote integrated service networks for that clientele . such a situation compromised network implementation and favoured a high degree of diversification among regions , most of the local districts having in fact barely implemented them . the degree of integration of mental health networks then relied mostly on the leadership of regional agencies and local providers . in quebec , mental health networks basically include : local community centres ( literally known as clscs : local community services centres ) , community organizations , private medical clinics and intersectoral resources . depending on the area , they may involve of general hospital psychiatric departments and psychiatric hospitals . local community centres offer primary care on a short - term basis , homemaker assistance , and health promotion programs . some of them have recently added specialized services on a long - term basis such as community follow - up and treatment programs for people suffering from serious mental health disorders . community organizations provide programs like follow - up , self - help for peers or family , support for workforce integration , short or medium term housing or help in finding housing , hot - line services or crisis counselling , rehabilitation services such as workshops and day centres , and recreational activities . as for private medical clinics , an ontario study reveals that fifty percent of people with mental health problems are treated exclusively by a general practitioner . the primarily solicited intersectoral resources for mental health networking are municipalities ( for welfare housing ) , school boards , the justice system , the police force , employment centres , food banks and soup kitchens , and so on . specifically found in general hospital psychiatric departments are short - term hospitalizations , day clinics , outpatient clinics and community treatment programs usually assertive . what psychiatric hospitals add to that is the ultra - specialized care handling of the most serious cases , long - term hospitalizations , co - morbidity programs , ultra - specialized clinics , training and advanced research . this article is based on three interrelated research projects on mental health integrated service networks in the province of quebec ( canada ) funded by the canadian health services research foundation , the canadian institute of health research , the fonds de recherche en sant du qubec and the quebec ministry of health and social services ( 20022004 ) . the projects were divided into three phases and conducted simultaneously . the first phase was designed to assess the implementation level of mental health integrated service networks and identify examples viable in different implementation contexts . lengthy interviews ( n=18 ) were conducted with managers of the mental health program in each regional agency . the second phase aimed at outlining the most effective strategies to operate integrated networks and the elements that facilitate or hinder their development . in that phase , the case study method was considered the most appropriate because it allows for a deep understanding of a phenomenon and takes into account its multidimensional aspects : political , cultural , financial and so on [ 36 , 37 ] . a case study is an empirical inquiry in which the boundaries between phenomenon and context are not clearly evident . six local networks in five of the health and social services regions in quebec were selected based on the results of the first phase . the six networks were chosen , by the research team including its decision - making partners at the provincial and regional levels , because of their representativeness of the province 's integrated service networks on the whole . the selection criteria were : the extent to which the networks reflected rural , urban or semi - urban , and metropolitan characteristics ; their high degree of implementation ; the existence or the absence of a psychiatric hospital in the area ; and the feasibility of the research : practical considerations such as the remoteness of the networks . the third phase , not considered in this article , is an evaluation of the effectiveness of the six local services networks in response to the needs of people with serious mental health disorders . the six cases , described largely in a recently published report , are grounded on extensive sources of qualitative and quantitative data . it is justified by its descriptive character , presenting ideal - types or models of integrated service networks suitable for different contexts and discussing their implementation and generalization . primary sources ( i.e. administrative documents at the organizational and local levels ; regional and ministerial policies ) , 2 . individual interviews conducted with 275 managers and clinical staff representative of all organizations belonging to the six networks , 50 service users and 25 of their relatives . the organizations considered are the ones involved in the mental health system mentioned in the previous section : local community centres , community organizations , intersectoral resources , general hospital departments , psychiatric hospitals and private medical clinics . the respondents were selected by using an intentional sampling strategy , and were interviewed from the winter of 2002 to the summer of 2004 . the interviews were recorded , and all the data collected were summarized using analytical grids that included , among others , the range of networks resources , the types of integration strategies , inter - organizational relationships and factors that facilitate or hinder network implementation . the cases were then studied by content analysis to construct models relevant to rural , semi - urban or urban and metropolitan contexts . the results were validated by the research team and its decision - making partners at the provincial and regional levels . the first two apply to rural settings , the third to an urban or semi - urban one and the last to a metropolitan area . depending on whether they are rural or metropolitan , the networks include approximately 20,000 to 200,000 people . in quebec , as elsewhere in the world , between 2 and 3% of the population face serious mental health disorders ( e.g. schizophrenia , bipolar disorder ) . that is the clientele targeted by the networks . each presentation begins with the local features determining the model to be adopted , and focuses on the governance structure and integration strategies . the population is more confined than in an urban or semi - urban environment . in addition , rural resources are not very specialized . more often than not there is no hospital . in rural quebec , the most available resources are a local community centre , community organizations , private medical clinics , and intersectoral resources ( e.g. police , the school board for training programs ) . the more manageable size of the network is also an advantage for implementing integrated service networks . the difference between them is the availability of specialized services offered by a hospital and the main integration strategy selected for the organization of the network . figure 1 illustrates the model without a hospital and in which the main integration strategy is a local mental health coordination committee . that committee brings together all the clinical staff from the local community centre , community organizations , intersectoral resources and private medical clinics involved in serving clients with serious mental health disorders . an elected official , usually from the local community centre , coordinates the committee 's activities . committee members may agree to jointly follow a client . to cover specialized services , a protocol ( memorandum of understanding ) is established between the local community centre and a psychiatric department in one of the region 's hospitals . the figure 2 model represents a more diversified service network because the district has a general hospital , although without a psychiatric department . this model is structured around a mobile mental health team made up of clinical staff administratively attached to a local community centre or a hospital , and coordinated by a local community centre officer . in this particular case , the team 's creation and operations were facilitated by the merger of the hospital and the local community centre . the mental health mobile team offers treatment and community follow - up of varying intensity . through protocols or informal coordinating links , it also ensures referrals and coordination of services with activities offered by other agencies such as community organizations , private medical clinics and intersectoral resources . as in figure 1 concerning emergencies and hospitalization , there are close ties between the mobile mental health team and the hospital team . a member of the mobile mental health team who is based at the hospital liaises with the hospital staff , the rest of the team and the general practitioners . to help the district 's mental health team in providing more specialized services , strong cooperation exists between the merged hospital - local community centre and the psychiatric hospital located in another network of the same region . a protocol allows for the transfer of clientele to the psychiatric hospital for medium or long - term hospitalization and for specialized services such as those given in a day clinic . it also allows a psychiatrist to come for one or two days per month to handle difficult cases and consult with the general practitioners and the mobile mental health team ( i.e. shared care practices ) . in the figure 1 and 2 models , integration strategies such as regional and local mental health planning and regional coordinating committees are also put forward so as to insure coherence and equity between the localities of a given region . compared to rural settings , semi - urban and urban local districts are characterized by a more extensive , diversified , and for urban territories , geographically concentrated range of resources . they have a general hospital with a psychiatric department , and there is usually no psychiatric hospital . people are less confined to a given territory and tend to shop around for services in adjacent areas . figure 3 introduces a model for organizing services in integrated networks in urban and semi - urban settings . as with the figure 1 model , the integrated service network is structured around a central strategy : a local mental health coordination committee that involves senior administrators from the hospital , the local community centre and community organizations involved in mental health . given the number of actors involved in the network , other integrating strategies are introduced . at the tactical level , service coordinators sit at sub - committees set up to work on specific health - related issues such as treatments , follow - up in the community and job integration . these sub - committees are required to develop strategies and common tools that facilitate work in an integrated network , for example common needs assessment grids , confidentiality agreements to transfer information between organizations and a resources directory . for large organizations such as hospitals and local community centres , liaison officers ensure the working of inter - organization links and clientele follow - up . at the operational level , the field staff is encouraged to elaborate individualized services plans and practices such as case management or assertive community treatment . it is also targeted for inter - organizational and inter - professional training , clinical coaching and internships or staff exchanges . the objective is to guarantee the acquisition of best practices knowledge , meet staff from the network , and instil a culture of partnership . the integrated strategies already outlined in the urban and semi - urban model apply to the first three points of entry in figure 3 the general hospital , the local community centre ( clsc ) and the community organizations . the last three points of entry : private medical clinics , pharmacies and intersectoral resources , are required to cooperate with the mental health network through informal coordination , protocols or shared - care initiatives . they are invited to the local coordination committee meetings on specific topics related to their mission and their expertise . a metropolitan setting is similar to an urban one , except that the overall features characterizing the latter are magnified : geographic concentration , diversification and proliferation of resources , and population mobility . they give the distinctive characters to the organization of services , because of the ultra - specialized care they offer . if in the vicinity there is also a general hospital with a psychiatric department they may regroup for rationalization purposes ( e.g. psychiatric emergency services at the general hospital and specialized clinics at the psychiatric hospital ) . compared to rural and urban or semi - urban districts , metropolitan areas have socio - demographic and economic features conducive to the organization of services encompassing several localities due to the supra - local mission of the psychiatric hospital . figure 4 presents a model similar to the one illustrated in figure 3 , except for an intermediary structure ( the small crossed circles in front of the oval in bold ) preceding the mental health coordination committee . the organization of the metropolitan model rests on a matrix organization of services that takes into account the strategic , tactical and operational levels of governance as well as distinct spheres of intervention such as treatment and hospitalization , support in the community , housing and employment . due to the large number of actors to mobilize , the intermediary structure brings coherence and coordination into those different spheres of intervention . moreover , a delegation of actors from each sphere is appointed to coordinate their own actions with the mental health coordination committee at the strategic level . in terms of complexity , the rural model is , not surprisingly , the easiest to implement . because of the small number of actors involved the scarcity of resources forces organizations to cooperate , favouring the recognition of interdependence , legitimacy and expertise of the partners , conditions required for the implementation of integrated service networks [ 28 , 40 ] . the collaborative culture of rural settings has been highlighted in other studies and in health integrated networks introduced in quebec [ 4143 ] . in rural settings , power between organizations is also more balanced than in urban or semi - urban and metropolitan areas , where a general or a psychiatric hospital plays a leading role . dispersion of power between organizations has been found to facilitate networking ; otherwise small organizations tend to benefit from the network , leaving larger units less interested in cooperation [ 27 , 38 , 40 , 44 ] . in quebec as in other settings , the result is that networks often essentially include local community centres and community organizations , with hospitals weakly linked to both . finally , in rural districts , the fact that clients can not easily shop around for services induces them to adhere to their local network . for the success of integrated networks implementation , the ways of organizing networks are crucial , but so are the utilization patterns of the clientele [ 4648 ] . consequently , two perspectives have to be taken into account in modelling integrated service networks : the organizations perspective and the population 's perspective . in this article the complexity of the health networks in urban or semi - urban and metropolitan districts encourages the development of more numerous integration strategies than in rural settings . the types of integration ( functional - administrative , clinical and physician - system ) which are related to the levels of governance help to manage the complexity of those settings , and are thus relevant to answer the first of the three questions put forward by leutz , cited in the introduction of this article : who should be in charge of integration ? . the strategic governing level of the system ( i.e. the ministry , the regional agencies and the local networks organizations upper management ) , addressing mostly the functional or administrative integration , is the one that can change the labour force rules and group organizations . it can put forward inter - organizational protocols , strategic planning and computerized information for the network . the tactical level of governing ( e.g. program coordinators and division heads ) is the most suitably positioned to ensure best practices for the clinical integration of the clientele , such as utilization of clinical protocols , liaison officer , community follow - up , shared care , etc . the health care system being a professional bureaucracy , the mobilization of the operational level ( i.e. the field staff ) is also critical in the implementation of integrated networks . training , clinical coaching , inter - organizational internships and staff involvement in decision making are facilitating elements for improving collaboration and system changes . therefore , all three levels of governance have to be interrelated in the management of a network . our integration models have pointed out the importance of the coordinating committee including sub - committees in playing such a role . the coordinating committee is the cornerstone of the network integration process by developing and monitoring integration strategies , enhancing a collaborative culture , and solving potential conflicts . but for networks without a history of collaborative culture and for complex networks such as in urban or semi - urban and metropolitan settings , our research projects underline that coordinating committees are inefficient if given an unclear mandate and little power . in that case the coordinating committees are restricted to a role of exchanging information while the organizations involved in the network continue to serve their own corporate objectives . for the development of complex networks , our observations then underscore the importance of enhancing substantially the decision - making power and control of the central coordinating committee . moreover , the most successful quebec regions in the implementation of integrated mental health services have benefited from firm policy directions promoting networks by the ministry and the regional agencies . which degree of financial and administrative integration is required to achieve effective clinical service integration ? first , total integration of the organizations administrative and financial structures means vertical integration of the system . the literature on the impact of mergers is not necessarily flattering : it describes a loss of innovation and flexibility , reduced diversification , heavier bureaucracy , and for the organizations forced to merge , major conflicts and discouragement among staff , and a transformation of the system in favour of the culture of the dominant organization [ 2 , 50 ] . ownership - based integration , however , can reduce transaction costs between separate production processes , produce economies of scope and scale , and facilitate imposition of common information and clinical practice standards . we have already mentioned that virtual integration is usually viewed as more effective for complex systems that present a high degree of diversification . in quebec , the health care organizations mode of financing , based on global budgeting , and the current ways of assessing their performance on an individual basis , do not help to integrate clinical services effectively . it is in the nature of an organization to grow without considering the efficiency of the whole system , and its performance indicators do not necessarily reflect a network mode of organization ( e.g. pressure to reduce length of stays in hospital may have the counter - effect of boosting premature departures and re - admissions , and do not necessarily take into account the essential resources for post - hospital follow - up ) . consequently , we believe that where there is a collaborative culture , a network can achieve quite a good clinical integration , specifically in rural districts without much degree of financial and administrative integration . but for urban or semi - urban and metropolitan settings , due to the complexity of such systems , financial integration between some organizations or by health sector , in this case mental health care , and administrative integration , are crucial for achieving a high degree of clinical integration . leutz 's third and last question : , what support structures and processes are needed to implement integrated service networks ? has been at the core of this article . while informal relations such as trust and commitment of the actors involved , play a fundamental role in structuring networks , we suggest that to succeed in complex settings , the development of integrated service networks involves orchestrating an extensive number of strategies in the administrative / functional , clinical and physician - system types of integration and at strategic , tactical and operational governing levels . the integration strategies encourage new interactions between parties that increase practices in favour of the reform . the integrated services intensity reached by a network depends on the coherence and scope covered by those strategies . finally , the complexity of the health care system , particularly in mental health , the scarcity of resources specifically offered in the community , and the overall difficulty in implementing reforms require to be addressed . the health care system is known as one of the most complex systems to manage . for implementing integrated service networks for instance , reforms have to be synchronized at different levels of governance : the ministry , regional agencies , local and organizational levels . several regulatory logics also interact , which make goals and lines of authority fluid : bureaucratic , professional , managerial , political , etc . . organizations are not necessarily divided by health sector or district territory , making it more difficult to implement integrated service networks . in mental health specifically , because of historical ideological clashes between psychiatry and some community organizations offering alternatives to psychiatry , the development of integrated service networks has not been easily achieved . the scarcity of resources offered in the community makes it also more difficult to integrate services into networks . milward and provan have underlined the crucial impact of the availability of resources in the integrate services networks effectiveness . lastly , change has to be carefully planned and strategies to support the implementation of reforms highly developed . authors in organizational change have increased our awareness on the importance of the phenomenon of resistance to change that must be overcome in a reform process [ 9 , 55 , 56 ] . for successfully implementing integrated service networks , all partners must for instance recognize common problems of system functioning , share a compatible vision , have interest in collaborating and acknowledge gains to be made from cooperation [ 9 , 28 , 57 ] . the population is more confined than in an urban or semi - urban environment . in addition , rural resources are not very specialized . more often than not there is no hospital . in rural quebec , the most available resources are a local community centre , community organizations , private medical clinics , and intersectoral resources ( e.g. police , the school board for training programs ) . the type of medical practice is not unlike the family medicine pattern . a tradition of collaboration and mutual assistance exists more than in other settings . the more manageable size of the network is also an advantage for implementing integrated service networks . the difference between them is the availability of specialized services offered by a hospital and the main integration strategy selected for the organization of the network . figure 1 illustrates the model without a hospital and in which the main integration strategy is a local mental health coordination committee . that committee brings together all the clinical staff from the local community centre , community organizations , intersectoral resources and private medical clinics involved in serving clients with serious mental health disorders . an elected official , usually from the local community centre , coordinates the committee 's activities . committee members may agree to jointly follow a client . to cover specialized services , a protocol ( memorandum of understanding ) is established between the local community centre and a psychiatric department in one of the region 's hospitals . the figure 2 model represents a more diversified service network because the district has a general hospital , although without a psychiatric department . this model is structured around a mobile mental health team made up of clinical staff administratively attached to a local community centre or a hospital , and coordinated by a local community centre officer . in this particular case , the team 's creation and operations were facilitated by the merger of the hospital and the local community centre . the mental health mobile team offers treatment and community follow - up of varying intensity . through protocols or informal coordinating links , it also ensures referrals and coordination of services with activities offered by other agencies such as community organizations , private medical clinics and intersectoral resources . as in figure 1 , protocols are signed with pharmacies for medication control . concerning emergencies and hospitalization , there are close ties between the mobile mental health team and the hospital team . a member of the mobile mental health team who is based at the hospital liaises with the hospital staff , the rest of the team and the general practitioners . to help the district 's mental health team in providing more specialized services , strong cooperation exists between the merged hospital - local community centre and the psychiatric hospital located in another network of the same region . a protocol allows for the transfer of clientele to the psychiatric hospital for medium or long - term hospitalization and for specialized services such as those given in a day clinic . it also allows a psychiatrist to come for one or two days per month to handle difficult cases and consult with the general practitioners and the mobile mental health team ( i.e. shared care practices ) . in the figure 1 and 2 models , integration strategies such as regional and local mental health planning and regional coordinating committees are also put forward so as to insure coherence and equity between the localities of a given region . compared to rural settings , semi - urban and urban local districts are characterized by a more extensive , diversified , and for urban territories , geographically concentrated range of resources . they have a general hospital with a psychiatric department , and there is usually no psychiatric hospital . people are less confined to a given territory and tend to shop around for services in adjacent areas . figure 3 introduces a model for organizing services in integrated networks in urban and semi - urban settings . as with the figure 1 model , the integrated service network is structured around a central strategy : a local mental health coordination committee that involves senior administrators from the hospital , the local community centre and community organizations involved in mental health . given the number of actors involved in the network , other integrating strategies are introduced . at the tactical level , service coordinators sit at sub - committees set up to work on specific health - related issues such as treatments , follow - up in the community and job integration . these sub - committees are required to develop strategies and common tools that facilitate work in an integrated network , for example common needs assessment grids , confidentiality agreements to transfer information between organizations and a resources directory . for large organizations such as hospitals and local community centres , liaison officers ensure the working of inter - organization links and clientele follow - up . at the operational level , the field staff is encouraged to elaborate individualized services plans and practices such as case management or assertive community treatment . it is also targeted for inter - organizational and inter - professional training , clinical coaching and internships or staff exchanges . the objective is to guarantee the acquisition of best practices knowledge , meet staff from the network , and instil a culture of partnership . the integrated strategies already outlined in the urban and semi - urban model apply to the first three points of entry in figure 3 the general hospital , the local community centre ( clsc ) and the community organizations . the last three points of entry : private medical clinics , pharmacies and intersectoral resources , are required to cooperate with the mental health network through informal coordination , protocols or shared - care initiatives . they are invited to the local coordination committee meetings on specific topics related to their mission and their expertise . a metropolitan setting is similar to an urban one , except that the overall features characterizing the latter are magnified : geographic concentration , diversification and proliferation of resources , and population mobility . they give the distinctive characters to the organization of services , because of the ultra - specialized care they offer . if in the vicinity there is also a general hospital with a psychiatric department they may regroup for rationalization purposes ( e.g. psychiatric emergency services at the general hospital and specialized clinics at the psychiatric hospital ) . compared to rural and urban or semi - urban districts , metropolitan areas have socio - demographic and economic features conducive to the organization of services encompassing several localities due to the supra - local mission of the psychiatric hospital . figure 4 presents a model similar to the one illustrated in figure 3 , except for an intermediary structure ( the small crossed circles in front of the oval in bold ) preceding the mental health coordination committee . the organization of the metropolitan model rests on a matrix organization of services that takes into account the strategic , tactical and operational levels of governance as well as distinct spheres of intervention such as treatment and hospitalization , support in the community , housing and employment . due to the large number of actors to mobilize , the intermediary structure brings coherence and coordination into those different spheres of intervention . moreover , a delegation of actors from each sphere is appointed to coordinate their own actions with the mental health coordination committee at the strategic level . in terms of complexity , the rural model is , not surprisingly , the easiest to implement . because of the small number of actors involved , the network is more manageable . the scarcity of resources forces organizations to cooperate , favouring the recognition of interdependence , legitimacy and expertise of the partners , conditions required for the implementation of integrated service networks [ 28 , 40 ] . the collaborative culture of rural settings has been highlighted in other studies and in health integrated networks introduced in quebec [ 4143 ] . in rural settings , power between organizations is also more balanced than in urban or semi - urban and metropolitan areas , where a general or a psychiatric hospital plays a leading role . dispersion of power between organizations has been found to facilitate networking ; otherwise small organizations tend to benefit from the network , leaving larger units less interested in cooperation [ 27 , 38 , 40 , 44 ] . in quebec as in other settings , the result is that networks often essentially include local community centres and community organizations , with hospitals weakly linked to both . finally , in rural districts , the fact that clients can not easily shop around for services induces them to adhere to their local network . for the success of integrated networks implementation , the ways of organizing networks are crucial , but so are the utilization patterns of the clientele [ 4648 ] . consequently , two perspectives have to be taken into account in modelling integrated service networks : the organizations perspective and the population 's perspective . in this article the complexity of the health networks in urban or semi - urban and metropolitan districts encourages the development of more numerous integration strategies than in rural settings . the types of integration ( functional - administrative , clinical and physician - system ) which are related to the levels of governance help to manage the complexity of those settings , and are thus relevant to answer the first of the three questions put forward by leutz , cited in the introduction of this article : who should be in charge of integration ? . the strategic governing level of the system ( i.e. the ministry , the regional agencies and the local networks organizations upper management ) , addressing mostly the functional or administrative integration , is the one that can change the labour force rules and group organizations . it can put forward inter - organizational protocols , strategic planning and computerized information for the network . the tactical level of governing ( e.g. program coordinators and division heads ) is the most suitably positioned to ensure best practices for the clinical integration of the clientele , such as utilization of clinical protocols , liaison officer , community follow - up , shared care , etc . the health care system being a professional bureaucracy , the mobilization of the operational level ( i.e. the field staff ) is also critical in the implementation of integrated networks . training , clinical coaching , inter - organizational internships and staff involvement in decision making are facilitating elements for improving collaboration and system changes . therefore , all three levels of governance have to be interrelated in the management of a network . our integration models have pointed out the importance of the coordinating committee including sub - committees in playing such a role . the coordinating committee is the cornerstone of the network integration process by developing and monitoring integration strategies , enhancing a collaborative culture , and solving potential conflicts . but for networks without a history of collaborative culture and for complex networks such as in urban or semi - urban and metropolitan settings , our research projects underline that coordinating committees are inefficient if given an unclear mandate and little power . in that case the coordinating committees are restricted to a role of exchanging information while the organizations involved in the network continue to serve their own corporate objectives . for the development of complex networks , our observations then underscore the importance of enhancing substantially the decision - making power and control of the central coordinating committee . moreover , the most successful quebec regions in the implementation of integrated mental health services have benefited from firm policy directions promoting networks by the ministry and the regional agencies . which degree of financial and administrative integration is required to achieve effective clinical service integration ? first , total integration of the organizations administrative and financial structures means vertical integration of the system . the literature on the impact of mergers is not necessarily flattering : it describes a loss of innovation and flexibility , reduced diversification , heavier bureaucracy , and for the organizations forced to merge , major conflicts and discouragement among staff , and a transformation of the system in favour of the culture of the dominant organization [ 2 , 50 ] . ownership - based integration , however , can reduce transaction costs between separate production processes , produce economies of scope and scale , and facilitate imposition of common information and clinical practice standards . we have already mentioned that virtual integration is usually viewed as more effective for complex systems that present a high degree of diversification . in quebec , the health care organizations mode of financing , based on global budgeting , and the current ways of assessing their performance on an individual basis , do not help to integrate clinical services effectively . it is in the nature of an organization to grow without considering the efficiency of the whole system , and its performance indicators do not necessarily reflect a network mode of organization ( e.g. pressure to reduce length of stays in hospital may have the counter - effect of boosting premature departures and re - admissions , and do not necessarily take into account the essential resources for post - hospital follow - up ) . consequently , we believe that where there is a collaborative culture , a network can achieve quite a good clinical integration , specifically in rural districts without much degree of financial and administrative integration . but for urban or semi - urban and metropolitan settings , due to the complexity of such systems , financial integration between some organizations or by health sector , in this case mental health care , and administrative integration , are crucial for achieving a high degree of clinical integration . leutz 's third and last question : , what support structures and processes are needed to implement integrated service networks ? has been at the core of this article . while informal relations such as trust and commitment of the actors involved , play a fundamental role in structuring networks , we suggest that to succeed in complex settings , the development of integrated service networks involves orchestrating an extensive number of strategies in the administrative / functional , clinical and physician - system types of integration and at strategic , tactical and operational governing levels . the integration strategies encourage new interactions between parties that increase practices in favour of the reform . the integrated services intensity reached by a network depends on the coherence and scope covered by those strategies . finally , the complexity of the health care system , particularly in mental health , the scarcity of resources specifically offered in the community , and the overall difficulty in implementing reforms require to be addressed . the health care system is known as one of the most complex systems to manage . for implementing integrated service networks for instance , reforms have to be synchronized at different levels of governance : the ministry , regional agencies , local and organizational levels . several regulatory logics also interact , which make goals and lines of authority fluid : bureaucratic , professional , managerial , political , etc . . organizations are not necessarily divided by health sector or district territory , making it more difficult to implement integrated service networks . in mental health specifically , because of historical ideological clashes between psychiatry and some community organizations offering alternatives to psychiatry , the development of integrated service networks has not been easily achieved . the scarcity of resources offered in the community makes it also more difficult to integrate services into networks . milward and provan have underlined the crucial impact of the availability of resources in the integrate services networks effectiveness . lastly , change has to be carefully planned and strategies to support the implementation of reforms highly developed . authors in organizational change have increased our awareness on the importance of the phenomenon of resistance to change that must be overcome in a reform process [ 9 , 55 , 56 ] . for successfully implementing integrated service networks , all partners must for instance recognize common problems of system functioning , share a compatible vision , have interest in collaborating and acknowledge gains to be made from cooperation [ 9 , 28 , 57 ] . integrated service networks are an organizational model that requires tight coordination among organizations and staff from a given health sector and district . they involve major interdependence between organizations and staffs that interact with a clientele whose health problems are complex and often chronic . this article has demonstrated the importance of structuring those networks to suit the context in which they are implemented : rural , urban or semi - urban and metropolitan . it has also stressed the relevance of mobilizing the health care staff according to functional / administrative , clinical and physician - system integration types and strategic , tactical , and operational governance levels . the importance of formalizing integration within a network , allowing for more enduring coordination , particularly by reinforcing the governance mechanisms at the local level , has been underscored . to reach such formalization , the integrated service networks face two issues : how to efficiently organize services to suit contexts and how to implement them . because of the high degree of intensity involved , integrated service networks are thus not suitable for all health care sectors . in quebec , they are mostly implemented in the following areas : serious mental health disorders , the frail elderly , youth with behavioural problems , physical or intellectual disabilities such as cranial - cerebral disorders and type 2 diabetes , and physical health problems for instance in oncology and palliative care . these networks may be more or less complex to organize depending on : ( a ) the number and type of organizations involved in the health and social fields ; ( b ) the network centrality , defined as the concentration of services given by , and inter - organizational transactions related to , one or some organizations in a network ; ( c ) the transformations required ; and ( d ) the consensus on intervention practices or the conflicting nature of the inter - organizational dynamics . serious mental health disorders offer an interesting illustration of integrated service networks , as it is , in our view , one of the most complex health sectors to manage not withstanding cases involving dual or triple health issues such as mental disorders , drug addiction and homelessness , or all of those . hutschemaekers , phd , professor at the radboud university of nijmegen 's academic centre for social sciences ( the netherlands ) and director of grip ( gelderse roos research institute for mental health care professionalization ) arnhem , the netherlands . mary e. wiktorowicz , phd , associate professor school of health policy and management , atkinson faculty of liberal & professional studies , york university , toronto , ontario , canada . jenny secker , phd , professor of mental health , anglia polytechnic university & south essex partnership nhs trust , united kingdom . marie - jose fleury , phd , associate professor , is assistant professor at mcgill university 's department of psychiatry . she is also adjunct professor at the universit de montral 's department of health administration and researcher at the douglas research center .

abstractpurposein the transformation of health care systems , the introduction of integrated service networks is considered to be one of the main solutions for enhancing efficiency . in the last few years , a wealth of literature has emerged on the topic of services integration . however , the question of how integrated service networks should be modelled to suit different implementation contexts has barely been touched . to fill that gap , this article presents four models for the organization of mental health integrated networks.data sourcesthe proposed models are drawn from three recently published studies on mental health integrated services in the province of quebec ( canada ) with the author as principal investigator.descriptionfollowing an explanation of the concept of integrated service network and a description of the quebec context for mental health networks , the models , applicable in all settings : rural , urban or semi - urban , and metropolitan , and summarized in four figures , are presented.discussion and conclusionto apply the models successfully , the necessity of rallying all the actors of a system , from the strategic , tactical and operational levels , according to the type of integration involved : functional / administrative , clinical and physician - system is highlighted . the importance of formalizing activities among organizations and actors in a network and reinforcing the governing mechanisms at the local level is also underlined . finally , a number of integration strategies and key conditions of success to operationalize integrated service networks are suggested .

Introduction Concept of integrated service networks Context underlying Quebec's mental health integrated service networks Methodology Models of integrated service networks Rural models Urban or semi-urban model Metropolitan model Discussion: key conditions for and main obstacles to the operationalization of the four models of integrated service networks Conclusion Reviewers Vitae

in highly developed countries , in order to enhance the efficiency of health care systems , the search for better practices and new organizational models is an issue . because of economic pressures and a succession of failed attempts to improve services coordination , the concept of integrated service networks has recently emerged as a way of structuring health care , particularly for chronic and complex problems . coordination and continuity of services is an old preoccupation , but what emerged mostly in the 1990s is the idea of creating a network of services linking autonomous health care and social service providers in a given district to treat specific health problems or clienteles such as serious mental health disorders or the frail elderly . since then , a wealth of literature has been published defining what integrated service networks , also known as organized delivery systems , integrated delivery systems or disease management , are or should be . despite the abundance of writings on integrated service networks , few empirical studies have been published , and still fewer have explored models to suit the various implementation contexts [ 25 ] . leutz 's questions such as who should be in charge of integration , the degree of financial and administrative integration needed to achieve clinical integration , and what support structures and processes are needed for integrated service networks require further investigation . this article explores the question of the organization of integrated service networks for the serious mental health disorders , based on the quebec ( canada ) context and on three recent research projects conducted in that province . first , the concept of integrated service network will be examined , focusing on fundamental dimensions relevant to its organization . after having outlined our methodology , the presentation of four mental health models of integrated service networks in rural , urban or semi - urban , and metropolitan areas will follow . we will then elaborate on key conditions of , and main obstacles to , the application of those models , referring to the three questions raised by leutz , cited in the preceding paragraph . to conclude , we will look at how these models of integrated service networks in mental health can be applied to other health care sectors and even generalized . as mentioned in the introduction , numerous works have been published on services integration and integrated service networks [ 5 , 1014 ] . services integration has three components : functional / administrative , clinical , and physician - system . the physician - system component stems from the importance of the physicians participation in the network , for they are ultimately clinically responsible for the care given [ 5 , 6 , 16 ] . it involves restructuring clinical practices for the clientele and the relationships between health care professionals , service lines and organizations at the different levels of governance : strategic ( upper management ) , tactical ( middle management ) and operational ( staff ) . the concept of network brings a territorial dimension to the idea of integration , and pertains to the organization of services for a specific health sector . depending on client and resources volume integrated service networks are based on an acknowledgement of considerable interdependence among the actors and organizations in a given district and sector of intervention . to implement functional / administrative , clinical and physician - system integration , integration strategies are mechanisms or processes to promote more coherent , coordinated and efficient services in a network [ 13 , 14 , 18 , 2123 ] . functional / administrative strategies aim at , for example , reducing services duplication , increasing flexibility in resources allocation so as to finance organizations in a network that offer more cost - effective interventions , improving and relating clinical information between organizations and better controlling the system . at the clinical level , the importance of creating a network is based on the actors interest in : ( a ) sharing skills and knowledge regarding a clientele difficult to treat and involving various types of clinical staff and organizations ; ( b ) building peer support that may encompass diversified skills ; ( c ) reinforcing consultation or sharing expertise and support among various lines of services ; ( d ) becoming more familiar with a district 's resources in order to better refer clientele ; ( e ) clarifying organizations missions and their referral processes , particularly appointing liaison officers to facilitate continuity ; ( f ) identifying key staff members to whom clients can be referred according to their needs ; ( g ) developing common tools for the network ; and ( h ) for more complex cases , facilitating joint interventions among professionals from different organizations . those integration strategies are crucial considering that integrated service networks involve sweeping changes in the organization of the health care system , in inter - organizational practices and in the delivery of services . in the province of quebec , control over that system is the responsibility of two regulatory bodies : the ministry of health and social services and the regional agencies . thus , the system is relatively decentralized at the regional level , despite the fact that on one side , the ministry holds strategic responsibilities and that on the other side , the local health care organizations retain a high degree of autonomy by handling such responsibilities as overall budget management . through mergers , the 917 quebec health care establishments that made up the system in 1990 implementation of integrated service networks went into full swing between 1997 and 2001 , owing to non - recurrent funding granted by the federal government to encourage innovation and system efficiency . owing to those funds , a number of integrated networks emerged for complex or chronic health problems , notably for the frail elderly , for type 2 diabetics and for cancer patients . in that context , the development of integrated service networks for people with serious mental health disorders was emphasized . the degree of integration of mental health networks then relied mostly on the leadership of regional agencies and local providers . the primarily solicited intersectoral resources for mental health networking are municipalities ( for welfare housing ) , school boards , the justice system , the police force , employment centres , food banks and soup kitchens , and so on . this article is based on three interrelated research projects on mental health integrated service networks in the province of quebec ( canada ) funded by the canadian health services research foundation , the canadian institute of health research , the fonds de recherche en sant du qubec and the quebec ministry of health and social services ( 20022004 ) . the first phase was designed to assess the implementation level of mental health integrated service networks and identify examples viable in different implementation contexts . the six networks were chosen , by the research team including its decision - making partners at the provincial and regional levels , because of their representativeness of the province 's integrated service networks on the whole . the selection criteria were : the extent to which the networks reflected rural , urban or semi - urban , and metropolitan characteristics ; their high degree of implementation ; the existence or the absence of a psychiatric hospital in the area ; and the feasibility of the research : practical considerations such as the remoteness of the networks . the interviews were recorded , and all the data collected were summarized using analytical grids that included , among others , the range of networks resources , the types of integration strategies , inter - organizational relationships and factors that facilitate or hinder network implementation . the cases were then studied by content analysis to construct models relevant to rural , semi - urban or urban and metropolitan contexts . the first two apply to rural settings , the third to an urban or semi - urban one and the last to a metropolitan area . each presentation begins with the local features determining the model to be adopted , and focuses on the governance structure and integration strategies . the population is more confined than in an urban or semi - urban environment . the more manageable size of the network is also an advantage for implementing integrated service networks . the difference between them is the availability of specialized services offered by a hospital and the main integration strategy selected for the organization of the network . that committee brings together all the clinical staff from the local community centre , community organizations , intersectoral resources and private medical clinics involved in serving clients with serious mental health disorders . to cover specialized services , a protocol ( memorandum of understanding ) is established between the local community centre and a psychiatric department in one of the region 's hospitals . a member of the mobile mental health team who is based at the hospital liaises with the hospital staff , the rest of the team and the general practitioners . in the figure 1 and 2 models , integration strategies such as regional and local mental health planning and regional coordinating committees are also put forward so as to insure coherence and equity between the localities of a given region . as with the figure 1 model , the integrated service network is structured around a central strategy : a local mental health coordination committee that involves senior administrators from the hospital , the local community centre and community organizations involved in mental health . the integrated strategies already outlined in the urban and semi - urban model apply to the first three points of entry in figure 3 the general hospital , the local community centre ( clsc ) and the community organizations . the last three points of entry : private medical clinics , pharmacies and intersectoral resources , are required to cooperate with the mental health network through informal coordination , protocols or shared - care initiatives . they give the distinctive characters to the organization of services , because of the ultra - specialized care they offer . compared to rural and urban or semi - urban districts , metropolitan areas have socio - demographic and economic features conducive to the organization of services encompassing several localities due to the supra - local mission of the psychiatric hospital . the organization of the metropolitan model rests on a matrix organization of services that takes into account the strategic , tactical and operational levels of governance as well as distinct spheres of intervention such as treatment and hospitalization , support in the community , housing and employment . moreover , a delegation of actors from each sphere is appointed to coordinate their own actions with the mental health coordination committee at the strategic level . because of the small number of actors involved the scarcity of resources forces organizations to cooperate , favouring the recognition of interdependence , legitimacy and expertise of the partners , conditions required for the implementation of integrated service networks [ 28 , 40 ] . in rural settings , power between organizations is also more balanced than in urban or semi - urban and metropolitan areas , where a general or a psychiatric hospital plays a leading role . for the success of integrated networks implementation , the ways of organizing networks are crucial , but so are the utilization patterns of the clientele [ 4648 ] . in this article the complexity of the health networks in urban or semi - urban and metropolitan districts encourages the development of more numerous integration strategies than in rural settings . the types of integration ( functional - administrative , clinical and physician - system ) which are related to the levels of governance help to manage the complexity of those settings , and are thus relevant to answer the first of the three questions put forward by leutz , cited in the introduction of this article : who should be in charge of integration ? the field staff ) is also critical in the implementation of integrated networks . therefore , all three levels of governance have to be interrelated in the management of a network . but for networks without a history of collaborative culture and for complex networks such as in urban or semi - urban and metropolitan settings , our research projects underline that coordinating committees are inefficient if given an unclear mandate and little power . for the development of complex networks , our observations then underscore the importance of enhancing substantially the decision - making power and control of the central coordinating committee . moreover , the most successful quebec regions in the implementation of integrated mental health services have benefited from firm policy directions promoting networks by the ministry and the regional agencies . the literature on the impact of mergers is not necessarily flattering : it describes a loss of innovation and flexibility , reduced diversification , heavier bureaucracy , and for the organizations forced to merge , major conflicts and discouragement among staff , and a transformation of the system in favour of the culture of the dominant organization [ 2 , 50 ] . it is in the nature of an organization to grow without considering the efficiency of the whole system , and its performance indicators do not necessarily reflect a network mode of organization ( e.g. but for urban or semi - urban and metropolitan settings , due to the complexity of such systems , financial integration between some organizations or by health sector , in this case mental health care , and administrative integration , are crucial for achieving a high degree of clinical integration . while informal relations such as trust and commitment of the actors involved , play a fundamental role in structuring networks , we suggest that to succeed in complex settings , the development of integrated service networks involves orchestrating an extensive number of strategies in the administrative / functional , clinical and physician - system types of integration and at strategic , tactical and operational governing levels . finally , the complexity of the health care system , particularly in mental health , the scarcity of resources specifically offered in the community , and the overall difficulty in implementing reforms require to be addressed . in mental health specifically , because of historical ideological clashes between psychiatry and some community organizations offering alternatives to psychiatry , the development of integrated service networks has not been easily achieved . authors in organizational change have increased our awareness on the importance of the phenomenon of resistance to change that must be overcome in a reform process [ 9 , 55 , 56 ] . the more manageable size of the network is also an advantage for implementing integrated service networks . the difference between them is the availability of specialized services offered by a hospital and the main integration strategy selected for the organization of the network . that committee brings together all the clinical staff from the local community centre , community organizations , intersectoral resources and private medical clinics involved in serving clients with serious mental health disorders . to cover specialized services , a protocol ( memorandum of understanding ) is established between the local community centre and a psychiatric department in one of the region 's hospitals . a member of the mobile mental health team who is based at the hospital liaises with the hospital staff , the rest of the team and the general practitioners . in the figure 1 and 2 models , integration strategies such as regional and local mental health planning and regional coordinating committees are also put forward so as to insure coherence and equity between the localities of a given region . figure 3 introduces a model for organizing services in integrated networks in urban and semi - urban settings . as with the figure 1 model , the integrated service network is structured around a central strategy : a local mental health coordination committee that involves senior administrators from the hospital , the local community centre and community organizations involved in mental health . the integrated strategies already outlined in the urban and semi - urban model apply to the first three points of entry in figure 3 the general hospital , the local community centre ( clsc ) and the community organizations . they give the distinctive characters to the organization of services , because of the ultra - specialized care they offer . compared to rural and urban or semi - urban districts , metropolitan areas have socio - demographic and economic features conducive to the organization of services encompassing several localities due to the supra - local mission of the psychiatric hospital . the organization of the metropolitan model rests on a matrix organization of services that takes into account the strategic , tactical and operational levels of governance as well as distinct spheres of intervention such as treatment and hospitalization , support in the community , housing and employment . moreover , a delegation of actors from each sphere is appointed to coordinate their own actions with the mental health coordination committee at the strategic level . the scarcity of resources forces organizations to cooperate , favouring the recognition of interdependence , legitimacy and expertise of the partners , conditions required for the implementation of integrated service networks [ 28 , 40 ] . in rural settings , power between organizations is also more balanced than in urban or semi - urban and metropolitan areas , where a general or a psychiatric hospital plays a leading role . for the success of integrated networks implementation , the ways of organizing networks are crucial , but so are the utilization patterns of the clientele [ 4648 ] . in this article the complexity of the health networks in urban or semi - urban and metropolitan districts encourages the development of more numerous integration strategies than in rural settings . the types of integration ( functional - administrative , clinical and physician - system ) which are related to the levels of governance help to manage the complexity of those settings , and are thus relevant to answer the first of the three questions put forward by leutz , cited in the introduction of this article : who should be in charge of integration ? the field staff ) is also critical in the implementation of integrated networks . therefore , all three levels of governance have to be interrelated in the management of a network . but for networks without a history of collaborative culture and for complex networks such as in urban or semi - urban and metropolitan settings , our research projects underline that coordinating committees are inefficient if given an unclear mandate and little power . for the development of complex networks , our observations then underscore the importance of enhancing substantially the decision - making power and control of the central coordinating committee . moreover , the most successful quebec regions in the implementation of integrated mental health services have benefited from firm policy directions promoting networks by the ministry and the regional agencies . the literature on the impact of mergers is not necessarily flattering : it describes a loss of innovation and flexibility , reduced diversification , heavier bureaucracy , and for the organizations forced to merge , major conflicts and discouragement among staff , and a transformation of the system in favour of the culture of the dominant organization [ 2 , 50 ] . it is in the nature of an organization to grow without considering the efficiency of the whole system , and its performance indicators do not necessarily reflect a network mode of organization ( e.g. but for urban or semi - urban and metropolitan settings , due to the complexity of such systems , financial integration between some organizations or by health sector , in this case mental health care , and administrative integration , are crucial for achieving a high degree of clinical integration . while informal relations such as trust and commitment of the actors involved , play a fundamental role in structuring networks , we suggest that to succeed in complex settings , the development of integrated service networks involves orchestrating an extensive number of strategies in the administrative / functional , clinical and physician - system types of integration and at strategic , tactical and operational governing levels . finally , the complexity of the health care system , particularly in mental health , the scarcity of resources specifically offered in the community , and the overall difficulty in implementing reforms require to be addressed . the health care system is known as one of the most complex systems to manage . in mental health specifically , because of historical ideological clashes between psychiatry and some community organizations offering alternatives to psychiatry , the development of integrated service networks has not been easily achieved . authors in organizational change have increased our awareness on the importance of the phenomenon of resistance to change that must be overcome in a reform process [ 9 , 55 , 56 ] . for successfully implementing integrated service networks , all partners must for instance recognize common problems of system functioning , share a compatible vision , have interest in collaborating and acknowledge gains to be made from cooperation [ 9 , 28 , 57 ] . integrated service networks are an organizational model that requires tight coordination among organizations and staff from a given health sector and district . this article has demonstrated the importance of structuring those networks to suit the context in which they are implemented : rural , urban or semi - urban and metropolitan . it has also stressed the relevance of mobilizing the health care staff according to functional / administrative , clinical and physician - system integration types and strategic , tactical , and operational governance levels . the importance of formalizing integration within a network , allowing for more enduring coordination , particularly by reinforcing the governance mechanisms at the local level , has been underscored . because of the high degree of intensity involved , integrated service networks are thus not suitable for all health care sectors . these networks may be more or less complex to organize depending on : ( a ) the number and type of organizations involved in the health and social fields ; ( b ) the network centrality , defined as the concentration of services given by , and inter - organizational transactions related to , one or some organizations in a network ; ( c ) the transformations required ; and ( d ) the consensus on intervention practices or the conflicting nature of the inter - organizational dynamics . serious mental health disorders offer an interesting illustration of integrated service networks , as it is , in our view , one of the most complex health sectors to manage not withstanding cases involving dual or triple health issues such as mental disorders , drug addiction and homelessness , or all of those . hutschemaekers , phd , professor at the radboud university of nijmegen 's academic centre for social sciences ( the netherlands ) and director of grip ( gelderse roos research institute for mental health care professionalization ) arnhem , the netherlands .

data on the pharmacology of darunavir / ritonavir during pregnancy are limited to case reports 18 , few of which examined pharmacokinetics 13,57,9 . the effects of pregnancy on the pharmacokinetics of darunavir remain unclear ; therefore , further pharmacokinetic studies are needed . maternal physiological changes during pregnancy ( e.g. blood volume expansion , increased glomerular filtration rate and alterations in hepatic metabolism ) may result in altered pharmacokinetics 10 . data support the conclusion that the pharmacokinetic parameters of hiv protease inhibitors ( pis ) are changed during pregnancy , leading to lower exposure in pregnant women 3,5,6,11,12 . studying darunavir / ritonavir in hiv - infected pregnant women may provide insights into population - specific pharmacokinetics that could inform treatment strategies and dosing recommendations . this nonrandomized study was designed to investigate the pharmacokinetic profile , antiviral activity and safety of darunavir / ritonavir [ 600/100 mg twice daily ( bid ) or 800/100 mg once daily ( qd ) ] , etravirine ( 200 mg bid ) and rilpivirine ( 25 mg qd ) in hiv - infected pregnant women . here we report only on the group that received darunavir / ritonavir 600/100 mg bid . hiv-1-infected pregnant women , aged 18 years or older , with pregnancies between 18 and 26 weeks of gestation were included in this multicentre , single - arm , open - label trial . subjects had to be on the study drug before screening and enrolment , and had to undergo an obstetric examination and have a normal level ii ultrasound . the study was approved by a centralized ethics committee [ western institutional review board ( irb ) ] or a site - specific irb . subjects with any obstetric complications , any neurological conditions requiring medication or any active disease that may have compromised patient safety or study outcomes were excluded from the study . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . eight blood draws were performed during each visit : predose and 1 , 2 , 3 , 4 , 6 , 9 and 12 h postdose . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . the unbound fraction of darunavir was calculated as the ratio of the unbound concentration in the filtrate to the total concentration in the plasma before centrifugation . human serum albumin and 1-acid glycoprotein ( aag ) were measured because darunavir is highly protein bound and the concentration of these proteins usually decreases during pregnancy as a result of haemodilution 14 . infants ' hiv-1 statuses were determined by polymerase chain reaction testing , reported within 16 weeks postpartum . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . the primary pharmacokinetic parameters used in the statistical analysis were auc12h , cmin and cmax on the logarithmic scale . the least squares means ( lsms ) of the primary parameters for each treatment were estimated with a linear mixed - effects model , controlling for period as a fixed effect and subject as a random effect . a 90% confidence interval ( ci ) both the difference between the lsms and the 90% ci were retransformed to the original scale . safety and antiviral activity were summarized using descriptive statistics ( sas / stat version 9.2 ; sas institute inc . , cary , nc ) . hiv-1-infected pregnant women , aged 18 years or older , with pregnancies between 18 and 26 weeks of gestation were included in this multicentre , single - arm , open - label trial . subjects had to be on the study drug before screening and enrolment , and had to undergo an obstetric examination and have a normal level ii ultrasound . the study was approved by a centralized ethics committee [ western institutional review board ( irb ) ] or a site - specific irb . subjects with any obstetric complications , any neurological conditions requiring medication or any active disease that may have compromised patient safety or study outcomes were excluded from the study . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . eight blood draws were performed during each visit : predose and 1 , 2 , 3 , 4 , 6 , 9 and 12 h postdose . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . the unbound fraction of darunavir was calculated as the ratio of the unbound concentration in the filtrate to the total concentration in the plasma before centrifugation . human serum albumin and 1-acid glycoprotein ( aag ) were measured because darunavir is highly protein bound and the concentration of these proteins usually decreases during pregnancy as a result of haemodilution 14 . infants ' hiv-1 statuses were determined by polymerase chain reaction testing , reported within 16 weeks postpartum . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . the primary pharmacokinetic parameters used in the statistical analysis were auc12h , cmin and cmax on the logarithmic scale . the least squares means ( lsms ) of the primary parameters for each treatment were estimated with a linear mixed - effects model , controlling for period as a fixed effect and subject as a random effect . a 90% confidence interval ( ci ) both the difference between the lsms and the 90% ci were retransformed to the original scale . safety and antiviral activity were summarized using descriptive statistics ( sas / stat version 9.2 ; sas institute inc . , cary , nc ) . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . the median age was 24 years ( range 1835 years ) , and 63% were black or african american . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . sixty - three per cent were previously treated with one protease inhibitor ( pi)-based regimen . of 16 subjects , 14 had at least one intensive pharmacokinetic visit and were included in the pharmacokinetic analysis , and 12 completed the treatment phase of the study . a total of five subjects discontinued ( four during treatment before delivery ; one during follow - up ) ( table s1 ) . two subjects discontinued because of adverse events ( aes ) : one for increased transaminases related to study drugs and one for unrelated premature delivery ( premature delivery led to an inability to fulfill protocol requirements as the patient missed the postpartum visit ) . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit ; third trimester adherence rate and pharmacokinetic data were not available ] . one subject was ineligible to continue the trial because of a failed drug screening . mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . data for unbound darunavir were not available for all patients as not all plasma samples were available for plasma protein binding analysis . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . the median tmax of unbound darunavir was approximately 3 h during pregnancy and 2 h postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . data for unbound darunavir were not available for all patients because of poor sample quality ( protein in the filtrate ) or low sample volume . n = 10 for c0h , n = 9 for cmin , and n = 7 for cmax and tmax . n = 11 for c0h , and n = 8 for cmin , cmax and tmax . n = 9 for cmin , and n = 7 for cmax . n = 12 for cmin and cmax . matched maternal and cord plasma concentrations were available for 10 subjects . excluding one outlier resulting from a possible switch of the samples , the mean [ standard deviation ( sd ) ] total darunavir concentrations were higher in maternal plasma vs. cord plasma [ 2324 ( 1056 ) ng / ml vs. 383 ( 322 ) ng / ml , respectively ] when assessed in nine women and their infants . the median cord : maternal plasma ratio was 0.1455 ( range 0.014070.3621 ) . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . excluding one outlier resulting from a possible switch of the samples , the mean ( sd ) total ritonavir concentrations were higher in maternal plasma vs. cord plasma [ 429 ng / ml vs. 17 ng / ml , respectively ) when assessed in seven women and their infants . the median cord : maternal plasma ratio was 0.1076 ( range 0.044320.114 ) . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig . three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . the third patient had detectable viral load during both trimesters ( 813 copies / ml in the second trimester and 59 copies / ml in the third trimester ) , with 100% reported adherence for both trimesters . of these three patients with detectable viral load during pregnancy , two had undetectable viral load in the postpartum and follow - up periods . cd4 count is significantly lower in hiv-1-negative women during pregnancy compared with the 12-week postdelivery period 15 ; therefore , the percentage of cd4 cells was plotted instead of absolute cd4 count . the percentage of cd4 cells increased from 30% at baseline to 35% at 25 weeks postpartum ( fig . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . the most common aes , occurring with an incidence of 25% , were infections and infestations ( 44% ) , gastrointestinal disorders ( 25% ) and premature labour ( 25% ) . of 12 infants , four were born prematurely ( at weeks 30 , 36 , 36 and 37 ) and no congenital abnormalities occurred . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . the median age was 24 years ( range 1835 years ) , and 63% were black or african american . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . sixty - three per cent were previously treated with one protease inhibitor ( pi)-based regimen . of 16 subjects , 14 had at least one intensive pharmacokinetic visit and were included in the pharmacokinetic analysis , and 12 completed the treatment phase of the study . a total of five subjects discontinued ( four during treatment before delivery ; one during follow - up ) ( table s1 ) . two subjects discontinued because of adverse events ( aes ) : one for increased transaminases related to study drugs and one for unrelated premature delivery ( premature delivery led to an inability to fulfill protocol requirements as the patient missed the postpartum visit ) . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . data for unbound darunavir were not available for all patients as not all plasma samples were available for plasma protein binding analysis . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . the median tmax of unbound darunavir was approximately 3 h during pregnancy and 2 h postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . data for unbound darunavir were not available for all patients because of poor sample quality ( protein in the filtrate ) or low sample volume . n = 10 for c0h , n = 9 for cmin , and n = 7 for cmax and tmax . n = 11 for c0h , and n = 8 for cmin , cmax and tmax . n = 9 for cmin , and n = 7 for cmax . n = 12 for cmin and cmax . matched maternal and cord plasma concentrations were available for 10 subjects . excluding one outlier resulting from a possible switch of the samples , the mean [ standard deviation ( sd ) ] total darunavir concentrations were higher in maternal plasma vs. cord plasma [ 2324 ( 1056 ) ng / ml vs. 383 ( 322 ) ng / ml , respectively ] when assessed in nine women and their infants . the median cord : maternal plasma ratio was 0.1455 ( range 0.014070.3621 ) . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . excluding one outlier resulting from a possible switch of the samples , the mean ( sd ) total ritonavir concentrations were higher in maternal plasma vs. cord plasma [ 429 ng / ml vs. 17 ng / ml , respectively ) when assessed in seven women and their infants . the median cord : maternal plasma ratio was 0.1076 ( range 0.044320.114 ) . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . the third patient had detectable viral load during both trimesters ( 813 copies / ml in the second trimester and 59 copies / ml in the third trimester ) , with 100% reported adherence for both trimesters . of these three patients with detectable viral load during pregnancy , two had undetectable viral load in the postpartum and follow - up periods . cd4 count is significantly lower in hiv-1-negative women during pregnancy compared with the 12-week postdelivery period 15 ; therefore , the percentage of cd4 cells was plotted instead of absolute cd4 count . the percentage of cd4 cells increased from 30% at baseline to 35% at 25 weeks postpartum ( fig . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . the most common aes , occurring with an incidence of 25% , were infections and infestations ( 44% ) , gastrointestinal disorders ( 25% ) and premature labour ( 25% ) . of 12 infants , four were born prematurely ( at weeks 30 , 36 , 36 and 37 ) and no congenital abnormalities occurred . lower exposures of total darunavir / ritonavir 600/100 mg bid were observed during pregnancy compared with postpartum . total darunavir cmax was 28 and 19% lower , and auc12h was 24 and 17% lower during the second and third trimesters , respectively , vs. postpartum . these data are consistent with published reports demonstrating a decrease in total drug exposure ( auc ) of darunavir / ritonavir 600/100 mg bid during pregnancy ranging from 29 1 to 36% 9 . lower levels of drug exposure during pregnancy vs. postpartum have been observed with other hiv pis 11,12,1619 . however , unlike previous reports , the current study also assessed free plasma darunavir levels , which were higher during pregnancy vs. postpartum . as a result , the difference in unbound cmax and auc12h at postpartum compared with values found during pregnancy was minimal . the small number of patients with pharmacokinetic data for unbound darunavir , six in the second trimester and seven in the third trimester , limited the ability to draw definitive conclusions ; however , we hypothesize that the lower total drug exposure of darunavir during pregnancy might be partially compensated for by higher free drug , resulting from lower protein binding to darunavir . the antiviral activity of darunavir / ritonavir in hiv - infected pregnant women was effective in preventing mother - to - child transmission and in suppressing hiv in pregnant women , consistent with findings in pregnant 1,3,5,9 and nonpregnant hiv - infected individuals 20 . despite lower total pi levels in studies in pregnant women , subjects achieved desired immunovirological responses and protection from transmission of hiv to their infants 3,5,6,9,11,12 . taken together , the findings that unbound concentrations of darunavir remained nearly unchanged during pregnancy relative to postpartum and that clinical responses were maintained suggest that no a priori dose adjustment is needed in darunavir / ritonavir when dosed bid in pregnant women . total ritonavir cmax was 34 and 37% lower , and auc12h was 28 and 33% lower during the second and third trimesters , respectively , vs. postpartum , similar to findings in previously published reports 11 . the levels of darunavir / ritonavir transferred from maternal to cord plasma were low , with median cord : maternal ratios of 0.1455 and 0.1076 , respectively , consistent with other reports demonstrating low transplacental passage of pis 3,7,9 . it is interesting to note that cmin values were on average higher during the second and third trimesters , respectively , vs. postpartum for both darunavir ( 43 and 86% higher , respectively ) and ritonavir ( 8 and 22% higher , respectively ) . plasma concentration values at 12 hours have also been presented in table 1 in order to provide additional context to these data . in this study , darunavir / ritonavir bid treatment was generally well tolerated in hiv - infected pregnant women , with few discontinuations because of aes ( 12% ) . the observed ae profile partially overlaps with that found previously in nonpregnant adults 20 , and it is unclear how many aes may actually have been associated with pregnancy independent of darunavir / ritonavir bid treatment . a limitation of this study may be the use of the 6- to 12-week postpartum levels as the reference . darunavir / ritonavir pharmacokinetic parameters after administration of darunavir / ritonavir 600/100 mg bid as part of an antiretroviral regimen during pregnancy and postpartum were not clinically different compared with historical controls . darunavir / ritonavir 600/100 mg bid administered with other antiretrovirals during pregnancy resulted in consistent unbound ( active ) drug exposure during pregnancy and postpartum , was effective in preventing mother - to - child transmission and in suppressing hiv and was generally well tolerated , with few discontinuations because of aes . data from this small pharmacokinetic study , while not definitive , suggest that darunavir / ritonavir 600/100 mg bid may be a treatment option for pregnant women in need of highly active antiretroviral therapy . additional supporting information may be found in the online version of this article at the publisher 's web - site : fig . s1 individual minimum plasma concentrations of total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . s2 mean ( standard error ) plasma concentration time curves for albumin ( a ) and 1-acid glycoprotein ( aag ) ( b ) assessed at various time - points throughout the study . fig . s3 antiviral activity expressed as percentage response rate ( a ) ( only patients who completed the study in its entirety are included ) and status of the immune system expressed as median percentage cd4 count ( b ) assessed at various time - points throughout the study .

objectivesantiretroviral therapy during pregnancy is recommended to reduce the risk of mother - to - child transmission of hiv and for maternal care management . physiological changes during pregnancy can affect pharmacokinetics , potentially altering pharmacological activity . we therefore evaluated the pharmacokinetics of twice - daily ( bid ) darunavir in hiv-1-infected pregnant women.methodshiv-1-infected pregnant women receiving an antiretroviral regimen containing darunavir / ritonavir 600/100 mg bid were enrolled in this study . total and unbound darunavir and total ritonavir plasma concentrations were obtained over 12 h during the second and third trimesters and postpartum . total darunavir and ritonavir plasma concentrations were determined using a validated high - performance liquid chromatography tandem mass spectrometry assay and unbound darunavir was determined using 14c - darunavir - fortified plasma . pharmacokinetic parameters were derived using noncompartmental analysis.resultsdata were available for 14 women . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) for total darunavir was 1724% lower during pregnancy than postpartum . the auc12h for unbound darunavir was minimally reduced during pregnancy vs. postpartum . the minimum plasma concentration ( cmin ) of total and unbound darunavir was on average 4386% and 1014% higher , respectively , during pregnancy vs. postpartum . the antiviral response ( < 50 hiv-1 rna copies / ml ) was 33% at baseline and increased to 7390% during treatment ; the percentage cd4 count increased over time . one serious adverse event was reported ( increased transaminase ) . all 12 infants born to women remaining in the study at delivery were hiv-1-negative ; four of these infants were premature.conclusionstotal darunavir exposure decreased during pregnancy . no clinically relevant change in unbound ( active ) darunavir occurred during pregnancy , suggesting that no dose adjustment is required for darunavir / ritonavir 600/100 mg bid in pregnant women .

Introduction Methods Study design and treatment Objectives Evaluations Statistical analysis Results Population and baseline characteristics Disposition Pharmacokinetics of total and unbound darunavir Pharmacokinetics of ritonavir Albumin and Antiviral activity Safety Discussion Supporting Information

data support the conclusion that the pharmacokinetic parameters of hiv protease inhibitors ( pis ) are changed during pregnancy , leading to lower exposure in pregnant women 3,5,6,11,12 . this nonrandomized study was designed to investigate the pharmacokinetic profile , antiviral activity and safety of darunavir / ritonavir [ 600/100 mg twice daily ( bid ) or 800/100 mg once daily ( qd ) ] , etravirine ( 200 mg bid ) and rilpivirine ( 25 mg qd ) in hiv - infected pregnant women . here we report only on the group that received darunavir / ritonavir 600/100 mg bid . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . the primary objective was to assess the effect of pregnancy on the pharmacokinetics of darunavir / ritonavir administered bid during the second and third trimesters of gestation and postpartum . the secondary objectives were to document antiviral activity , safety and tolerability of darunavir / ritonavir - based regimens during pregnancy and postpartum , to compare darunavir / ritonavir concentrations between maternal serum and cord blood at delivery and to assess the outcomes for infants of women treated with darunavir / ritonavir bid during pregnancy . intensive pharmacokinetic samplings over 12 h were performed at three study visits : during the second trimester ( 2428 weeks ) , during the third trimester ( 3438 weeks ) and 612 weeks postpartum . darunavir ( total and unbound ) and ritonavir ( total ) plasma concentrations were measured . cord blood and maternal plasma concentrations of total darunavir and ritonavir were also measured on the day of delivery . total darunavir and ritonavir plasma concentrations were determined using a previously validated high - performance liquid chromatography tandem mass spectrometry assay with a lower limit of quantification of 5.00 ng / ml for both compounds 13 . total and unbound pharmacokinetic parameters were derived from the plasma concentration actual time data using noncompartmental analysis ( winnonlin professional 4.1 ; pharsight , mountain view , ca ) . the minimum and maximum plasma concentrations ( cmin and cmax , respectively ) along with time to reach cmax ( tmax ) were obtained by inspection of the plasma concentration time profiles . the area under the plasma concentration time curve from 0 to 12 h ( auc12h ) was determined using the linear trapezoidal rule . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit ; third trimester adherence rate and pharmacokinetic data were not available ] . mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . data for unbound darunavir were not available for all patients as not all plasma samples were available for plasma protein binding analysis . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . excluding one outlier resulting from a possible switch of the samples , the mean [ standard deviation ( sd ) ] total darunavir concentrations were higher in maternal plasma vs. cord plasma [ 2324 ( 1056 ) ng / ml vs. 383 ( 322 ) ng / ml , respectively ] when assessed in nine women and their infants . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig . three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . the third patient had detectable viral load during both trimesters ( 813 copies / ml in the second trimester and 59 copies / ml in the third trimester ) , with 100% reported adherence for both trimesters . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . sixteen women were enrolled in this part of the trial and received darunavir / ritonavir 600/100 mg bid ( supplementary table s1 ) . most ( 80% ) subjects had a cd4 count 350 cells/l , 33% had a viral load < 50 hiv-1 rna copies / ml , 53% had a viral load between 50 and 400 copies / ml and 14% had a viral load 400 copies / ml . two subjects discontinued because of virological failure related to suboptimal adherence as documented by the principal investigator [ one patient reported 87.5% adherence ( auc12h 69 970 ng / h / ml ; cmin 4140 ng / ml ) in the 4 days prior to the second trimester visit and 75% adherence in the 4 days prior to the third trimester visit ( no pharmacokinetic data were available ) , and the other patient reported 62.5% adherence in the 4 days prior to the second trimester visit mean total and unbound darunavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . mean ( standard deviation ) plasma concentration time curves for total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum . the maximum plasma concentration of total darunavir during the second and third trimesters was 28 and 19% lower , respectively , vs. postpartum based on the lsm ratios . total darunavir cmin was on average 43 and 86% higher during the second and third trimesters , respectively , vs. postpartum . total darunavir auc12h during pregnancy was 24 and 17% lower in the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmax was 22 and 18% lower during the second and third trimesters , respectively , vs. postpartum . unbound darunavir cmin was 10 and 14% higher during the second and third trimesters , respectively , vs. postpartum . unbound darunavir auc12h was 8 and 7% lower during the second and third trimesters , respectively , vs. postpartum . the free fraction of darunavir ( ratio of unbound plasma concentration vs. total plasma concentration ) was slightly higher during pregnancy vs. postpartum ( data not shown ) . mean ( standard deviation ) * pharmacokinetic parameters and statistical results for total and unbound darunavir and total ritonavir , after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum lsm , least squares mean ; ci , confidence interval ; c0h , predose plasma concentration ; nd , not determined ; cmin , minimum plasma concentration ; cmax , maximum plasma concentration ; c12h , plasma concentration at 12 hours ; tmax , time to reach the maximum plasma concentration ; auc12h , area under the plasma concentration time curve over 12 h ; cl / f , apparent clearance . mean total ritonavir plasma concentrations were higher during the postpartum period compared with the second and third trimesters of pregnancy ( fig . median total ritonavir plasma concentrations peaked 4 h after drug administration during pregnancy and at 6 h in the postpartum period ( table 1 ) . total ritonavir cmax during the second and third trimesters of pregnancy was 34 and 37% lower , respectively , vs. postpartum based on the lsm ratios . total ritonavir cmin was on average 8 and 22% higher during the second and third trimesters , respectively , vs. postpartum . total ritonavir auc12h during pregnancy was 28 and 33% lower in the second and third trimesters , respectively , vs. postpartum . excluding one outlier resulting from a possible switch of the samples , the mean ( sd ) total ritonavir concentrations were higher in maternal plasma vs. cord plasma [ 429 ng / ml vs. 17 ng / ml , respectively ) when assessed in seven women and their infants . mean baseline albumin and aag concentrations were 31 and 617 g / l and postpartum concentrations were 38 and 790 g / l , respectively , resulting in a 22 to 28% decrease during pregnancy vs. postpartum ( fig . the response rate ( < 50 copies / ml ) increased significantly from 33% ( five of 15 ) at baseline ( table s1 ) to 73% ( eight of 11 ) and 90% ( nine of 10 ) during the second and third trimesters , respectively , and was 80% ( eight of 10 ) at 12 weeks postpartum ( fig three patients had detectable viral loads during pregnancy ; two patients had detectable viral load during the second but not the third trimester [ 72 copies / ml in one patient ( 100% reported adherence in both trimesters ) and 123 copies / ml in the other ( 87.5% reported adherence in the second trimester , and 100% reported adherence in the third ) ] . all 12 infants born to women who stayed on treatment until delivery were hiv negative . one of these infants was born to a patient who discontinued the study but remained on study treatment during follow - up . lower exposures of total darunavir / ritonavir 600/100 mg bid were observed during pregnancy compared with postpartum . total darunavir cmax was 28 and 19% lower , and auc12h was 24 and 17% lower during the second and third trimesters , respectively , vs. postpartum . these data are consistent with published reports demonstrating a decrease in total drug exposure ( auc ) of darunavir / ritonavir 600/100 mg bid during pregnancy ranging from 29 1 to 36% 9 . the small number of patients with pharmacokinetic data for unbound darunavir , six in the second trimester and seven in the third trimester , limited the ability to draw definitive conclusions ; however , we hypothesize that the lower total drug exposure of darunavir during pregnancy might be partially compensated for by higher free drug , resulting from lower protein binding to darunavir . the antiviral activity of darunavir / ritonavir in hiv - infected pregnant women was effective in preventing mother - to - child transmission and in suppressing hiv in pregnant women , consistent with findings in pregnant 1,3,5,9 and nonpregnant hiv - infected individuals 20 . taken together , the findings that unbound concentrations of darunavir remained nearly unchanged during pregnancy relative to postpartum and that clinical responses were maintained suggest that no a priori dose adjustment is needed in darunavir / ritonavir when dosed bid in pregnant women . total ritonavir cmax was 34 and 37% lower , and auc12h was 28 and 33% lower during the second and third trimesters , respectively , vs. postpartum , similar to findings in previously published reports 11 . it is interesting to note that cmin values were on average higher during the second and third trimesters , respectively , vs. postpartum for both darunavir ( 43 and 86% higher , respectively ) and ritonavir ( 8 and 22% higher , respectively ) . in this study , darunavir / ritonavir bid treatment was generally well tolerated in hiv - infected pregnant women , with few discontinuations because of aes ( 12% ) . darunavir / ritonavir pharmacokinetic parameters after administration of darunavir / ritonavir 600/100 mg bid as part of an antiretroviral regimen during pregnancy and postpartum were not clinically different compared with historical controls . darunavir / ritonavir 600/100 mg bid administered with other antiretrovirals during pregnancy resulted in consistent unbound ( active ) drug exposure during pregnancy and postpartum , was effective in preventing mother - to - child transmission and in suppressing hiv and was generally well tolerated , with few discontinuations because of aes . data from this small pharmacokinetic study , while not definitive , suggest that darunavir / ritonavir 600/100 mg bid may be a treatment option for pregnant women in need of highly active antiretroviral therapy . s1 individual minimum plasma concentrations of total darunavir ( a ) , unbound darunavir ( b ) and total ritonavir ( c ) after administration of darunavir / ritonavir 600/100 mg twice daily ( bid ) , during the second and third trimesters and postpartum .

plasmacytoid dendritic cells ( pdcs ) are the main producers of type i interferons ( ifn - i ) in response to foreign nucleic acids , thereby indirectly influencing immunity . they can also differentiate into antigen presenting cells ( apcs ) to directly stimulate and modulate t cell responses . pdcs have been implicated in the pathogenesis of many human inflammatory diseases and their corresponding mouse models . for example , chronic pdc activation and subsequent ifn - i production can promote autoimmune diseases , such as lupus erythematosus ( sle ) and psoriasis . multiple sclerosis ( ms ) is a chronic inflammatory disease of the central nervous system ( cns ) . in ms patients , pdcs are present in the cerebrospinal fluid ( csf ) , leptomeninges and demyelinating lesions , . the exact role of pdcs in the pathogenesis of ms is controversial , as they might have both pathogenic and protective functions . in one hand , pdc - derived cytokines , including type - i ifn and il-6 induces pro - pathogenic th1 and th17 cells , which are implicated in ms pathogenesis , . on the other hand , pdcs can exert a protective role in ms through the production of type - i ifn . for instance , relapsing patients treated with ifn- exhibit reduced disease severity . in experimental autoimmune encephalomyelitis ( eae ) , the rodent model of ms , ifn- and ifnar - deficiency exacerbates disease severity , . furthermore , one study suggests that pdcs inhibit pro - pathogenic conventional dcs ( cdcs ) functions in the cns and , consequently locally inhibit encephalitogenic th17 cells . in addition to a controversial local role in the cns , pdcs have also been implicated in the modulation of autoimmune th1 and th17 priming in secondary lymphoid organs ( slos ) during early phases of eae development . it was suggested that pdcs promote th17 priming , whereas we have demonstrated that the selective abrogation of mhcii expression by pdcs leads to increased mog - specific th1 and th17 and impaired treg proliferation . as a result , lack of mhcii on pdcs correlates with exacerbated eae . thus , although many questions remain to be elucidated , modulation of pdc functions as an approach to treat ms is currently an axis of intense investigation . we have previously demonstrated that the adoptive transfer of mhcii - sufficient pdcs prior to eae induction significantly dampened disease severity , whereas mchii - deficient pdc had no effect . here we investigated whether and how pdcs may modulate the course of eae after disease onset . we found that when transferred during acute eae phase , pdcs led to dramatic disease remission . protection was pdc - dependent , correlated with reduced cns inflammation , and decreased encephalitogenic th1 and th17 cells . our results demonstrated that pdc transfer induced the recruitment of resting endogenous pdcs to the cns via a chemerin dependent mechanism , and confer a tolerogenic environment , which , together with local myelin peptide delivery , inhibits encephalitogenic t cells and results in disease improvement . therefore , modulation of cns inflammation by pdcs in eae mice is an interesting axis of investigation to ameliorate disease clinical outcome . up to date , almost all current therapies in autoimmune diseases are based on the systemic suppression of immune functions and are not curative . our work reinforces and validates the relevance of emerging therapeutic concepts regarding the use of cell therapies for autoimmune diseases . h2-aa ( mhcii ) , ubiquitin - gfp , ifn- , ifnar , bdca2-dtr , cd45.1 ( charles river , france ) and 2d2 mice were in a c57bl/6 background . mice were bred and maintained under spf conditions at geneva medical school animal facility and under eops conditions at charles river , france . bone marrow ( bm ) chimeric mice all procedures were approved by and performed in accordance with the guidelines of the animal research committee of geneva . active eae was induced as described by immunizing mice with 100 g of mog35 - 55 peptide ( biotrend ) emulsified in incomplete freund 's adjuvant ( bd diagnosis ) supplemented with 500 g / ml mycobacterium tuberculosis h37ra ( bd diagnosis ) . at the time of immunization and 48 h later , mice also received 300 ng of pertussis toxin ( sigma aldrich ) into the tail vein . for passive eae induction , encephalitogenic cd4 t cells were generated in vitro from ln and spleen cells of 2d2 mice as described . mice received 67 ng of pertussis toxin at the day of cell injection and 48 h later . mice were monitored daily for disease clinical symptoms , and blindly scored as follows . 1 , flaccid tail ; 2 , impaired righting reflex and hind limb weakness ; 3 , complete hind limb paralysis ; 4 , complete hind limb paralysis with partial fore limb paralysis ; 5,moribund . in some experiments , eae mice were treated at indicated time points with -neta ( abcam ) ( 10 mg / kg / daily ) , dt ( 100 ng / mouse ) , or 10 g / ml of mog35 - 55 peptide in pbs . cdcs and pdcs were generated as described from bm of wt for cdcs and wt , mhcii , ubi - gfp , cd45.1 , and ifn- mice for pdcs . for some experiments , pdcs were treated with 1 g / ml cpg - b ( invivogen ) for the last 24 h of culture . pdcs were enriched from bm cell cultures after 7 days or purified ex - vivo from bm using a pdc isolation kit ( mylteniy biotec ) according to manufacturer 's instructions . cdcs were purified by cell sorting from bm - cell cultures as cd11cpdca-1 , using a moflowastrios ( beckman coulter ) . cdcs and pdcs were loaded or not with 10 g / ml mog35 - 55 or ova323 - 339 peptide and 10 10 cells were injected i.v . into eae dcs were isolated from lymph nodes ( ln ) , spleen , liver and spinal cord ( sc ) . by digesting organ fragments with an enzymatic mix containing collagenase d ( 1 mg / ml ) and dnase i ( 10 g / ml ) ( roche ) in hbss . for liver and sc , single cell suspensions were further centrifuged through a discontinuous 30:70% percoll ( invitrogen ) gradient . monoclonal antibodies used for flow cytometry were from : biolegend ; anti - cd11c ( n418 ) , anti i - ab ( af6 - 120.1 ) , anti - cd11b ( m1/70 ) , anti - cd86 ( p03 ) , anti - pd-1 ( 10f.9g2 ) ( pmp1 - 30 ) ; anti - cd8 ( 536.7 ) ; anti - vla-4 ( hmb1 - 1 ) , anti - ly6c ( hk1.4 ) , anti - icos - l ( hk5.3 ) ; from ebioscience : anti - cd4 ( gk1.5 ) , anti - cd69 ( h1.2f3 ) , vegfr2 ( flk1 ) , anti - ter119 ( ter-119 ) ; anti - cmklr1 ( bz194 ) , anti - foxp3 ( fjk-16s ) , anti il-17 ( ebio17b7 ) , anti - flt3 ( cd135 ) ( a2f10 ) , anti - siglec h ( ebio440c ) ; from bd : anti pdca-1 ( bst-2 , cd317 ) , anti - cd45 ( 30f11 ) , anti - cd16/32 fcyriii ( clone 2.4g2 ) , anti - cd45r / b220 ( ra3 - 6b2 ) , anti - cd19 ( 1d3 ) , anti - cd3 ( 145 - 2c11 ) , anti - c - kit ( cd117 ) ( 2b8 ) , anti - sca-1 ( d7 ) , ki67 ( b56 ) and anti ifn- ( xmg1.2 ) . anti - ly49q ( clone 2e6 ) was from mbl . for flow cytometry analysis of dcs , single cell suspensions were incubated with fcblock ( anti - cd16/32 fcriii ) for 10 min , at 4 c and stained using antibodies against cd11c and pdca-1 or siglec - h . cdcs were defined as cd11cpdca-1 and pdcs as cd11cpdca-1 or cd11csiglec - h . for hematopoietic progenitor analysis , bm cells were stained with the following biotinylated lineage markers : anti - cd3 , anti - b220 , anti - cd19 , anti - cd11b , anti - ter119 , anti- ly6c followed by streptavidin fitc conjugated staining . data were acquired in a cyan adp ( beckman coulter ) and analysed using flowjo software ( tree star ) . intracellular cytokine stainings were done with the cytofix / cytoperm kit ( bd ) for ifn- and il-17 staining . cell proliferation was assessed by flow cytometry using anti human ki67 and respective isotype control . for ifn- and il-17 staining , sc , ln and spleen cells were cultured in rpmi containing 10% heat - inactivated fetal bovine serum , 50 mm 2-mercaptoethanol , 100 mm sodium pyruvate , and 100 m penicillin / streptomycin at 37 c , 5% co2 . cells were stimulated for 18 h with pma / ionomycin and golgi stop solution ( bd ) was added to the last 4 h of culture . cryopreserved sc , post - fixed with 4% pfa and embedded in oct ( sakura finetek ) , were cut into 10 m - thick sections , and stained using primary antibodies against cd45 and pdca-1 appropriate species - specific alexa555-labelled , cy3- , or cy5-conjugated secondary antibodies ( all from jackson immunoresearch laboratories , inc . ) with dapi ( sigma aldrich ) counterstaining . images were acquired with a confocal microscope ( lsm 700 ; carl zeiss , inc . ) mice were transcardiacally perfused with pbs followed by 4% pfa and post - fixed overnight . brain and sc , dehydrated and embedded in paraffin , were cut into 2 m thick sections and stained with mayer 's hemalum , differentiated in acidic - alcohol and co - stained with eosin and coverslipped with depex mounting medium ( serva electrophoretics gmbh , heidelberg , germany ) using tissue tek prisma slide stainer ( sakura seiki c.o . , statistical significance was assessed by the two - tailed unpaired student 's t test or 1-way anova with bonferroni post hoc test . eae incidence was analysed using the 2-way anova with bonferroni post hoc test , using prism 5.0 software ( graphpad software ) . in order to explore a potential therapeutic role for pdcs in eae , we adoptively transferred in vitro bone marrow ( bm ) derived wt mog35 - 55 loaded pdcs into c57bl/6 mice after disease onset , 12 days after immunization with mog35 - 55 + cfa . strikingly , whereas clinical scores of control non pdc - injected mice continued to rise , those of pdc - injected mice stayed stable for few days post - pdc transfer , and then rapidly decreased , resulting in a significant inhibition of eae ( fig . moreover , mice presenting a clinical score of 3 at the time of pdc transfer either significantly recovered or exhibited stabilized disease development , whereas scores of control animals maintained their progression ( supplementary fig . consistent with an amelioration of clinical scores , as early as four days post - pdc transfer , inflammatory foci were reduced in spinal cord ( sc ) of pdc - transferred mice , compared to control eae mice ( fig . 1b ) . in agreement with reduced cns inflammation , microglial cells downmodulated the expression of mhcii molecules ( fig . 1c ) . in spite of this , four days after pdc transfer , we did not observe any differences regarding the frequency of cd4 and cd8 t cells infiltrating the sc ( supplementary fig . 2a ) , nor in the expression of vla-4 by t cells ( supplementary fig . consequently , t cell migration from slos to sc was not altered upon pdc transfer . however , the expression of pd-1 , a molecule that delivers negative signals to t cells , was increased on sc - infiltrating cd4 t cells one day after pdc transfer ( fig . remarkably , encephalitogenic th1 ( at day 4 and 9 after pdc transfer ) and th17 frequencies ( at day 9 after pdc transfer ) were substantially decreased in sc of pdc - transferred mice ( fig . these data demonstrate that adoptive transfer of bm - derived pdcs after eae onset induces a strong and rapid improvement of disease clinical symptoms and cns inflammation . this effect appeared to be pdc - specific , since transfer of mog35 - 55-loaded cdcs did not significantly impact eae development ( supplementary fig . in addition , although most of our experiments were done using 510 10 of transferred pdcs , a similarly efficient therapeutical effect was observed by using only 1 10 cells ( supplementary fig . importantly , adoptively transferred mog35 - 55 loaded ex vivo bm resident pdcs efficiently induced eae amelioration ( fig . 1f ) , demonstrating that in vivo , terminally differentiated pdcs efficiently inhibit the disease . to determine the mechanisms accounting for pdc - mediated eae amelioration , we analysed the body distribution of injected bm - pdcs derived from ubiquitin - gfp mice . two days post - injection , gfp pdcs were detectable in ln , spleen and inflamed sc ( fig . in addition , a very low frequency of gfp pdcs was observed in the liver and the bm ( data not shown ) . in situ immunofluorescence staining of sc revealed that , gfp pdcs localized within eae lesions ( fig . 2b , top ) , expressed the pdc - specific marker pdca-1 ( yellow arrows ) and were surrounded by endogenous infiltrating pdcs ( white arrows ) ( fig . 2b , bottom ) . while the frequencies of cdcs , macrophages and microglial cells were similar in sc of pdc - transferred and control eae mice ( supplementary fig . 4 ) , we observed a massive ( six - fold ) increase of total pdc frequencies in sc of pdc - transferred eae mice ( fig . 2c ) . injected pdcs ( gfp ) represented only 25% of total sc pdcs ( fig . 2d ) , suggesting that endogenous pdcs were recruited to the sc following exogenous pdc transfer . in agreement , increased endogenous pdc frequencies in sc of pdc - transferred animals did not result from local pdc proliferation ( data not shown ) . therefore , we hypothesized that they would be recruited from the bm , where pdcs originate from . accordingly , four days post - pdc transfer into eae mice , we observed in the bm a substantial increase of total pdc frequencies , with only a minor proportion consisting of transferred gfp pdcs ( supplementary fig . these results suggest that the increase in endogenous pdc frequency could be due to active proliferation or enhanced de novo generation . ki67 staining revealed a negligible proliferation rate of pdcs in the bm , in both control and pdc - transferred eae mice ( supplementary fig . in contrast , we observed a significant increase in lsk ( lineage - negative , sca1 , c - kit ) progenitor 's frequencies in the bm , one - day post - pdc transfer ( supplementary fig . in line with this notion , both cdc and pdc frequencies were significantly increased in ln , 4 days after pdc - transfer ( supplementary fig . furthermore , at the same time point , bm - resident pdcs , from pdc - transferred mice , exhibited an upregulation of ly49q , a marker of fully differentiated pdcs compared to control mice ( supplementary fig . altogether , these data show that pdc transfer in eae mice increases hematopoietic cell generation in the bm , leading to enhanced cdc and pdc frequencies in slos , and resulting in the selective recruitment of pdcs to the cns . increased pdc frequencies in sc of eae mice following pdc transfer suggests that these cells might play a protective role locally in the cns . to test this hypothesis , we induced passive eae by injecting in vitro differentiated mog35 - 55-specific 2d2 cd4 t cell effectors . upon both mild ( 1 10 2d2 effectors , fig . 2e , left ) and strong ( 2 10 2d2 effectors , fig . 2e , right ) eae induction , pdcs injected at early disease clinical symptoms onset were able to inhibit disease development , with significantly reduced eae incidence and clinical scores . our data strongly suggests that endogenous pdcs recruited to the cns following pdc transfer are implicated in eae amelioration . indeed , exogenous mog35 - 55 loaded cdcs , which did not significantly ameliorate eae upon transfer ( supplementary fig . 3a ) , also did not induce endogenous pdc recruitment to sc ( fig . 3b ) . to firmly demonstrate that endogenous pdc recruitment to sc was necessary for disease amelioration following exogenous pdc transfer , we used a genetic mouse model to deplete endogenous pdcs . for this purpose , cd45.1 wt pdcs were transferred into bdca-2 dtr eae mice , and mice were injected or not with dt at the same time pdcs were transferred . endogenous pdcs were efficiently depleted in the spleens of dt - treated mice ( not shown ) . however , exogenous ( cd45.1 ) pdc numbers reaching the sc were not affected ( fig . 3c , left ) , but as a consequence of bdca-2 dtr pdc depletion , endogenous pdc recruitment to the sc was substantially impaired in dt treated , compared to untreated animals ( fig . importantly , eae amelioration following mog35 - 55 loaded pdc transfer was totally abrogated in dt treated mice ( fig . 3d ) . therefore , endogenous pdc recruitment to sc after pdc transfer is mandatory to confer eae protection . although a specific receptor mediating pdc migration to inflamed tissues has not yet been identified , different chemokines and chemotactic factors , as well as its receptors have been related to pdc homing to peripheral tissues under pathological conditions . in order to identify possible mechanism(s ) responsible for pdc recruitment to cns , we analysed the implication of chemerin / cmklr1 ( chemerin receptor ) , an axis linked to pdc migration in humans . first , and as previously observed , we found that sc of eae mice contained increased chemerin mrna levels , compared to naive sc ( fig . in addition , in spleens of eae mice , pdcs expressed high levels of cmklr1 , compared to cdcs , irrespective of pdc transfer ( fig . interestingly , the frequency of pdcs expressing cmklr1 was substantially increased in sc as early as one day after pdc transfer , and was sustained four days after transfer ( fig . in contrast , cmklr1 expression remained low and unaltered on sc infiltrating cdcs from both pdc - transferred and control eae mice ( fig . in order to test a possible involvement of the chemerin / cmklr1 axis in the recruitment of endogenous pdcs to the sc following pdc transfer , we treated pdc transferred or control eae mice with a cmklr1 small molecule antagonist , -naphthoyl ethyltrimethylammonium iodide ( -neta ) , known to block cmklr1 cell migration . -neta treatment , for 3 consecutive days following pdc transfer , significantly blocked pdc recruitment to sc of eae mice ( fig . these results demonstrate that pdc recruitment to sc following pdc transfer is mediated by the chemerin - cmklr1 axis . however , given that this molecule not only interferes with pdc homing but also affects migration of additional key effector cells in eae , such as macrophages and microglial cells ( supplementary fig . 6 ) , we could not use this experimental approach to evaluate the impact of blocking endogenous pdc recruitment to the cns on disease evolution . however , the precise mechanisms involved in ifn- mediated disease amelioration are still incompletely understood . in order to check whether ifn- was involved in pdc - mediated protection in eae , we transferred ifn- or wt mog35 - 55 loaded pdcs into eae recipient mice . both wt and ifn- pdcs mediated the same level of disease inhibition , thus excluding a role for ifn- production by exogenous pdcs in disease amelioration ( fig . 5b , left ) , as well as a similar increase in sc pdc frequencies ( fig . 5b , right ) , when transferred into either wt or ifn - i receptor deficient ( ifnar ) eae recipient mice . altogether , these results firmly rule out a role for ifn - i in disease amelioration induced by pdc transfer . we have previously showed that when transferred prior to eae induction , pdc - mediated protection was mhcii dependent . however , in post - eae settings , disease inhibition was as efficient using either mhcii sufficient or deficient pdcs ( fig . 5c ) , suggesting a mhcii independent role for injected mog35 - 55 loaded pdcs in regulating cns - inflammation . a requirement for mhcii expression by endogenous pdcs , that are recruited to the sc after pdc injection , was also ruled out , since eae protection after wt pdc transfer was not altered in mice selectively lacking mhcii expression by pdcs ( mtxpiii + iv : wt bm chimeric mice , as described before ) ( supplementary fig . surprisingly , eae protection was abolished when transferred pdcs were either unloaded or loaded with an irrelevant peptide ( ova323 - 339 ) , prior to their injection ( fig . importantly , in contrast to high peptide concentrations ( 100200 g / ml ) that have been described to confer eae amelioration , intravenously injection of 10 g / ml of mog35 - 55 peptide , the concentration used to load pdcs , did not confer any disease improvement ( supplementary fig . notably , disease amelioration in eae mice injected with mog35 - 55-loaded wt or mhcii pdcs correlated with pdc recruitment to sc ( fig . in contrast , pdc frequencies were not increased in sc of eae mice injected with unloaded pdcs , in which no disease protection was observed ( fig . 5d , e ) , again supporting the need of endogenous pdc recruitment to sc for pdc therapeutic effect . in order to investigate the features related to pdc tolerogenic effect , we analysed pdc phenotype in sc of eae mice . irrespective of pdc transfer , cns pdcs expressed lower levels of cd86 , compared to cdcs ( fig . conversely , the expression of cd69 by endogenous pdcs was substantially downregulated upon pdc transfer ( fig . these findings suggest that the inflammatory status of pdcs in sc from eae mice was markedly altered following pdc transfer . to determine whether the lack of pdc activation is involved in the pdc transfer - therapeutic effect , we treated pdcs with cpg - b prior to their injection . cpg - treated pdcs did not induce in situ upregulation of cmklr1 by pdcs ( fig . 6c ) , neither the recruitment of endogenous pdcs to sc of eae mice ( fig . importantly , we further observed that injection of cpg - b activated mog35 - 55 loaded pdcs did not confer any significant disease amelioration , compared to untreated mog35 - 55 loaded pdcs ( fig . 6e ) , supporting a role for steady - state pdcs in disease amelioration . altogether , these results demonstrate that steady - state pdc - transfer promotes a pdc - dependent tolerogenic environment in sc of eae mice , resulting in the general suppression of eae pathogenicity and clinical symptoms . in eae and ms , whether pdcs have a prominent pathogenic or tolerogenic role is still controversial . activated pdcs , through the production of pro - inflammatory cytokines , such as tnf and il-6 , have been claimed to be pathogenic and promote disease progression , both in mice and humans . in agreement , on the other hand , constitutive or antibody mediated pdc depletion during peak disease correlates with eae exacerbation , . we have previously demonstrated that pdcs can present myelin - derived autoantigens via mhcii to cd4 t cells in an eae context and promote the expansion of suppressive treg cells . in the present work , in order to explore pdc tolerogenic potential in a therapeutic eae setting , we performed adoptive transfer of syngenic pdcs after disease onset . strikingly , pdcs induced a rapid and substantial amelioration of both cns inflammation and eae clinical scores upon transfer into sick mice . transferred pdcs rapidly reached the sc , localized in lesion areas in the cns , and inhibited already primed encephalitogenic mog35 - 55-specific 2d2 cd4 t effector cells , pointing out to a local immunoregulatory role of these cells during eae effector phase . however , we can not rule out a possible systemic impact on immune cells in other organs following pdc transfer , and future studies will determine whether this might be beneficial in other inflammatory diseases . the idea that few exogenous pdcs reaching the sc could confer such strong disease amelioration seemed rather puzzling . when investigating the cellular compartments in the sc after pdc transfer , we observed a tremendous increase in total pdc frequency , but not of other cell types such as cdcs , macrophages or microglial cells , suggesting a specific recruitment of endogenous pdcs to the sc . importantly , selective depletion of endogenous pdcs completely abrogated disease amelioration following pdc adoptive transfer . thus , the accumulation of endogenous pdcs in the cns of eae mice following pdc transfer accounts for eae protection . interestingly , we observed a significant increase in de novo hsc progenitors ' generation in the bm of pdc transferred compared to control eae mice . one possibility is that upon transfer , few exogenous pdcs reach the bm and locally produce a factor promoting hsc generation . in agreement , it has been recently demonstrated that angiopoietin - like 7 , which is produced by pdcs , regulates the expansion and repopulation of human hematopoietic stem and progenitor cells . alternatively , lsk progenitor increase in bm might reflect a physiologic demand for pdc generation , as an indirect consequence of pdc - recruitment to the cns following pdc transfer . newly generated bm - pdcs would then be specifically recruited to the inflamed cns through chemerin / cmklr1 axis , an inflammatory chemotactic factor that is involved in pdc migration . in vivo , chemerin expression correlated with pdc infiltration into peripheral tissues during autoimmune diseases , such as sle . most importantly , chemerin was detected in intralesional cerebrovascular endothelial cells of ms patients , and its receptor is expressed by pdcs . in agreement , we observed an increase in chemerin expression in the sc of eae mice compared to nave mice . furthermore , we found a significant and specific increase in the expression of cmklr1 ( or chem23r ) by pdcs in the sc of pdc - transferred mice . blockade of cmklr1 induced cell migration abrogated pdc recruitment to the sc , demonstrating that following pdc transfer , endogenous pdc homing to inflamed cns is mediated by the chemerin / cmklr1 axis . however , microglia cells and cns - infiltrating myeloid dendritic cells also express cmklr1 , and eae clinical and histological disease was found less severe in cmklr1 deficient mice when compared to wt counterparts . one possibility is that under inflammatory eae condition , activated cns pdcs indeed promote disease pathogenesis , and that in cmklr-1 deficient mice , pdc recruitment to the cns is impaired and mice consequently develop attenuated eae . in our settings of pdc transfer in eae mice , endogenous pdcs would be deflected from pro - pathogenic to pro - tolerogenic functions after downmodulation of their activation state . in our system , rather , a non - activated state for pdcs seemed to be required . accordingly , cpg - b activated pdcs did not induce endogenous pdc recruitment to the cns of eae mice , neither did they conferred any significant level of disease amelioration , when compared to steady - state pdcs . notably , expression of cd69 , a marker for activated pdcs , was substantially reduced on endogenous pdcs recruited to sc after pdc transfer . therefore , the combination of several pdc - specific features in the cns : i ) increased pdc frequencies ; ii ) modulation of pdc activation status ; iii ) ability to provide myelin antigen ; iv ) production of a state permissive for tolerance , altogether likely contribute to pdc protective role in eae . mhcii deficient pdcs , were able to induce the same degree of disease inhibition compared to wt pdcs , ruling out a role for mhcii mediated antigen presentation by exogenous pdcs in this protective setting . our data support a local inhibition of encephalitogenic effector t cells in the cns of eae mice after pdc transfer . first , as early as 1 day post - pdc injection , t cells up - regulated the inhibitory molecule pd-1 , and second , after 4 days , we observed reduced frequencies of encephalitogenic th1 and th17 cells in the sc . current available antibody based therapies target various immune cell populations mediating the autoimmune chronic reaction for the treatment of ms , rheumatoid arthritis and other autoimmune diseases . autologous stem cell transplantations are currently tested as immunotherapy for autoimmune diseases , including ms . although visionary , therapies using terminally differentiated cells for the treatment of autoimmune diseases are emerging and are up to date mainly focused on the adoptive transfer of tregs . both preclinical and clinical data in support for treg infusional therapy suggest that ex vivo expanded , autologous tregs might have a beneficial effect in patients for t1d and sle , . in ms , indeed , while tregs infused prior eae induction confer protection , their therapeutic value is considerably diminished when infused after disease initiation , . whether tregs acquire suppressive functions and are directly linked to eae protection after pdc - transfer will require further investigation . if these approaches efficiently suppress eae when injections were performed prior disease induction , tolerogenic apcs however impact mainly the priming of encephalitogenic t cells , and no efficacy was consequently proven when effector t cell responses and disease symptoms are already established . these observations make our results even more attractive in the sense that pdc suppressive effect on established eae is not apc dependent , and directly impact disease effector phase in the cns . together , our results highlight pdcs as important mediators in ms immunotherapy . manipulation of pdc numbers as well as enhancement of pdc potential immunoregulatory properties could be exploited in order to treat ms patients .

plasmacytoid dendritic cells ( pdcs ) exhibit both innate and adaptive functions . in particular they are the main source of type i ifns and directly impact t cell responses through antigen presentation . we have previously demonstrated that during experimental autoimmune encephalomyelitis ( eae ) initiation , myelin - antigen presentation by pdcs is associated with suppressive treg development and results in attenuated eae . here , we show that pdcs transferred during acute disease phase confer recovery from eae . clinical improvement is associated with migration of injected pdcs into inflamed cns and is dependent on the subsequent and selective chemerin - mediated recruitment of endogenous pdcs to the cns . the protective effect requires pdc pre - loading with myelin antigen , and is associated with the modulation of cns - infiltrating pdc phenotype and inhibition of cns encephalitogenic t cells . this study may pave the way for novel pdc - based cell therapies in autoimmune diseases , aiming at specifically modulating pathogenic cells that induce and sustain autoimmune inflammation .

Introduction Materials and methods Results Discussion Competing interests

plasmacytoid dendritic cells ( pdcs ) are the main producers of type i interferons ( ifn - i ) in response to foreign nucleic acids , thereby indirectly influencing immunity . they can also differentiate into antigen presenting cells ( apcs ) to directly stimulate and modulate t cell responses . for example , chronic pdc activation and subsequent ifn - i production can promote autoimmune diseases , such as lupus erythematosus ( sle ) and psoriasis . in one hand , pdc - derived cytokines , including type - i ifn and il-6 induces pro - pathogenic th1 and th17 cells , which are implicated in ms pathogenesis , . on the other hand , pdcs can exert a protective role in ms through the production of type - i ifn . in experimental autoimmune encephalomyelitis ( eae ) , the rodent model of ms , ifn- and ifnar - deficiency exacerbates disease severity , . furthermore , one study suggests that pdcs inhibit pro - pathogenic conventional dcs ( cdcs ) functions in the cns and , consequently locally inhibit encephalitogenic th17 cells . in addition to a controversial local role in the cns , pdcs have also been implicated in the modulation of autoimmune th1 and th17 priming in secondary lymphoid organs ( slos ) during early phases of eae development . it was suggested that pdcs promote th17 priming , whereas we have demonstrated that the selective abrogation of mhcii expression by pdcs leads to increased mog - specific th1 and th17 and impaired treg proliferation . thus , although many questions remain to be elucidated , modulation of pdc functions as an approach to treat ms is currently an axis of intense investigation . we have previously demonstrated that the adoptive transfer of mhcii - sufficient pdcs prior to eae induction significantly dampened disease severity , whereas mchii - deficient pdc had no effect . we found that when transferred during acute eae phase , pdcs led to dramatic disease remission . protection was pdc - dependent , correlated with reduced cns inflammation , and decreased encephalitogenic th1 and th17 cells . our results demonstrated that pdc transfer induced the recruitment of resting endogenous pdcs to the cns via a chemerin dependent mechanism , and confer a tolerogenic environment , which , together with local myelin peptide delivery , inhibits encephalitogenic t cells and results in disease improvement . therefore , modulation of cns inflammation by pdcs in eae mice is an interesting axis of investigation to ameliorate disease clinical outcome . up to date , almost all current therapies in autoimmune diseases are based on the systemic suppression of immune functions and are not curative . our work reinforces and validates the relevance of emerging therapeutic concepts regarding the use of cell therapies for autoimmune diseases . bone marrow ( bm ) chimeric mice all procedures were approved by and performed in accordance with the guidelines of the animal research committee of geneva . for passive eae induction , encephalitogenic cd4 t cells were generated in vitro from ln and spleen cells of 2d2 mice as described . cdcs and pdcs were generated as described from bm of wt for cdcs and wt , mhcii , ubi - gfp , cd45.1 , and ifn- mice for pdcs . for hematopoietic progenitor analysis , bm cells were stained with the following biotinylated lineage markers : anti - cd3 , anti - b220 , anti - cd19 , anti - cd11b , anti - ter119 , anti- ly6c followed by streptavidin fitc conjugated staining . intracellular cytokine stainings were done with the cytofix / cytoperm kit ( bd ) for ifn- and il-17 staining . for ifn- and il-17 staining , sc , ln and spleen cells were cultured in rpmi containing 10% heat - inactivated fetal bovine serum , 50 mm 2-mercaptoethanol , 100 mm sodium pyruvate , and 100 m penicillin / streptomycin at 37 c , 5% co2 . cryopreserved sc , post - fixed with 4% pfa and embedded in oct ( sakura finetek ) , were cut into 10 m - thick sections , and stained using primary antibodies against cd45 and pdca-1 appropriate species - specific alexa555-labelled , cy3- , or cy5-conjugated secondary antibodies ( all from jackson immunoresearch laboratories , inc . ) in order to explore a potential therapeutic role for pdcs in eae , we adoptively transferred in vitro bone marrow ( bm ) derived wt mog35 - 55 loaded pdcs into c57bl/6 mice after disease onset , 12 days after immunization with mog35 - 55 + cfa . strikingly , whereas clinical scores of control non pdc - injected mice continued to rise , those of pdc - injected mice stayed stable for few days post - pdc transfer , and then rapidly decreased , resulting in a significant inhibition of eae ( fig . consistent with an amelioration of clinical scores , as early as four days post - pdc transfer , inflammatory foci were reduced in spinal cord ( sc ) of pdc - transferred mice , compared to control eae mice ( fig . in spite of this , four days after pdc transfer , we did not observe any differences regarding the frequency of cd4 and cd8 t cells infiltrating the sc ( supplementary fig . 2a ) , nor in the expression of vla-4 by t cells ( supplementary fig . consequently , t cell migration from slos to sc was not altered upon pdc transfer . however , the expression of pd-1 , a molecule that delivers negative signals to t cells , was increased on sc - infiltrating cd4 t cells one day after pdc transfer ( fig . remarkably , encephalitogenic th1 ( at day 4 and 9 after pdc transfer ) and th17 frequencies ( at day 9 after pdc transfer ) were substantially decreased in sc of pdc - transferred mice ( fig . this effect appeared to be pdc - specific , since transfer of mog35 - 55-loaded cdcs did not significantly impact eae development ( supplementary fig . in addition , although most of our experiments were done using 510 10 of transferred pdcs , a similarly efficient therapeutical effect was observed by using only 1 10 cells ( supplementary fig . to determine the mechanisms accounting for pdc - mediated eae amelioration , we analysed the body distribution of injected bm - pdcs derived from ubiquitin - gfp mice . 2b , top ) , expressed the pdc - specific marker pdca-1 ( yellow arrows ) and were surrounded by endogenous infiltrating pdcs ( white arrows ) ( fig . while the frequencies of cdcs , macrophages and microglial cells were similar in sc of pdc - transferred and control eae mice ( supplementary fig . 4 ) , we observed a massive ( six - fold ) increase of total pdc frequencies in sc of pdc - transferred eae mice ( fig . injected pdcs ( gfp ) represented only 25% of total sc pdcs ( fig . 2d ) , suggesting that endogenous pdcs were recruited to the sc following exogenous pdc transfer . in agreement , increased endogenous pdc frequencies in sc of pdc - transferred animals did not result from local pdc proliferation ( data not shown ) . therefore , we hypothesized that they would be recruited from the bm , where pdcs originate from . in contrast , we observed a significant increase in lsk ( lineage - negative , sca1 , c - kit ) progenitor 's frequencies in the bm , one - day post - pdc transfer ( supplementary fig . in line with this notion , both cdc and pdc frequencies were significantly increased in ln , 4 days after pdc - transfer ( supplementary fig . altogether , these data show that pdc transfer in eae mice increases hematopoietic cell generation in the bm , leading to enhanced cdc and pdc frequencies in slos , and resulting in the selective recruitment of pdcs to the cns . to test this hypothesis , we induced passive eae by injecting in vitro differentiated mog35 - 55-specific 2d2 cd4 t cell effectors . our data strongly suggests that endogenous pdcs recruited to the cns following pdc transfer are implicated in eae amelioration . to firmly demonstrate that endogenous pdc recruitment to sc was necessary for disease amelioration following exogenous pdc transfer , we used a genetic mouse model to deplete endogenous pdcs . for this purpose , cd45.1 wt pdcs were transferred into bdca-2 dtr eae mice , and mice were injected or not with dt at the same time pdcs were transferred . endogenous pdcs were efficiently depleted in the spleens of dt - treated mice ( not shown ) . 3c , left ) , but as a consequence of bdca-2 dtr pdc depletion , endogenous pdc recruitment to the sc was substantially impaired in dt treated , compared to untreated animals ( fig . in order to identify possible mechanism(s ) responsible for pdc recruitment to cns , we analysed the implication of chemerin / cmklr1 ( chemerin receptor ) , an axis linked to pdc migration in humans . first , and as previously observed , we found that sc of eae mice contained increased chemerin mrna levels , compared to naive sc ( fig . interestingly , the frequency of pdcs expressing cmklr1 was substantially increased in sc as early as one day after pdc transfer , and was sustained four days after transfer ( fig . in contrast , cmklr1 expression remained low and unaltered on sc infiltrating cdcs from both pdc - transferred and control eae mice ( fig . in order to test a possible involvement of the chemerin / cmklr1 axis in the recruitment of endogenous pdcs to the sc following pdc transfer , we treated pdc transferred or control eae mice with a cmklr1 small molecule antagonist , -naphthoyl ethyltrimethylammonium iodide ( -neta ) , known to block cmklr1 cell migration . these results demonstrate that pdc recruitment to sc following pdc transfer is mediated by the chemerin - cmklr1 axis . however , given that this molecule not only interferes with pdc homing but also affects migration of additional key effector cells in eae , such as macrophages and microglial cells ( supplementary fig . 6 ) , we could not use this experimental approach to evaluate the impact of blocking endogenous pdc recruitment to the cns on disease evolution . in order to check whether ifn- was involved in pdc - mediated protection in eae , we transferred ifn- or wt mog35 - 55 loaded pdcs into eae recipient mice . we have previously showed that when transferred prior to eae induction , pdc - mediated protection was mhcii dependent . 5c ) , suggesting a mhcii independent role for injected mog35 - 55 loaded pdcs in regulating cns - inflammation . a requirement for mhcii expression by endogenous pdcs , that are recruited to the sc after pdc injection , was also ruled out , since eae protection after wt pdc transfer was not altered in mice selectively lacking mhcii expression by pdcs ( mtxpiii + iv : wt bm chimeric mice , as described before ) ( supplementary fig . 5d , e ) , again supporting the need of endogenous pdc recruitment to sc for pdc therapeutic effect . in order to investigate the features related to pdc tolerogenic effect , we analysed pdc phenotype in sc of eae mice . conversely , the expression of cd69 by endogenous pdcs was substantially downregulated upon pdc transfer ( fig . to determine whether the lack of pdc activation is involved in the pdc transfer - therapeutic effect , we treated pdcs with cpg - b prior to their injection . cpg - treated pdcs did not induce in situ upregulation of cmklr1 by pdcs ( fig . 6c ) , neither the recruitment of endogenous pdcs to sc of eae mice ( fig . importantly , we further observed that injection of cpg - b activated mog35 - 55 loaded pdcs did not confer any significant disease amelioration , compared to untreated mog35 - 55 loaded pdcs ( fig . altogether , these results demonstrate that steady - state pdc - transfer promotes a pdc - dependent tolerogenic environment in sc of eae mice , resulting in the general suppression of eae pathogenicity and clinical symptoms . in agreement , on the other hand , constitutive or antibody mediated pdc depletion during peak disease correlates with eae exacerbation , . we have previously demonstrated that pdcs can present myelin - derived autoantigens via mhcii to cd4 t cells in an eae context and promote the expansion of suppressive treg cells . in the present work , in order to explore pdc tolerogenic potential in a therapeutic eae setting , we performed adoptive transfer of syngenic pdcs after disease onset . strikingly , pdcs induced a rapid and substantial amelioration of both cns inflammation and eae clinical scores upon transfer into sick mice . transferred pdcs rapidly reached the sc , localized in lesion areas in the cns , and inhibited already primed encephalitogenic mog35 - 55-specific 2d2 cd4 t effector cells , pointing out to a local immunoregulatory role of these cells during eae effector phase . however , we can not rule out a possible systemic impact on immune cells in other organs following pdc transfer , and future studies will determine whether this might be beneficial in other inflammatory diseases . when investigating the cellular compartments in the sc after pdc transfer , we observed a tremendous increase in total pdc frequency , but not of other cell types such as cdcs , macrophages or microglial cells , suggesting a specific recruitment of endogenous pdcs to the sc . importantly , selective depletion of endogenous pdcs completely abrogated disease amelioration following pdc adoptive transfer . thus , the accumulation of endogenous pdcs in the cns of eae mice following pdc transfer accounts for eae protection . interestingly , we observed a significant increase in de novo hsc progenitors ' generation in the bm of pdc transferred compared to control eae mice . in agreement , it has been recently demonstrated that angiopoietin - like 7 , which is produced by pdcs , regulates the expansion and repopulation of human hematopoietic stem and progenitor cells . alternatively , lsk progenitor increase in bm might reflect a physiologic demand for pdc generation , as an indirect consequence of pdc - recruitment to the cns following pdc transfer . newly generated bm - pdcs would then be specifically recruited to the inflamed cns through chemerin / cmklr1 axis , an inflammatory chemotactic factor that is involved in pdc migration . in vivo , chemerin expression correlated with pdc infiltration into peripheral tissues during autoimmune diseases , such as sle . most importantly , chemerin was detected in intralesional cerebrovascular endothelial cells of ms patients , and its receptor is expressed by pdcs . in agreement , we observed an increase in chemerin expression in the sc of eae mice compared to nave mice . furthermore , we found a significant and specific increase in the expression of cmklr1 ( or chem23r ) by pdcs in the sc of pdc - transferred mice . blockade of cmklr1 induced cell migration abrogated pdc recruitment to the sc , demonstrating that following pdc transfer , endogenous pdc homing to inflamed cns is mediated by the chemerin / cmklr1 axis . however , microglia cells and cns - infiltrating myeloid dendritic cells also express cmklr1 , and eae clinical and histological disease was found less severe in cmklr1 deficient mice when compared to wt counterparts . one possibility is that under inflammatory eae condition , activated cns pdcs indeed promote disease pathogenesis , and that in cmklr-1 deficient mice , pdc recruitment to the cns is impaired and mice consequently develop attenuated eae . in our settings of pdc transfer in eae mice , endogenous pdcs would be deflected from pro - pathogenic to pro - tolerogenic functions after downmodulation of their activation state . accordingly , cpg - b activated pdcs did not induce endogenous pdc recruitment to the cns of eae mice , neither did they conferred any significant level of disease amelioration , when compared to steady - state pdcs . notably , expression of cd69 , a marker for activated pdcs , was substantially reduced on endogenous pdcs recruited to sc after pdc transfer . therefore , the combination of several pdc - specific features in the cns : i ) increased pdc frequencies ; ii ) modulation of pdc activation status ; iii ) ability to provide myelin antigen ; iv ) production of a state permissive for tolerance , altogether likely contribute to pdc protective role in eae . mhcii deficient pdcs , were able to induce the same degree of disease inhibition compared to wt pdcs , ruling out a role for mhcii mediated antigen presentation by exogenous pdcs in this protective setting . our data support a local inhibition of encephalitogenic effector t cells in the cns of eae mice after pdc transfer . first , as early as 1 day post - pdc injection , t cells up - regulated the inhibitory molecule pd-1 , and second , after 4 days , we observed reduced frequencies of encephalitogenic th1 and th17 cells in the sc . current available antibody based therapies target various immune cell populations mediating the autoimmune chronic reaction for the treatment of ms , rheumatoid arthritis and other autoimmune diseases . autologous stem cell transplantations are currently tested as immunotherapy for autoimmune diseases , including ms . although visionary , therapies using terminally differentiated cells for the treatment of autoimmune diseases are emerging and are up to date mainly focused on the adoptive transfer of tregs . in ms , indeed , while tregs infused prior eae induction confer protection , their therapeutic value is considerably diminished when infused after disease initiation , . whether tregs acquire suppressive functions and are directly linked to eae protection after pdc - transfer will require further investigation . if these approaches efficiently suppress eae when injections were performed prior disease induction , tolerogenic apcs however impact mainly the priming of encephalitogenic t cells , and no efficacy was consequently proven when effector t cell responses and disease symptoms are already established . these observations make our results even more attractive in the sense that pdc suppressive effect on established eae is not apc dependent , and directly impact disease effector phase in the cns .

atherosclerosis is a main cause of cardiovascular disease ( cvd ) which is a leading factor in determining both morbidity and mortality in developed countries . atherosclerosis is characterized as an inflammatory process which occurs as a result of complex cellular and environmental interactions . atherosclerotic lesion development involves the expression of cytokines and growth factors by infiltrating inflammatory cells such as macrophages and by structural local cells of the vessel wall . a strong emphasis has been placed on the roles of endothelial cells ( ecs ) and smooth muscle cells ( smcs ) of the intima and media , respectively ; however , there is growing evidence to suggest that cells of the adventitia may also play a role in the generation of atherosclerotic plaques . adventitial fibroblasts have been shown to be involved in neointima formation following vascular insult [ 2 , 3 ] and the generation of reactive oxygen species and have the capacity to attract leukocytes and release cytokines , chemokines , and growth factors [ 5 , 6 ] . inflammatory molecules such as mcp-1 , il-6 , vegf , and il-8 have been implicated in atherosclerosis ; however , their regulation and expression by adventitial fibroblasts remain to be fully defined . among the network of molecules known to modulate inflammation in general are several within the gp130 family of cytokines that include interleukin ( il)-6 , il-11 , and il-31 , oncostatin m ( osm ) , leukemia inhibitory factor ( lif ) , ciliary neurotrophic factor ( cntf ) , il-27 , and others . there is some evidence that suggests that gp130 cytokine members osm , and il-6 , may play a role in atherosclerosis . osm and il-6 have been detected within apoe deficient mouse and human atherosclerotic plaques [ 1214 ] . furthermore , stimulation of human endothelial cells in vitro with either osm or il-6 resulted in increased expression of leukocyte adhesion molecules and chemokines [ 15 , 16 ] , which have the potential to promote leukocyte infiltration . in addition , osm can amplify the effects of other inflammatory mediators in other systems ; osm has been shown to synergize with various cytokines such as tnf , il-1 , and with il-17 to induce cartilage breakdown [ 1719 ] . in human airway smooth muscle cells , the synergistic actions of osm with il-1 resulted in increased chemokine , growth factor , and cytokine expression . the effects of osm on aortic smooth muscle cells are only recently emerging , and effects on aortic adventitial fibroblasts are not known . toll - like receptors ( tlr ) are established as important sensing molecules of the innate immune system , and cells that express tlrs can respond with inflammatory signalling pathways . interestingly , tlr-4 has been detected on numerous cell types within atherosclerotic lesions , and its agonists have also been detected in atheromas ( reviewed by den dekker et al . ) . in addition , double knockout apoe / tlr-4 / mice demonstrated a significant reduction in atherosclerosis compared to apoe / mice , and the administration of lps ( a typical tlr-4 ligand ) to the adventitial surface of murine arteries increased atherosclerosis compared to control , implicating the adventitia in lesion progression . however , precise mechanisms are not yet clear , and how tlr systems and gp130 cytokines interact is not known . it is therefore of interest to elucidate the effects of osm on mesenchymal cells of the aorta , including adventitial fibroblasts and smooth muscle cells , in context of gp130 cytokines or other stimuli , in order to delineate potential roles of regulation of structural cell activation in plaque development . we here used in vitro cell culture systems of human aortic cells and showed induction of synergistic responses , cell signalling , and receptor regulation by osm and tlr-4 ligand in aortic adventitial fibroblasts and smooth muscle cells . human aortic adventitial fibroblasts ( haoafs ) and human aortic smooth muscle cells ( haosmcs ) were primary cells purchased from lonza group ltd . ( basel , switzerland ) and were cultured in stromal cell growth medium or smooth muscle growth medium-2 ( lonza group ltd . ) , respectively , according to the manufacturer 's instructions in 5% fbs , at 37c in 5% co2 conditions . cultures were serum deprived in media containing 2% fbs for three hours prior to stimulation and then stimulated with media containing 2% fbs for designated time points with recombinant osm , lif , il-6 , il-31 , or il-11 ( r&d systems , minneapolis , mn ) and/or with lipopolysaccharide ( sigma aldrich corp . , st . lps from escherichia coli 026:b6 ( highly purified , free of other tlr ligands ) was purchased from sigma - aldrich canada ( oakville , on ) . elisas were performed for il-6 , vegf , and il-8 using duoset kits purchased from r&d systems . elisas were performed according to the manufacturer 's instructions . for analysis of cell signalling pathways , haoaf and haosmc cultures were stimulated for 20 minutes , and cell lysates were generated , and immunoblots were performed as published previously . primary antibodies for pstat-1 ( tyr 701 ) , total stat-1 , pstat-3 ( tyr 705 and ser 727 ) , total stat-3 , pstat-5 ( tyr 694 ) , p - p38 ( thr 180/tyr 182 ) , total p38 , p - sapk / jnk ( thr 183/tyr 185 ) , p - nfb p65 ( ser 536 ) , total nfb p65 , pakt ( thr 308 ) , and total akt were purchased from cell signaling technology ( danvers , ma ) . antibodies for p - erk 1/2 ( e4 ) , total erk 1/2 ( k23 ) , total jnk ( d-2 ) , total stat-5 ( c-17 ) , and total actin ( i-19 ) were purchased from santa cruz biotechnology inc . mouse anti - goat igg - hrp goat anti - rabbit igg - hrp , or goat anti - mouse igg - hrp were used as secondary antibodies and were purchased from santa cruz biotechnology inc . relative protein levels were determined by densitometry using imagej software ( nih , bethesda , md ) . all western blot densitometry analyses were normalized to a loading control and compared relatively to untreated / control samples . pure link rna mini kit ( life technologies , carlsbad , ca ) was used according to the manufacturer 's instructions to isolate and purify the rna . rna was reversed transcribed using superscript ii ( life technologies ) , and 25 ng of the resultant cdna was used to measure expression of il-6 , il-8 , vegf , mcp-1 , tlr-4 , cd14 , osmr- , gp130 , and -actin using taqman predeveloped assay reagents purchased from ambion ( life technologies ) . mrna expression levels for each sample were performed in triplicate and normalized to -actin levels in that sample and expressed relative to control ( media alone ) values . taqman was performed using the 7900ht fast real - time pcr system ( life technologies ) . all statistical tests were performed using graphpad prism 5 software ( graphpad software inc . , san diego , ca ) . a p value less than 0.05 was considered statistically significant . for the elisa data , two - way analysis of variance ( anova ) was used to determine significance between lps alone and lps combined with one of the gp130 cytokines . for the mrna data , human aortic adventitial fibroblasts were stimulated with the tlr-4 ligand lps alone , gp130 cytokines alone , or combinations as indicated ( figure 1 ) to determine responses in vitro . mcp-1 levels were measured in the supernatants by elisa , and significant increases ( to over 5000 pg / ml ) were detected upon stimulation with lps or osm alone ( p < 0.0001 ) , but not with lif , il-31 , or il-6 compared to control levels ( figures 1(a ) and 1(b ) ) . upon stimulation with the combination of lps and osm , there was a synergistic induction of mcp-1 ( to over 15 000 pg / ml ) with the concentrations of 10 and 100 ng / ml lps ( figure 1(a ) ) , which was not observed with the other gp130 cytokines tested ( figures 1(b ) and 2 ) . in contrast , lif , il-31 , il-11 , and il-6 showed a trend to suppress the lps - induced mcp-1 ( figure 1 ) , but this was consistently observed with lif and il-11 ( and not il-31 or il-6 ) and only at 10 ng / ml ( but not other concentrations ) of lps ( figure 2 ) . collectively , the observations emphasize the stimulatory effect of osm and not other gp130 cytokines on mcp-1 expression . haoafs responded to osm or lps alone with increases in il-6 levels detected ( figures 1(c ) and 1(d ) ) in the supernatants after 18 hours ( p < 0.01 versus control ) . furthermore , osm synergized with lps in terms of il-6 induction when the two were used in combination to stimulate haoaf cells , which was evident at lps concentrations as low as 0.1 and 1 ng / ml . il-6 levels were lower in response to other gp130 cytokines tested ( figures 1(d ) and 2 ) . in a similar trend , stimulation of haoafs with osm or lps alone was able to induce significantly increased ( p < 0.0001 ) vegf levels in the supernatants compared to control ( figures 1(e ) and 1(f ) ) , and when lps and osm were combined , vegf levels in haoaf supernatants were enhanced compared to either treatment alone . combinations of lps with lif , il-31 , il-11 , or il-6 did not have similar effects ( figures 1(f ) and 3 ) . il-8 levels were also measured by elisa in haoaf supernatants and were significantly elevated in response to 1 , 10 , or 100 ng / ml lps compared to control ( p < 0.0001 ) ( figure 1(g ) ) . stimulation with osm alone did not cause an increase in detected il-8 above control levels ( figure 1(h ) ) . moreover , when combined with lps , osm was able to significantly decrease the il-8 levels that were detected in haoafs supernatants compared to lps alone ( p < 0.0001 ) ( figure 1(g ) ) . effects of other gp130 cytokine that were tested were not statistically significant ( figures 1(h ) and 3 ) . western blots were generated in order to determine whether there was amplification of intracellular signaling cascades with the combined lps and osm stimulation compared to either stimulation alone ( figure 4(a ) shows the images , and figure 4(b ) shows the densitometry analysis ) . at 20 minutes of stimulation , p - tyr - stat-1 , p - tyr - stat-3 and p - ser - stat-3 , p - tyr - stat-5 , pjnk , perk , p - p38 , and p - akt showed increased signal compared to control upon stimulation with osm . when the two stimuli were combined , 20 minute signals included similar alterations in of all of these pathways , and a further elevation of p - p38 was evident over that in response to either osm or lps alone . we next examined aortic smooth muscle cells to assess whether they respond in a similar fashion to aortic fibroblasts in vitro . human aortic smooth muscle cell cultures ( haosmcs ) were thus stimulated with lps and/or gp130 cytokines in a similar fashion to the experiments with haoaf . the combined treatment with lps and osm resulted in synergistically elevated levels of mcp-1 detected in the supernatants compared to either treatment alone ( figure 5(a ) ) . this effect was unique to cells stimulated with osm , compared to when lps was combined with other gp130 cytokines ( figure 5(b ) ) . upon examining il-6 levels , osm alone was able to induce the greatest levels of il-6 compared to the other gp130 cytokines tested ( p < 0.0001 ) ( figure 5(d ) ) . osm synergized with lps at doses of 1 , 10 , and 100 ng / ml lps in il-6 induction , when the two stimuli were used in combination ( figure 5(c ) ) , resulting in detection of approximately 3-fold elevation over levels induced by either agent alone ( figure 5(d ) ) . while lps alone had a small but significant effect on vegf levels in haosmc supernatants compared to control levels ( p < 0.001 ) , osm alone induced increased levels of vegf in a dose dependent manner ( figure 5(e ) ) . in addition , there was a synergistic increase in vegf levels detected upon stimulation with the combination of lps and ( either 0.5 or 5 ng / ml ) osm . lps alone induces significant levels of il-8 in haosmc supernatants ( 0.1 ng / ml lps p < 0.01 , 1 , 10 and 100 ng / ml lps p < 0.0001 versus control ) ( figure 5 ( g ) ) , while osm alone had no effect on detectable levels of il-8 ( figure 5 ( h ) ) . osm was able to inhibit the il-8 levels that were induced by lps when the two stimuli were combined ( p < 0.0001 ) ( figures 5(g ) and 5(h ) ) . the steady state mrna levels in haoafs and haosmcs were also analyzed following stimulation in vitro for 6 hours . the combined treatment of lps and osm resulted in augmented steady state mcp-1 mrna levels compared to either treatment alone in haoafs and haosmcs ( figures 6(a ) and 6(e ) ) . as the mcp-1 mrna levels did , the elevation of il-6 mrna also correlated with the data obtained by elisa . in haoafs and haosmcs , lps or osm alone was able to induce an increase in the steady state il-6 mrna levels compared to control , and these levels were further increased upon stimulation with the combination of lps and osm ( figures 6(b ) and 6(f ) ) . in both cell types , osm alone was able to induce a greater fold induction of vegf mrna than lps alone , and the combined lps and osm treatment resulted in still further elevation of vegf mrna steady state levels ( figures 6(c ) and 6(g ) ) . as was observed with the il-8 protein levels , lps was able to induce an increase in the steady state il-8 mrna that was detected compared to control levels in both haoafs and haosmcs . when lps was combined with osm , there was a reduction of the il-8 mrna levels detected ( figures 6(d ) and 6(h ) ) . to compare the cell signaling of osm to that of other gp130 cells in haoaf and haosmc , we assessed stat-3 activation by western blots as shown in figure 7(a ) . only osm stimulation resulted in elevated p - stat3 levels when the cytokines were used at 5 ng / ml concentrations . when haoafs were assessed for mrna levels for mcp-1 at the 6-hour time point ( figure 7(b ) ) , osm elevated mcp-1 mrna alone and further in combination with lps , as previously observed in figure 6 ; however , lif , il-11 , il-31 , or il-6 did not . to explore additional mechanisms by which synergistic responses may occur in these cell types in the haoafs and haosmcs upon stimulation with lps and osm , expression of receptors for lps and osm was assessed after 5 hours of stimulation . for components of the lps receptor , tlr-4 and cd14 steady state mrna levels were measured . lps stimulation caused an approximately 2-fold induction of tlr-4 expression in both haoafs and haosmcs , while osm had no effect compared to control ( figures 8(a ) and 8(e ) ) . when cd14 mrna levels were measured , 5 ng / ml osm was able to induce a 2-fold increase in steady state levels compared to control in haoaf cells ( figure 8(b ) ) and a 1.6-fold increase in haosmcs ( figure 8(f ) ) . to determine if lps modulated the expression of osm receptor subunits , mrna levels for the osmr chain and gp130 chain were assessed . lps induced approximately 2-fold increases in steady state mrna levels of both the osmr and gp130 receptor subunits in haoaf ( figures 8(c ) and 8(d ) ) where haosmcs were less responsive to lps in this regard ( figures 8(g ) and 8(h ) ) . osm was able to induce increases ( greater than 2 fold ) in osmr chain mrnas in both cell types . the principal observations made in this study support novel roles of oncostatin m in regulation of inflammatory responses in vessel wall cells and thus atherosclerotic plaques . osm , but not other gp130 cytokines , induced responses itself and synergized with lps in regulation of mcp-1 , il-6 , and vegf expression in haoaf ( figures 13 ) . we also observed similar regulation of mcp-1 , il-6 and vegf in aortic smooth muscle cells ( figure 5 ) , suggesting that simultaneous presence of lps and osm engages these cells to potentially contribute to the chemokine and growth factor elevations seen in atherosclerotic plaques . although smc activation clearly participates in atherosclerosis , in our in vitro studies here the levels of mcp-1 , il-6 , vegf , and il-8 protein detected in the supernatants of haoafs were similar or higher on a cell / cell basis than those detected in haosmc supernatants . in addition , lps induced the expression of receptor chains osmr and gp130 , while osm induced lps receptor components tlr4 and cd14 in adventitial fibroblasts ( figure 8) . the interplay of tlr - ligands , osm , and osm receptors ( osmr ) represent activities not previously described . fibroblast activation occurs in numerous chronic inflammatory diseases , and there is accumulating evidence implicating adventitial fibroblasts in vascular inflammation . xu et al . showed that adventitial fibroblasts were among the first cells to proliferate and express mcp-1 in aortas of atherosclerotic apoe knockout mice . . demonstrated that il-6 and mcp-1 were present in the aortic adventitia in close proximity to fibroblasts upon subcutaneous infusion of mice with angiotensin ii , which resulted in macrophage recruitment , adventitial growth , and aortic dissections . vink et al . have shown that haoafs express tlr-4 and can respond to lps at the mrna level . the data presented here confirms these results with respect to the expression of mcp-1 , il-6 , and il-8 but further demonstrates expression at the protein level and documents synergy with osm stimulation . the demonstration that mcp-1 and macrophages were present in atherosclerotic plaques and that mcp-1 causes monocyte / macrophage chemoattraction and extravasation has implicated mcp-1 in the atherosclerotic process . mice lacking both the ldl receptor and the mcp-1 genes were found to be protected from atherosclerosis compared to ldl receptor knockout mice . given the functions of mcp-1 , the increased expression by adventitial fibroblasts / smc as a result of a tlr-4 ligand and osm in the arterial wall could influence atherosclerotic plaque development . the role of il-6 in atherosclerosis is less well defined than that of mcp-1 ; however , there is evidence that il-6 can also contribute to vascular inflammation in mouse models . il-6 has been shown to increase the expression of leukocyte adhesion molecules and chemokines by endothelial cells , which could facilitate leukocyte entry into the vessel wall , and thus , synergistic induction of il-6 by lps and osm could also contribute to arterial lesion development . our observations that il-6 did not regulate haoaf or haosmc in our system in vitro could be explained by a lack of cellular il-6r expression by these particular cell types . as shown in other systems , the soluble form ( sil-6r ) , as long as in sufficient concentrations , can also enable responses of smooth muscle cells to il-6 in vitro . whether sil-6r can enable responses by haoaf , or is present in atherosclerotic lesions at sufficient amounts , needs further study . vegf , another factor implicated in the development of atherosclerosis , has been detected in atherosclerotic , but not healthy arteries , and localized to macrophages , ecs , smcs , and within microvessels of advanced lesions . in addition , atherosclerotic mice administered low doses of vegf presented with larger lesions containing increased macrophage infiltration were compared to control mice . our findings that lps and osm synergized to enhance vegf levels in the supernatants of haoafs and haosmcs suggest another mechanism by which osm or lps could impact inflammation and remodelling in the vascular wall by modulating local levels of vegf . il-8 is a potent neutrophil chemoattractant , and there is support for the participation of neutrophils in atherosclerosis . our findings that lps - induced il-8 was inhibited by osm ( figures 1 , 5 , and 6 ) were in contrast to the effect of osm on induction of mcp-1 , il-6 , and vegf , indicating selective regulation of genes by osm . in other systems , osm has been shown to inhibit il-8 expression in response to il-1 , where human synovial and lung fibroblasts , as well as peritoneal mesothelial cells , stimulated with il-1 showed reduced il-8 expression upon costimulation with osm but enhanced il-6 and mcp-1 expression [ 31 , 32 ] . the mechanism is not clear but may involve transcriptional factors that regulate the il-8 promoter differently from promoters of mcp-1 or other chemokines . osm does induce stat-6 activation in lung fibroblasts , and stat6 has been shown to negatively regulate the il-8 promoter in tnf - induced keratinocytes . however , whether osm induces stat-6 in haoaf or haosmc has ( to our knowledge ) not yet been tested . despite such inhibitory effects on il-8 , osm can induce the expression of other neutrophil cxc chemokines including ena-78 , gro , and , and induce neutrophil chemotaxis across ec monolayers . thus , osm may participate in neutrophil accumulation through regulation of such other neutrophil chemoattractants . analysis of activation of stat3 showed that only osm , and not lif , il-31 , il-11 , nor il-6 stimulation , resulted in elevated pstat3 signal , with or without added lps , in either the adventitial fibroblast or the aortic smooth muscle cell cultures ( figure 7(a ) ) . furthermore , only osm augmented haoaf mrna levels of mcp-1 ( figure 7(b ) ) . thus , the activation of stat3 is associated with selective effects by osm and not other gp130 cytokines in these cells . although osm can engage both the lifr ( gp130:lifr- complex ) and the specific osmr ( gp130:osmr- complex ) in human cells , the lack of comparable function by lif in this system indicates that osm is acting through the osmr in both haoafs and haosmcs . the combination of osm and lps engaged multiple cell signalling pathways in haoafs ( figure 4 ) , and coactivation of various combinations of pathways could potentiate rna transcription or stability mechanisms in regulation of target genes mcp-1 , il-6 , or vegf . at a short term of stimulation ( 20 minutes ) , we did observe enhanced phosphorylation of p38 compared to either lps or osm treatment alone , and the activation of p38 has been shown to be necessary for maximal mcp-1 , vegf , and il-6 expression in other systems . in addition , shc , which can initiate mapk signaling , has been shown to be activated by signaling through the osmr chain , while not being activated by signaling through the receptors of other the gp130 cytokines tested . however , definitive proof as a key mechanism would need further study , for example , with pharmacological or sirna inhibitors , as our observations associate stat3 and augmented p38 activation with synergistic effects but do not define molecular mechanism . at a later time point ( 6 hr ) , osm induced increases in cd14 mrna ( figure 7 ) . since augmented cd14 has been shown to enhance lps signalling , this may indicate that osm can sensitize cells to subsequent tlr - ligand stimulation . indeed in airway smooth muscle cells , osm has been shown to synergize with il-1 and il-4 by inducing their receptor components [ 20 , 39 ] . in parallel , tlr - ligand induced both the osmr and gp130 chains at the mrna level ( figure 7 ) , and if the mrna elevation translates into cell surface expression , this may implicate further sensitization of haoaf to osm ligand present extracellularly . however , receptor change associations with the synergy require further study using molecular inhibitors to determine if they cause the altered responses . alternatively , the role of autocrine stimulation initiated by lps may contribute to the mechanisms of synergy with osm . synergistic responses to osm in combination with il-1 are evident in vitro in a variety of cells including lung fibroblasts , airway smooth muscle cells , and chondrocytes . faffe et al . showed that osm upregulates the il-1 receptor in airway smooth muscle cells . further analysis would be required to determine if vascular smc or adventitial fibroblasts respond to lps with either il-1 or its receptor expression , to test this possibility . several tlr-4 ligands have been discovered in atherosclerotic lesions including danger associated molecular patterns such as fibronectin - eda and mm - ldl , as well as pathogen associated molecular patterns such as lps from gram - negative bacteria and chlamydia pneumoniae hsp-60 , and it has been hypothesized that tlr-4 activation could be a component of atherosclerotic lesion development . in addition , activation of tlr-4 has been shown to induce osm expression in monocytes , t cells , and dendritic cells , and since osm is detected in atherosclerotic plaques , such tlr activation may contribute to osm expression locally in lesions . osm is detected by immunolocalization in apoe/ mouse atherosclerotic lesions strongly in the intima earlier ( 20 weeks ) , but later ( 30 , 54 weeks ) osm was detected more broadly in the lesion and vessel wall . thus , osm regulation of smc and adventitial fibroblasts may be most prominent later in the development of atherosclerotic plaque , as its levels increase over time across the vessel . our study suggests that its presence would activate these cells to a much higher degree that other gp130 cytokines and thus contribute uniquely to pathogenic mechanisms . we suggest that exogenous or endogenous tlr - ligands , in tandem with osm function , represent novel activities separate from other gp130 cytokines and contribute to the severity of atherosclerotic plaque development .

accumulating evidence suggests that adventitial fibroblasts play a significant role in contributing to inflammation of the arterial wall and pathogenesis of atherosclerosis . the effects of gp130 cytokines on these cells ( including oncostatin m-[osm ] and il-6 ) , some of which have been implicated in atherosclerosis , are currently unknown . experiments were performed to determine whether gp130 cytokines regulate human aortic adventitial fibroblasts ( haoafs ) or smooth muscle cells ( haosmcs ) alone or in context of tlr-4 ligands ( also implicated in atherosclerosis ) . haoafs and haosmcs were stimulated with lps and/or one of osm , il-6 , il-11 , il-31 , or lif . elisas performed on cell supernatants showed that stimulation with osm alone caused increased mcp-1 , il-6 , and vegf levels . when combined , lps and osm synergized to increase mcp-1 , il-6 , vegf protein , and mrna expression as assessed by qrt - pcr , in both haoafs and haosmcs , while lps - induced il-8 levels were reduced . such effects were not observed with other gp130 cytokines . signalling pathways including stats , mapkinases , and nfb were activated , and lps induced steady state mrna levels of the osm receptor chains osmr and gp130 . the results suggest that osm is able to synergize with tlr-4 ligands to induce proinflammatory responses by haoafs and haosmcs , supporting the notion that osm regulation of these cells contributes to the pathogenesis of atherosclerosis .

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

a strong emphasis has been placed on the roles of endothelial cells ( ecs ) and smooth muscle cells ( smcs ) of the intima and media , respectively ; however , there is growing evidence to suggest that cells of the adventitia may also play a role in the generation of atherosclerotic plaques . adventitial fibroblasts have been shown to be involved in neointima formation following vascular insult [ 2 , 3 ] and the generation of reactive oxygen species and have the capacity to attract leukocytes and release cytokines , chemokines , and growth factors [ 5 , 6 ] . inflammatory molecules such as mcp-1 , il-6 , vegf , and il-8 have been implicated in atherosclerosis ; however , their regulation and expression by adventitial fibroblasts remain to be fully defined . among the network of molecules known to modulate inflammation in general are several within the gp130 family of cytokines that include interleukin ( il)-6 , il-11 , and il-31 , oncostatin m ( osm ) , leukemia inhibitory factor ( lif ) , ciliary neurotrophic factor ( cntf ) , il-27 , and others . there is some evidence that suggests that gp130 cytokine members osm , and il-6 , may play a role in atherosclerosis . in addition , osm can amplify the effects of other inflammatory mediators in other systems ; osm has been shown to synergize with various cytokines such as tnf , il-1 , and with il-17 to induce cartilage breakdown [ 1719 ] . in human airway smooth muscle cells , the synergistic actions of osm with il-1 resulted in increased chemokine , growth factor , and cytokine expression . the effects of osm on aortic smooth muscle cells are only recently emerging , and effects on aortic adventitial fibroblasts are not known . toll - like receptors ( tlr ) are established as important sensing molecules of the innate immune system , and cells that express tlrs can respond with inflammatory signalling pathways . in addition , double knockout apoe / tlr-4 / mice demonstrated a significant reduction in atherosclerosis compared to apoe / mice , and the administration of lps ( a typical tlr-4 ligand ) to the adventitial surface of murine arteries increased atherosclerosis compared to control , implicating the adventitia in lesion progression . however , precise mechanisms are not yet clear , and how tlr systems and gp130 cytokines interact is not known . it is therefore of interest to elucidate the effects of osm on mesenchymal cells of the aorta , including adventitial fibroblasts and smooth muscle cells , in context of gp130 cytokines or other stimuli , in order to delineate potential roles of regulation of structural cell activation in plaque development . we here used in vitro cell culture systems of human aortic cells and showed induction of synergistic responses , cell signalling , and receptor regulation by osm and tlr-4 ligand in aortic adventitial fibroblasts and smooth muscle cells . human aortic adventitial fibroblasts ( haoafs ) and human aortic smooth muscle cells ( haosmcs ) were primary cells purchased from lonza group ltd . cultures were serum deprived in media containing 2% fbs for three hours prior to stimulation and then stimulated with media containing 2% fbs for designated time points with recombinant osm , lif , il-6 , il-31 , or il-11 ( r&d systems , minneapolis , mn ) and/or with lipopolysaccharide ( sigma aldrich corp . elisas were performed for il-6 , vegf , and il-8 using duoset kits purchased from r&d systems . for analysis of cell signalling pathways , haoaf and haosmc cultures were stimulated for 20 minutes , and cell lysates were generated , and immunoblots were performed as published previously . rna was reversed transcribed using superscript ii ( life technologies ) , and 25 ng of the resultant cdna was used to measure expression of il-6 , il-8 , vegf , mcp-1 , tlr-4 , cd14 , osmr- , gp130 , and -actin using taqman predeveloped assay reagents purchased from ambion ( life technologies ) . for the elisa data , two - way analysis of variance ( anova ) was used to determine significance between lps alone and lps combined with one of the gp130 cytokines . for the mrna data , human aortic adventitial fibroblasts were stimulated with the tlr-4 ligand lps alone , gp130 cytokines alone , or combinations as indicated ( figure 1 ) to determine responses in vitro . mcp-1 levels were measured in the supernatants by elisa , and significant increases ( to over 5000 pg / ml ) were detected upon stimulation with lps or osm alone ( p < 0.0001 ) , but not with lif , il-31 , or il-6 compared to control levels ( figures 1(a ) and 1(b ) ) . upon stimulation with the combination of lps and osm , there was a synergistic induction of mcp-1 ( to over 15 000 pg / ml ) with the concentrations of 10 and 100 ng / ml lps ( figure 1(a ) ) , which was not observed with the other gp130 cytokines tested ( figures 1(b ) and 2 ) . in contrast , lif , il-31 , il-11 , and il-6 showed a trend to suppress the lps - induced mcp-1 ( figure 1 ) , but this was consistently observed with lif and il-11 ( and not il-31 or il-6 ) and only at 10 ng / ml ( but not other concentrations ) of lps ( figure 2 ) . collectively , the observations emphasize the stimulatory effect of osm and not other gp130 cytokines on mcp-1 expression . in a similar trend , stimulation of haoafs with osm or lps alone was able to induce significantly increased ( p < 0.0001 ) vegf levels in the supernatants compared to control ( figures 1(e ) and 1(f ) ) , and when lps and osm were combined , vegf levels in haoaf supernatants were enhanced compared to either treatment alone . combinations of lps with lif , il-31 , il-11 , or il-6 did not have similar effects ( figures 1(f ) and 3 ) . il-8 levels were also measured by elisa in haoaf supernatants and were significantly elevated in response to 1 , 10 , or 100 ng / ml lps compared to control ( p < 0.0001 ) ( figure 1(g ) ) . moreover , when combined with lps , osm was able to significantly decrease the il-8 levels that were detected in haoafs supernatants compared to lps alone ( p < 0.0001 ) ( figure 1(g ) ) . western blots were generated in order to determine whether there was amplification of intracellular signaling cascades with the combined lps and osm stimulation compared to either stimulation alone ( figure 4(a ) shows the images , and figure 4(b ) shows the densitometry analysis ) . at 20 minutes of stimulation , p - tyr - stat-1 , p - tyr - stat-3 and p - ser - stat-3 , p - tyr - stat-5 , pjnk , perk , p - p38 , and p - akt showed increased signal compared to control upon stimulation with osm . when the two stimuli were combined , 20 minute signals included similar alterations in of all of these pathways , and a further elevation of p - p38 was evident over that in response to either osm or lps alone . human aortic smooth muscle cell cultures ( haosmcs ) were thus stimulated with lps and/or gp130 cytokines in a similar fashion to the experiments with haoaf . the combined treatment with lps and osm resulted in synergistically elevated levels of mcp-1 detected in the supernatants compared to either treatment alone ( figure 5(a ) ) . this effect was unique to cells stimulated with osm , compared to when lps was combined with other gp130 cytokines ( figure 5(b ) ) . upon examining il-6 levels , osm alone was able to induce the greatest levels of il-6 compared to the other gp130 cytokines tested ( p < 0.0001 ) ( figure 5(d ) ) . osm synergized with lps at doses of 1 , 10 , and 100 ng / ml lps in il-6 induction , when the two stimuli were used in combination ( figure 5(c ) ) , resulting in detection of approximately 3-fold elevation over levels induced by either agent alone ( figure 5(d ) ) . while lps alone had a small but significant effect on vegf levels in haosmc supernatants compared to control levels ( p < 0.001 ) , osm alone induced increased levels of vegf in a dose dependent manner ( figure 5(e ) ) . lps alone induces significant levels of il-8 in haosmc supernatants ( 0.1 ng / ml lps p < 0.01 , 1 , 10 and 100 ng / ml lps p < 0.0001 versus control ) ( figure 5 ( g ) ) , while osm alone had no effect on detectable levels of il-8 ( figure 5 ( h ) ) . the steady state mrna levels in haoafs and haosmcs were also analyzed following stimulation in vitro for 6 hours . the combined treatment of lps and osm resulted in augmented steady state mcp-1 mrna levels compared to either treatment alone in haoafs and haosmcs ( figures 6(a ) and 6(e ) ) . in haoafs and haosmcs , lps or osm alone was able to induce an increase in the steady state il-6 mrna levels compared to control , and these levels were further increased upon stimulation with the combination of lps and osm ( figures 6(b ) and 6(f ) ) . in both cell types , osm alone was able to induce a greater fold induction of vegf mrna than lps alone , and the combined lps and osm treatment resulted in still further elevation of vegf mrna steady state levels ( figures 6(c ) and 6(g ) ) . as was observed with the il-8 protein levels , lps was able to induce an increase in the steady state il-8 mrna that was detected compared to control levels in both haoafs and haosmcs . when lps was combined with osm , there was a reduction of the il-8 mrna levels detected ( figures 6(d ) and 6(h ) ) . when haoafs were assessed for mrna levels for mcp-1 at the 6-hour time point ( figure 7(b ) ) , osm elevated mcp-1 mrna alone and further in combination with lps , as previously observed in figure 6 ; however , lif , il-11 , il-31 , or il-6 did not . to explore additional mechanisms by which synergistic responses may occur in these cell types in the haoafs and haosmcs upon stimulation with lps and osm , expression of receptors for lps and osm was assessed after 5 hours of stimulation . for components of the lps receptor , tlr-4 and cd14 steady state mrna levels were measured . lps stimulation caused an approximately 2-fold induction of tlr-4 expression in both haoafs and haosmcs , while osm had no effect compared to control ( figures 8(a ) and 8(e ) ) . when cd14 mrna levels were measured , 5 ng / ml osm was able to induce a 2-fold increase in steady state levels compared to control in haoaf cells ( figure 8(b ) ) and a 1.6-fold increase in haosmcs ( figure 8(f ) ) . to determine if lps modulated the expression of osm receptor subunits , mrna levels for the osmr chain and gp130 chain were assessed . lps induced approximately 2-fold increases in steady state mrna levels of both the osmr and gp130 receptor subunits in haoaf ( figures 8(c ) and 8(d ) ) where haosmcs were less responsive to lps in this regard ( figures 8(g ) and 8(h ) ) . osm was able to induce increases ( greater than 2 fold ) in osmr chain mrnas in both cell types . osm , but not other gp130 cytokines , induced responses itself and synergized with lps in regulation of mcp-1 , il-6 , and vegf expression in haoaf ( figures 13 ) . we also observed similar regulation of mcp-1 , il-6 and vegf in aortic smooth muscle cells ( figure 5 ) , suggesting that simultaneous presence of lps and osm engages these cells to potentially contribute to the chemokine and growth factor elevations seen in atherosclerotic plaques . although smc activation clearly participates in atherosclerosis , in our in vitro studies here the levels of mcp-1 , il-6 , vegf , and il-8 protein detected in the supernatants of haoafs were similar or higher on a cell / cell basis than those detected in haosmc supernatants . in addition , lps induced the expression of receptor chains osmr and gp130 , while osm induced lps receptor components tlr4 and cd14 in adventitial fibroblasts ( figure 8) . the interplay of tlr - ligands , osm , and osm receptors ( osmr ) represent activities not previously described . showed that adventitial fibroblasts were among the first cells to proliferate and express mcp-1 in aortas of atherosclerotic apoe knockout mice . the data presented here confirms these results with respect to the expression of mcp-1 , il-6 , and il-8 but further demonstrates expression at the protein level and documents synergy with osm stimulation . given the functions of mcp-1 , the increased expression by adventitial fibroblasts / smc as a result of a tlr-4 ligand and osm in the arterial wall could influence atherosclerotic plaque development . il-6 has been shown to increase the expression of leukocyte adhesion molecules and chemokines by endothelial cells , which could facilitate leukocyte entry into the vessel wall , and thus , synergistic induction of il-6 by lps and osm could also contribute to arterial lesion development . as shown in other systems , the soluble form ( sil-6r ) , as long as in sufficient concentrations , can also enable responses of smooth muscle cells to il-6 in vitro . vegf , another factor implicated in the development of atherosclerosis , has been detected in atherosclerotic , but not healthy arteries , and localized to macrophages , ecs , smcs , and within microvessels of advanced lesions . our findings that lps and osm synergized to enhance vegf levels in the supernatants of haoafs and haosmcs suggest another mechanism by which osm or lps could impact inflammation and remodelling in the vascular wall by modulating local levels of vegf . our findings that lps - induced il-8 was inhibited by osm ( figures 1 , 5 , and 6 ) were in contrast to the effect of osm on induction of mcp-1 , il-6 , and vegf , indicating selective regulation of genes by osm . analysis of activation of stat3 showed that only osm , and not lif , il-31 , il-11 , nor il-6 stimulation , resulted in elevated pstat3 signal , with or without added lps , in either the adventitial fibroblast or the aortic smooth muscle cell cultures ( figure 7(a ) ) . thus , the activation of stat3 is associated with selective effects by osm and not other gp130 cytokines in these cells . although osm can engage both the lifr ( gp130:lifr- complex ) and the specific osmr ( gp130:osmr- complex ) in human cells , the lack of comparable function by lif in this system indicates that osm is acting through the osmr in both haoafs and haosmcs . the combination of osm and lps engaged multiple cell signalling pathways in haoafs ( figure 4 ) , and coactivation of various combinations of pathways could potentiate rna transcription or stability mechanisms in regulation of target genes mcp-1 , il-6 , or vegf . at a short term of stimulation ( 20 minutes ) , we did observe enhanced phosphorylation of p38 compared to either lps or osm treatment alone , and the activation of p38 has been shown to be necessary for maximal mcp-1 , vegf , and il-6 expression in other systems . indeed in airway smooth muscle cells , osm has been shown to synergize with il-1 and il-4 by inducing their receptor components [ 20 , 39 ] . in parallel , tlr - ligand induced both the osmr and gp130 chains at the mrna level ( figure 7 ) , and if the mrna elevation translates into cell surface expression , this may implicate further sensitization of haoaf to osm ligand present extracellularly . synergistic responses to osm in combination with il-1 are evident in vitro in a variety of cells including lung fibroblasts , airway smooth muscle cells , and chondrocytes . showed that osm upregulates the il-1 receptor in airway smooth muscle cells . several tlr-4 ligands have been discovered in atherosclerotic lesions including danger associated molecular patterns such as fibronectin - eda and mm - ldl , as well as pathogen associated molecular patterns such as lps from gram - negative bacteria and chlamydia pneumoniae hsp-60 , and it has been hypothesized that tlr-4 activation could be a component of atherosclerotic lesion development . in addition , activation of tlr-4 has been shown to induce osm expression in monocytes , t cells , and dendritic cells , and since osm is detected in atherosclerotic plaques , such tlr activation may contribute to osm expression locally in lesions . thus , osm regulation of smc and adventitial fibroblasts may be most prominent later in the development of atherosclerotic plaque , as its levels increase over time across the vessel . our study suggests that its presence would activate these cells to a much higher degree that other gp130 cytokines and thus contribute uniquely to pathogenic mechanisms . we suggest that exogenous or endogenous tlr - ligands , in tandem with osm function , represent novel activities separate from other gp130 cytokines and contribute to the severity of atherosclerotic plaque development .

autism is a complex developmental disorder which has lifelong effects on several aspects of an individual . although , the autism spectrum disorder ( asd ) is known to be neurogenetic in origin , its diagnosis is primarily based on behavioral and clinical signs and symptoms . according to diagnostic and statistical manual of mental disorders , 4 edition , text - revision ( dsm - iv - tr ) , and international classification of diseases ( icd ) , there are three main diagnostic criteria for autistic disorders : impairments in social interaction , impairments in communication and language and restricted , stereotyped behaviors , interests and activities . regarding the neurologic and genetic basis of autism , however , so far regularly standard diagnostic tests are based on psychological and behavioral assessments such as autism diagnostic interview - revised ( adi - r ) , autism diagnostic observation schedule ( ados - g ) and childhood autism rating scale ( cars ) . recently , autism studies have shown an increased interest in examining the effect of different developmental , educational and behavioral interventions on children with asd after their diagnosis is confirmed by diagnostic instruments . several researchers have administered longitudinal studies to monitor the long lasting improvement of autistic children . as an example glen et al evaluated outcomes including cognitive , language , adaptive , social and academic measures after 4 years of intensive behavioral treatment . they found that about half of their autistic children achieved average post treatment scores and succeeded in regular education classrooms by showing rapid learning . however , till recent , to choose a valid and proper measure assessing the changes and progress in autistic populations is a major controversial issue . for years , there was no specific measure while instead , measures such as ados , adi - r or cars have being used to examine the symptoms , evaluate changes and improvement in response to different interventions . although these measures display overall stability over time , they are primarily designed as diagnostic tools but not sensitive enough to examine symptom severity and also intra - subject changes . moreover , the assessment tools provided for typically developing ( td ) children have been used to evaluate autistic individuals . however , these measures are not often suitable for autistic children since they commonly show a too far different developmental pattern compared to their td peers . another problem in assessing long - term changes is the lack of an instrument which can evaluate autism severity along with developmental deficits . although , some of them such as vineland adaptive behavior scales ( vabs ) as an informant based measure do cover a wide age range , it is not appropriate for comparing autistic with normative data . hence this schedule focuses mainly on developmental profile which in autism is very delayed and deviant . increasing number of children classified as asd over the past decade , alarms a vital need for early life interventions . hence , in order to establish a reliable baseline for tracking the trajectory of early treatments , sensitive monitoring tools are becoming more mandatory . however , given several constraints of families , schools and service providers of individuals with asd , it is important to provide symptom severity profile in a time - efficient , economic and practical way . autism treatment evaluation checklist ( atec ) is a one paged checklist designed to be completed by parents , teachers or caretakers and is a simple but effective tool to assess the severity of symptoms as well as developmental aspects of autism . in other words atec also fulfills the need for a valid , easy to administer and sensitive to change instrument . the atec which totally contains 77 items , covers four main impairment areas of asds including communication , sociability , sensory - cognitive awareness and health - physical - behavior ( for more details on the scale , see methods ) . the atec is freely available and can be scored online with minimal training and resources . a considerable amount of literature administered autism treatment evaluation checklist , has described that atec is sensitive to intra - subject changes after treatment programs . in a recent investigation , megiati given their findings , they introduce atec as a potentially useful and promising tool for gathering reliable data on current behaviors and skills as well as general functioning of children with autism spectrum disorders . moreover , other findings have shown that atec data were significantly correlated with other equivalent diagnostic tools ( ( e.g . pervasive developmental disorders behavior inventory ( pdd - bi ) : r=0.87 or severity of autism scale ( sas ) : r=0.7 ) ) . given together , one can argue that atec is a potentially reliable and valid tool for monitoring progress over time . it is noteworthy that exploring the standardization of a questionnaire is a continuing dynamic process and different investigations with different samples are supposed to examine the validity and reliability of the instrument . in addition , concerned for the fact that most of the health related questionnaires and scales have been developed in english speaking countries , the need to adapt the questionnaires in other than the source language has grown rapidly . cross - cultural adaptation is a valid process through which reliable health status measures may be obtained in order to be used in different countries in spite of different sociocultural conditions . the process of adaptation provides a ground to measure a same phenomenon using a same instrument across different cultures . so far , however , there have been few studies applying atec in different countries in order to be implemented in other languages than english as well as monitoring the validity of the instrument . thus , the purpose of the present study is to investigate the cross - cultural adaptation , validity and reliability of atec questionnaire in an iranian autistic sample . this project used a convenience sample of 134 children and adolescents with autism spectrum disorders ( 111 boys and 23 girls ) aged 6 - 15 years ( mean : 9.6 , sd : 1.97 ) . all children met criteria for a diagnosis of autism on both the dsm - iv , and adi - r , while the diagnosis was established in a previous assessment by either a child psychiatrist or psychologist . the child 's parent or caregiver completed written informed consent before they were assigned to the study . the study was approved by the medical ethics committee of tehran university of medical sciences . the current study used the essential methodological steps suggested by internationally recognized publications for the procedures involved in the cultural adaption of measurement instruments . cross - cultural adaption stages were followed as below : forward translation : in this stage , the questionnaire was translated from original language ( english ) to target language ( persian ) by 2 bilingual translators whose mother tongue was persian . one of the translators was aware of the concepts examined by the instrument and had translated such medical questionnaires before . the other translator ( also called native translator ) was neither aware nor informed of the concepts and aims of the measurement instrument and had no medical background . in order to approach to a conclusive data , the results produced by both translators were compared with each other by the translators and a recording observer ( one of the researchers involved in the present study ) . back translation : the final persian translation was again back translated into english by 2 bilingual , native english - speaking translators who were totally blind to the original version . they were neither aware of the concepts and aims of the questionnaire nor had academic training in autism . expert committee : the final persian translation and the back translation were compared and reviewed by a multidisciplinary , expert committee to obtain a final version . the committee was composed of the translators , a psychologist , a methodologist , and a medical doctor . on the way to guarantee accurate comprehension , the members of the committee evaluated and reviewed the topics of each section while taking into account the semantic , idiomatic and cultural equivalents and the intelligibility of the items . the committee also reviews all the atec drafts ( i.e. the original , forward and back translation drafts ) and reveals their suggestions about each item ; finally the last version of the draft was produced . a sample of 20 primary caregivers of autistic children and adolescents was pretested in order to verify the comprehensibility of the statements and questions , to assess the equivalence of the instrument within the iranian culture and to recognize the errors in the final version . in order to assess the psychometric properties of the translated version of the instrument , the standardized factors were evaluated : content validity : the content quality of the autism treatment evaluation checklist was reviewed by the expert committee all through the cultural adaptation procedure . the items or questions would also have been revised if 15% of the participants had difficulty in the comprehension of the items in the pretest stage . construct validity : the construct quality of the questionnaire was evaluated in order to ascertain that the persian version of the atec really measures what it is expected to measure by comparison of the adapted version of the instrument with similar tests that assess similar factors . each subscale of atec was examined with its equivalent from adi - r ; for example atec subscale 1 entered the analysis paired with verbal subscale of adi - r . in this way , there was more assurance that the adapted version is measuring a construct comparable to the original . reliability : the reliability of the questionnaire was evaluated by measuring the internal consistency of all the items within each subscale of the questionnaire and stability of the instrument as well ( test - retest ) . measures : autism treatment evaluation checklist atec consists of 4 subscales : 1 : speech / language/ communication ( 14 items ; maximum score : 28 ) ; 2 : sociability ( 20 items ; maximum score : 14 ) ; 3 : sensory / cognitive / awareness ( 18 items ; maximum score : 36 ) , and 4 : health / physical/ behavior ( 25 items , maximum score : 75 ) . items on subscales 1 - 3 are scored from 0 ( not descriptive ) to 2 ( very descriptive ) . scoring on subscale 4 ranges from 0 ( not a problem ) to 3 ( serious problem ) . the total maximum score is 179 with a higher score representing higher severity of autistic behaviors and poorer social developmental skills , a decrease in scores indicates progress and improvement in autistic problems . adi - r which provides a comprehensive assessment of individuals suspected to have autism spectrum disorders , was also used in this project . adi - r consists of 93 items and focuses mainly on three functional domains : language and communication , reciprocal social interactions , restricted , repetitive and stereotyped behaviors and interests . the interview also addresses other clinical factors like aggression , self - injury and possible epileptic features . for administrating adi - r an experienced interviewer data were scored and interpreted by using a diagnostic algorithm or a current behavior algorithm . construct validity of the instrument was evaluated by demonstrating the correlation between atec and adi - r according to pearson correlation ; the set point for p. value was 0.05 . internal consistency ( reliability ) of the instrument was confirmed by cronbach 's coefficient alpha and guttman split - half coefficient ; an acceptable internal consistency was defined as a value > 0.7 . furthermore , the stability of the instrument was evaluated using the test - retest reliability method ; the data obtained in first test session and retest session ( separated by 2 weeks ) were analyzed using the intraclass correlation coefficient to assess the reliability of all the scales measured . this project used a convenience sample of 134 children and adolescents with autism spectrum disorders ( 111 boys and 23 girls ) aged 6 - 15 years ( mean : 9.6 , sd : 1.97 ) . all children met criteria for a diagnosis of autism on both the dsm - iv , and adi - r , while the diagnosis was established in a previous assessment by either a child psychiatrist or psychologist . the child 's parent or caregiver completed written informed consent before they were assigned to the study . the study was approved by the medical ethics committee of tehran university of medical sciences . the current study used the essential methodological steps suggested by internationally recognized publications for the procedures involved in the cultural adaption of measurement instruments . cross - cultural adaption stages were followed as below : forward translation : in this stage , the questionnaire was translated from original language ( english ) to target language ( persian ) by 2 bilingual translators whose mother tongue was persian . one of the translators was aware of the concepts examined by the instrument and had translated such medical questionnaires before . the other translator ( also called native translator ) was neither aware nor informed of the concepts and aims of the measurement instrument and had no medical background . in order to approach to a conclusive data , the results produced by both translators were compared with each other by the translators and a recording observer ( one of the researchers involved in the present study ) . back translation : the final persian translation was again back translated into english by 2 bilingual , native english - speaking translators who were totally blind to the original version . they were neither aware of the concepts and aims of the questionnaire nor had academic training in autism . expert committee : the final persian translation and the back translation were compared and reviewed by a multidisciplinary , expert committee to obtain a final version . the committee was composed of the translators , a psychologist , a methodologist , and a medical doctor . on the way to guarantee accurate comprehension , the members of the committee evaluated and reviewed the topics of each section while taking into account the semantic , idiomatic and cultural equivalents and the intelligibility of the items . the committee also reviews all the atec drafts ( i.e. the original , forward and back translation drafts ) and reveals their suggestions about each item ; finally the last version of the draft was produced . a sample of 20 primary caregivers of autistic children and adolescents was pretested in order to verify the comprehensibility of the statements and questions , to assess the equivalence of the instrument within the iranian culture and to recognize the errors in the final version . in order to assess the psychometric properties of the translated version of the instrument , the standardized factors were evaluated : content validity : the content quality of the autism treatment evaluation checklist was reviewed by the expert committee all through the cultural adaptation procedure . the items or questions would also have been revised if 15% of the participants had difficulty in the comprehension of the items in the pretest stage . construct validity : the construct quality of the questionnaire was evaluated in order to ascertain that the persian version of the atec really measures what it is expected to measure by comparison of the adapted version of the instrument with similar tests that assess similar factors . each subscale of atec was examined with its equivalent from adi - r ; for example atec subscale 1 entered the analysis paired with verbal subscale of adi - r . in this way , there was more assurance that the adapted version is measuring a construct comparable to the original . reliability : the reliability of the questionnaire was evaluated by measuring the internal consistency of all the items within each subscale of the questionnaire and stability of the instrument as well ( test - retest ) . measures : autism treatment evaluation checklist atec consists of 4 subscales : 1 : speech / language/ communication ( 14 items ; maximum score : 28 ) ; 2 : sociability ( 20 items ; maximum score : 14 ) ; 3 : sensory / cognitive / awareness ( 18 items ; maximum score : 36 ) , and 4 : health / physical/ behavior ( 25 items , maximum score : 75 ) . items on subscales 1 - 3 are scored from 0 ( not descriptive ) to 2 ( very descriptive ) . scoring on subscale 4 ranges from 0 ( not a problem ) to 3 ( serious problem ) . the total maximum score is 179 with a higher score representing higher severity of autistic behaviors and poorer social developmental skills , a decrease in scores indicates progress and improvement in autistic problems . adi - r which provides a comprehensive assessment of individuals suspected to have autism spectrum disorders , was also used in this project . adi - r consists of 93 items and focuses mainly on three functional domains : language and communication , reciprocal social interactions , restricted , repetitive and stereotyped behaviors and interests . the interview also addresses other clinical factors like aggression , self - injury and possible epileptic features . for administrating adi - r an experienced interviewer data were scored and interpreted by using a diagnostic algorithm or a current behavior algorithm . construct validity of the instrument was evaluated by demonstrating the correlation between atec and adi - r according to pearson correlation ; the set point for p. value was 0.05 . internal consistency ( reliability ) of the instrument was confirmed by cronbach 's coefficient alpha and guttman split - half coefficient ; an acceptable internal consistency was defined as a value > 0.7 . furthermore , the stability of the instrument was evaluated using the test - retest reliability method ; the data obtained in first test session and retest session ( separated by 2 weeks ) were analyzed using the intraclass correlation coefficient to assess the reliability of all the scales measured . descriptive data and internal consistency coefficients for each atec subscale and total scores atec : autism treatment evaluation checklist / sd : standard deviation the procedures of translation , back translation and submission of the instrument to the expert committee showed that there was no need for significant changing of the meaning of items or adding and removing the statements . during the pretest stage , a direct interview with the participants was performed in order to appraise the difficulties in completing the questionnaires or to identify any misunderstanding in items or statements . participants who were interviewed in this stage reported no difficulties in comprehending the content of each items ; however two complained that they had difficulties in recognizing the extent to which their child had problems during answering to some items ( in language and communication subscale ) . following further discussion with the expert committee , authors found no need to significant change in wording and the reported problem seemed to be related to the information reminiscence situation . however , according to the expert committee discretion , only the statements of two items were refined by adding a word . in the sociability subscale , the word " his / her inside " was added to the statement of the item 12 in parentheses moreover , in the sensory / cognitive/ awareness subscale an exemplification for the word " tuning in " was added to the statement of the item 17 . the internal consistency was evaluated by means of cronbach 's alpha and guttman split - half methods . the results showed a high internal consistency of the instrument for total score in both methods ( cronbach 's coefficient alpha : 0.93 ; guttman split - half : 0.77 ) . internal consistency of the four atec subscales was also excellent in both methods ( table 1 ) . stability ( test - retest ) test - retest reliability of the atec was calculated using intraclass correlation coefficient . the stability was excellent for all the subscales and also for total scores ( table 2 ) . test - retest reliability scores of the atec ( persian version ) atec : autism treatment evaluation checklist / ci : confidence interval data obtained for construct validation were submitted to statistical analysis using pearson correlation . the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the procedures of translation , back translation and submission of the instrument to the expert committee showed that there was no need for significant changing of the meaning of items or adding and removing the statements . during the pretest stage , a direct interview with the participants was performed in order to appraise the difficulties in completing the questionnaires or to identify any misunderstanding in items or statements . participants who were interviewed in this stage reported no difficulties in comprehending the content of each items ; however two complained that they had difficulties in recognizing the extent to which their child had problems during answering to some items ( in language and communication subscale ) . following further discussion with the expert committee , authors found no need to significant change in wording and the reported problem seemed to be related to the information reminiscence situation . however , according to the expert committee discretion , only the statements of two items were refined by adding a word . in the sociability subscale , the word " his / her inside " was added to the statement of the item 12 in parentheses moreover , in the sensory / cognitive/ awareness subscale an exemplification for the word " tuning in " was added to the statement of the item 17 . the internal consistency was evaluated by means of cronbach 's alpha and guttman split - half methods . the results showed a high internal consistency of the instrument for total score in both methods ( cronbach 's coefficient alpha : 0.93 ; guttman split - half : 0.77 ) . internal consistency of the four atec subscales was also excellent in both methods ( table 1 ) . stability ( test - retest ) test - retest reliability of the atec was calculated using intraclass correlation coefficient . the stability was excellent for all the subscales and also for total scores ( table 2 ) . test - retest reliability scores of the atec ( persian version ) atec : autism treatment evaluation checklist / ci : confidence interval data obtained for construct validation were submitted to statistical analysis using pearson correlation . the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the current study was set out to adapt " autism treatment evaluation checklist " to persian language . given the complex nature of autism spectrum disorders , there is a subsequent need for a comprehensive battery of diagnostic and monitoring instruments . hence , cross - cultural adaptation of the asd questionnaires is worthy to be investigated . in such a way , a wide range of data can be provided using different language versions of a questionnaire for asd in different societies . alongside other formal evaluation tools for autism spectrum disorders , however , to our knowledge there were already few published studies investigating cultural adaptation of the atec in languages other than english . although a few asd scales have been translated into persian and being used in autism schools and clinics ( e.g. autism scaling questionnaire ( asq ) or childhood autism rating scale ( cars ) ) ; we were never informed about their validation procedure through the literature . given together , the current project provides a reliable and valid translation of atec which remained stable via the cross - cultural and cross - linguistic processes . small changes made in the questionnaire by the expert committee , were introduced to smooth the progress of items comprehension and the association of the responses . regarding the psychometric properties of an instrument , the confirmation of its validity in other cultures boosts the validation of the original one . in relation to the construct validity of the persian version of atec , our results showed that atec measures were significantly correlated with the data obtained in adi - r interview . results from adi - r have been proven to support a thorough evaluation of core symptoms of autistic disorders listed in diagnostic and statistic manual of mental disorders ( dsm iv - tr ) . thus the current study indicated that the persian version of the atec had acceptable properties for evaluating autistic symptoms in individuals with asd . moreover , these results accord with the findings of magiati et al , which showed that atec is a promising instrument for gathering reliable and valid information on autistic individuals functioning . furthermore , there are several studies in which atec has been used as a tool for measuring severity of asd and the authors reported that this questionnaire was successfully able to do so . despite the popularity of atec to evaluate and monitor progress in autistic individuals over time , to date few data on the reliability ( internal consistency ) of the questionnaire have been published . rimland and edelson cite a few data ( reliability : 0.94 for the total scores and 0.8 - 0.9 for subscale scores ) on over 1300 online completed atecs . moreover , recently in a cohort investigation , magiati et al supplemented the previous limited literature on the value of atec and reported a high internal consistency at their two time points of assessment ( when the children were aged about 5.5 years and 5 - 6 years later ) . our study also confirms the previous findings by showing high values of internal consistency for atec . all subscales of the questionnaire as well as total scores had similar range of high internal consistency ( cronbach 's alpha > 0.80 ) . given the primary role of atec which is to measure factors that are expected partially to change over long term period or under treatment conditions , megiati et al reported that atec total scores obtained in the baseline significantly predicted the extent of improvement after 5 - 6 years . on the other hand , providing additional value to megiati findings , current study showed excellent stability ( i.e. , test - retest reliability ) for all the subscales over a short time period . nevertheless , there are a few limitations and remained questions on atec validation that current study could not address . , a larger sample study is needed to conduct a factor analysis on atec . to address the discriminant validity of the questionnaire , it should be used to differentiate asd from normally developing children or other developmental disorders . furthermore , future studies could use other standard measures of autism symptom severity along with atec to examine other possible association among subscales . a few studies which used atec to examine severity and monitor symptoms in individuals with asd indicated the value of atec in autism research . in conclusion the current study also indicated that the persian version of the atec is a reliable and valid tool for evaluating asd symptoms in an iranian sample with asd . this finding has important implication for developing effective therapeutic programs as well as ongoing longitudinal research projects in asds .

objectivethe objectives of the current study were to translate and adapt autism treatment evaluation checklist ( atec ) into persian language and to investigate its reliability and validity in an iranian autistic sample.methodsa total sample of 134 children with autism spectrum disorders aged 6 - 15 years were assigned to the study . the process of cross - cultural adaptation was performed according to international methodological steps as following : translation , back - translation , revision by an expert committee and pretest . a sample of 20 primary caregivers of autistic children were pretested . the content validity of the atec was reviewed by the expert committee all through the stages . the construct quality of the questionnaire was evaluated by comparison of the adapted version of the instrument with similar tests assessed similar factors . moreover , the reliability of the questionnaire was evaluated through stability and homogeneity assessments.findingsthe results showed good content validity and internal consistency ( cronbach 's alpha : 0.86 - 0.93 ) . in relation to construct validity , there was significant correlation between atec subscales and raw data obtained from autism diagnostic interview - revised ( adi - r ) ( r=0.38 - 0.79 ) . the intraclass correlation coefficient for the test retest reliability was excellent for all the subscales and also for total scores ( icc : 0.79 - 0.93).conclusioncross - cultural adaptation of atec was successful . the psychometric properties were verified and indicated that the adapted questionnaire is valid and reliable to use in iranian culture .

Introduction Subjects and Methods Participants Procedure Data analysis Findings Cross-cultural adaptation process Reliability-Internal consistency (Homogeneity) Validity Discussion Conclusion Conflict of Interest

although , the autism spectrum disorder ( asd ) is known to be neurogenetic in origin , its diagnosis is primarily based on behavioral and clinical signs and symptoms . according to diagnostic and statistical manual of mental disorders , 4 edition , text - revision ( dsm - iv - tr ) , and international classification of diseases ( icd ) , there are three main diagnostic criteria for autistic disorders : impairments in social interaction , impairments in communication and language and restricted , stereotyped behaviors , interests and activities . regarding the neurologic and genetic basis of autism , however , so far regularly standard diagnostic tests are based on psychological and behavioral assessments such as autism diagnostic interview - revised ( adi - r ) , autism diagnostic observation schedule ( ados - g ) and childhood autism rating scale ( cars ) . several researchers have administered longitudinal studies to monitor the long lasting improvement of autistic children . for years , there was no specific measure while instead , measures such as ados , adi - r or cars have being used to examine the symptoms , evaluate changes and improvement in response to different interventions . moreover , the assessment tools provided for typically developing ( td ) children have been used to evaluate autistic individuals . autism treatment evaluation checklist ( atec ) is a one paged checklist designed to be completed by parents , teachers or caretakers and is a simple but effective tool to assess the severity of symptoms as well as developmental aspects of autism . a considerable amount of literature administered autism treatment evaluation checklist , has described that atec is sensitive to intra - subject changes after treatment programs . in a recent investigation , megiati given their findings , they introduce atec as a potentially useful and promising tool for gathering reliable data on current behaviors and skills as well as general functioning of children with autism spectrum disorders . it is noteworthy that exploring the standardization of a questionnaire is a continuing dynamic process and different investigations with different samples are supposed to examine the validity and reliability of the instrument . in addition , concerned for the fact that most of the health related questionnaires and scales have been developed in english speaking countries , the need to adapt the questionnaires in other than the source language has grown rapidly . cross - cultural adaptation is a valid process through which reliable health status measures may be obtained in order to be used in different countries in spite of different sociocultural conditions . the process of adaptation provides a ground to measure a same phenomenon using a same instrument across different cultures . so far , however , there have been few studies applying atec in different countries in order to be implemented in other languages than english as well as monitoring the validity of the instrument . thus , the purpose of the present study is to investigate the cross - cultural adaptation , validity and reliability of atec questionnaire in an iranian autistic sample . this project used a convenience sample of 134 children and adolescents with autism spectrum disorders ( 111 boys and 23 girls ) aged 6 - 15 years ( mean : 9.6 , sd : 1.97 ) . all children met criteria for a diagnosis of autism on both the dsm - iv , and adi - r , while the diagnosis was established in a previous assessment by either a child psychiatrist or psychologist . the child 's parent or caregiver completed written informed consent before they were assigned to the study . the study was approved by the medical ethics committee of tehran university of medical sciences . the current study used the essential methodological steps suggested by internationally recognized publications for the procedures involved in the cultural adaption of measurement instruments . cross - cultural adaption stages were followed as below : forward translation : in this stage , the questionnaire was translated from original language ( english ) to target language ( persian ) by 2 bilingual translators whose mother tongue was persian . one of the translators was aware of the concepts examined by the instrument and had translated such medical questionnaires before . in order to approach to a conclusive data , the results produced by both translators were compared with each other by the translators and a recording observer ( one of the researchers involved in the present study ) . expert committee : the final persian translation and the back translation were compared and reviewed by a multidisciplinary , expert committee to obtain a final version . on the way to guarantee accurate comprehension , the members of the committee evaluated and reviewed the topics of each section while taking into account the semantic , idiomatic and cultural equivalents and the intelligibility of the items . the committee also reviews all the atec drafts ( i.e. the original , forward and back translation drafts ) and reveals their suggestions about each item ; finally the last version of the draft was produced . a sample of 20 primary caregivers of autistic children and adolescents was pretested in order to verify the comprehensibility of the statements and questions , to assess the equivalence of the instrument within the iranian culture and to recognize the errors in the final version . in order to assess the psychometric properties of the translated version of the instrument , the standardized factors were evaluated : content validity : the content quality of the autism treatment evaluation checklist was reviewed by the expert committee all through the cultural adaptation procedure . construct validity : the construct quality of the questionnaire was evaluated in order to ascertain that the persian version of the atec really measures what it is expected to measure by comparison of the adapted version of the instrument with similar tests that assess similar factors . each subscale of atec was examined with its equivalent from adi - r ; for example atec subscale 1 entered the analysis paired with verbal subscale of adi - r . in this way , there was more assurance that the adapted version is measuring a construct comparable to the original . reliability : the reliability of the questionnaire was evaluated by measuring the internal consistency of all the items within each subscale of the questionnaire and stability of the instrument as well ( test - retest ) . measures : autism treatment evaluation checklist atec consists of 4 subscales : 1 : speech / language/ communication ( 14 items ; maximum score : 28 ) ; 2 : sociability ( 20 items ; maximum score : 14 ) ; 3 : sensory / cognitive / awareness ( 18 items ; maximum score : 36 ) , and 4 : health / physical/ behavior ( 25 items , maximum score : 75 ) . adi - r which provides a comprehensive assessment of individuals suspected to have autism spectrum disorders , was also used in this project . adi - r consists of 93 items and focuses mainly on three functional domains : language and communication , reciprocal social interactions , restricted , repetitive and stereotyped behaviors and interests . for administrating adi - r an experienced interviewer data were scored and interpreted by using a diagnostic algorithm or a current behavior algorithm . construct validity of the instrument was evaluated by demonstrating the correlation between atec and adi - r according to pearson correlation ; the set point for p. value was 0.05 . internal consistency ( reliability ) of the instrument was confirmed by cronbach 's coefficient alpha and guttman split - half coefficient ; an acceptable internal consistency was defined as a value > 0.7 . furthermore , the stability of the instrument was evaluated using the test - retest reliability method ; the data obtained in first test session and retest session ( separated by 2 weeks ) were analyzed using the intraclass correlation coefficient to assess the reliability of all the scales measured . this project used a convenience sample of 134 children and adolescents with autism spectrum disorders ( 111 boys and 23 girls ) aged 6 - 15 years ( mean : 9.6 , sd : 1.97 ) . all children met criteria for a diagnosis of autism on both the dsm - iv , and adi - r , while the diagnosis was established in a previous assessment by either a child psychiatrist or psychologist . the child 's parent or caregiver completed written informed consent before they were assigned to the study . the study was approved by the medical ethics committee of tehran university of medical sciences . the current study used the essential methodological steps suggested by internationally recognized publications for the procedures involved in the cultural adaption of measurement instruments . cross - cultural adaption stages were followed as below : forward translation : in this stage , the questionnaire was translated from original language ( english ) to target language ( persian ) by 2 bilingual translators whose mother tongue was persian . one of the translators was aware of the concepts examined by the instrument and had translated such medical questionnaires before . in order to approach to a conclusive data , the results produced by both translators were compared with each other by the translators and a recording observer ( one of the researchers involved in the present study ) . they were neither aware of the concepts and aims of the questionnaire nor had academic training in autism . expert committee : the final persian translation and the back translation were compared and reviewed by a multidisciplinary , expert committee to obtain a final version . on the way to guarantee accurate comprehension , the members of the committee evaluated and reviewed the topics of each section while taking into account the semantic , idiomatic and cultural equivalents and the intelligibility of the items . the committee also reviews all the atec drafts ( i.e. the original , forward and back translation drafts ) and reveals their suggestions about each item ; finally the last version of the draft was produced . a sample of 20 primary caregivers of autistic children and adolescents was pretested in order to verify the comprehensibility of the statements and questions , to assess the equivalence of the instrument within the iranian culture and to recognize the errors in the final version . in order to assess the psychometric properties of the translated version of the instrument , the standardized factors were evaluated : content validity : the content quality of the autism treatment evaluation checklist was reviewed by the expert committee all through the cultural adaptation procedure . construct validity : the construct quality of the questionnaire was evaluated in order to ascertain that the persian version of the atec really measures what it is expected to measure by comparison of the adapted version of the instrument with similar tests that assess similar factors . each subscale of atec was examined with its equivalent from adi - r ; for example atec subscale 1 entered the analysis paired with verbal subscale of adi - r . in this way , there was more assurance that the adapted version is measuring a construct comparable to the original . reliability : the reliability of the questionnaire was evaluated by measuring the internal consistency of all the items within each subscale of the questionnaire and stability of the instrument as well ( test - retest ) . measures : autism treatment evaluation checklist atec consists of 4 subscales : 1 : speech / language/ communication ( 14 items ; maximum score : 28 ) ; 2 : sociability ( 20 items ; maximum score : 14 ) ; 3 : sensory / cognitive / awareness ( 18 items ; maximum score : 36 ) , and 4 : health / physical/ behavior ( 25 items , maximum score : 75 ) . adi - r which provides a comprehensive assessment of individuals suspected to have autism spectrum disorders , was also used in this project . adi - r consists of 93 items and focuses mainly on three functional domains : language and communication , reciprocal social interactions , restricted , repetitive and stereotyped behaviors and interests . for administrating adi - r an experienced interviewer data were scored and interpreted by using a diagnostic algorithm or a current behavior algorithm . construct validity of the instrument was evaluated by demonstrating the correlation between atec and adi - r according to pearson correlation ; the set point for p. value was 0.05 . internal consistency ( reliability ) of the instrument was confirmed by cronbach 's coefficient alpha and guttman split - half coefficient ; an acceptable internal consistency was defined as a value > 0.7 . furthermore , the stability of the instrument was evaluated using the test - retest reliability method ; the data obtained in first test session and retest session ( separated by 2 weeks ) were analyzed using the intraclass correlation coefficient to assess the reliability of all the scales measured . descriptive data and internal consistency coefficients for each atec subscale and total scores atec : autism treatment evaluation checklist / sd : standard deviation the procedures of translation , back translation and submission of the instrument to the expert committee showed that there was no need for significant changing of the meaning of items or adding and removing the statements . participants who were interviewed in this stage reported no difficulties in comprehending the content of each items ; however two complained that they had difficulties in recognizing the extent to which their child had problems during answering to some items ( in language and communication subscale ) . following further discussion with the expert committee , authors found no need to significant change in wording and the reported problem seemed to be related to the information reminiscence situation . however , according to the expert committee discretion , only the statements of two items were refined by adding a word . in the sociability subscale , the word " his / her inside " was added to the statement of the item 12 in parentheses moreover , in the sensory / cognitive/ awareness subscale an exemplification for the word " tuning in " was added to the statement of the item 17 . the internal consistency was evaluated by means of cronbach 's alpha and guttman split - half methods . the results showed a high internal consistency of the instrument for total score in both methods ( cronbach 's coefficient alpha : 0.93 ; guttman split - half : 0.77 ) . internal consistency of the four atec subscales was also excellent in both methods ( table 1 ) . stability ( test - retest ) test - retest reliability of the atec was calculated using intraclass correlation coefficient . the stability was excellent for all the subscales and also for total scores ( table 2 ) . test - retest reliability scores of the atec ( persian version ) atec : autism treatment evaluation checklist / ci : confidence interval data obtained for construct validation were submitted to statistical analysis using pearson correlation . the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the procedures of translation , back translation and submission of the instrument to the expert committee showed that there was no need for significant changing of the meaning of items or adding and removing the statements . participants who were interviewed in this stage reported no difficulties in comprehending the content of each items ; however two complained that they had difficulties in recognizing the extent to which their child had problems during answering to some items ( in language and communication subscale ) . following further discussion with the expert committee , authors found no need to significant change in wording and the reported problem seemed to be related to the information reminiscence situation . however , according to the expert committee discretion , only the statements of two items were refined by adding a word . in the sociability subscale , the word " his / her inside " was added to the statement of the item 12 in parentheses moreover , in the sensory / cognitive/ awareness subscale an exemplification for the word " tuning in " was added to the statement of the item 17 . the internal consistency was evaluated by means of cronbach 's alpha and guttman split - half methods . the results showed a high internal consistency of the instrument for total score in both methods ( cronbach 's coefficient alpha : 0.93 ; guttman split - half : 0.77 ) . internal consistency of the four atec subscales was also excellent in both methods ( table 1 ) . stability ( test - retest ) test - retest reliability of the atec was calculated using intraclass correlation coefficient . the stability was excellent for all the subscales and also for total scores ( table 2 ) . test - retest reliability scores of the atec ( persian version ) atec : autism treatment evaluation checklist / ci : confidence interval data obtained for construct validation were submitted to statistical analysis using pearson correlation . the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the achieved values of the atec subscales and related adi - r subscales are shown in table 3 . results showed significant positive association between each pair variables , language and behavioral subscale indicated highest interrelation ( r=0.7 and 0.79 ) . construct validity of the atec : correlations between atec subscales and adi - r raw data atec : autism treatment evaluation checklist / adi - r : autism diagnostic interview - revised the current study was set out to adapt " autism treatment evaluation checklist " to persian language . given the complex nature of autism spectrum disorders , there is a subsequent need for a comprehensive battery of diagnostic and monitoring instruments . hence , cross - cultural adaptation of the asd questionnaires is worthy to be investigated . alongside other formal evaluation tools for autism spectrum disorders , however , to our knowledge there were already few published studies investigating cultural adaptation of the atec in languages other than english . given together , the current project provides a reliable and valid translation of atec which remained stable via the cross - cultural and cross - linguistic processes . small changes made in the questionnaire by the expert committee , were introduced to smooth the progress of items comprehension and the association of the responses . regarding the psychometric properties of an instrument , the confirmation of its validity in other cultures boosts the validation of the original one . in relation to the construct validity of the persian version of atec , our results showed that atec measures were significantly correlated with the data obtained in adi - r interview . results from adi - r have been proven to support a thorough evaluation of core symptoms of autistic disorders listed in diagnostic and statistic manual of mental disorders ( dsm iv - tr ) . thus the current study indicated that the persian version of the atec had acceptable properties for evaluating autistic symptoms in individuals with asd . furthermore , there are several studies in which atec has been used as a tool for measuring severity of asd and the authors reported that this questionnaire was successfully able to do so . despite the popularity of atec to evaluate and monitor progress in autistic individuals over time , to date few data on the reliability ( internal consistency ) of the questionnaire have been published . rimland and edelson cite a few data ( reliability : 0.94 for the total scores and 0.8 - 0.9 for subscale scores ) on over 1300 online completed atecs . moreover , recently in a cohort investigation , magiati et al supplemented the previous limited literature on the value of atec and reported a high internal consistency at their two time points of assessment ( when the children were aged about 5.5 years and 5 - 6 years later ) . all subscales of the questionnaire as well as total scores had similar range of high internal consistency ( cronbach 's alpha > 0.80 ) . given the primary role of atec which is to measure factors that are expected partially to change over long term period or under treatment conditions , megiati et al reported that atec total scores obtained in the baseline significantly predicted the extent of improvement after 5 - 6 years . , test - retest reliability ) for all the subscales over a short time period . nevertheless , there are a few limitations and remained questions on atec validation that current study could not address . to address the discriminant validity of the questionnaire , it should be used to differentiate asd from normally developing children or other developmental disorders . in conclusion the current study also indicated that the persian version of the atec is a reliable and valid tool for evaluating asd symptoms in an iranian sample with asd .

wiskott - aldrich syndrome ( was ) is an x - linked primary immunodeficiency disease resulting from mutations within the was gene that result in the loss of was protein ( wasp ) or function . subjects with was suffer from combined immunodeficiency , thrombocytopenia , and eczema and also frequently develop autoimmunity and lymphoid malignancies.1 , 2 , 3 , 4 wasp is a scaffold protein involved in signal transduction pathways that activate the actin cytoskeleton downstream of multiple cell surface receptors , including the b and t cell antigen receptors.5 , 6 , 7 although the disease phenotype can be alleviated with hematopoietic stem cell transplantation ( hsct ) , the success of this therapy is variable , depending on factors such as the patient s age , donor compatibility , conditioning regimen , and the extent of reconstitution . in the absence of a histocompatibility leukocyte antigen ( hla)-matched donor , transplantation with a mismatched donor has a reduced survival rate.3 , 8 , 9 since the phenotype of was deficiency impacts only hematopoietic cells , gene therapy is a possible alternative . in this approach , a wasp expression cassette is stably integrated into the chromatin of autologous hematopoietic stem cells ( hscs ) using viral - based gene delivery . previous and ongoing clinical trials have demonstrated the efficacy of gene therapy for alleviating the pathologies of was.10 , 11 , 12 importantly , following development of t cell leukemia due to insertional mutagenesis in -retroviral gene therapy trials for both severe combined immunodeficiency ( scid ) and was,13 , 14 , 15 much research has focused on strategies for eliminating this risk . the use of self - inactivating ( sin ) lentiviruses ( lvs ) for gene transfer is one critical improvement , combining a safer integration profile ( less affinity for insertions near promoters than -retroviruses16 , 17 , 18 ) with the ability to select internal promoters that optimize transgene expression and safety . because of the association between internal promoter strength and transformation potential , internal promoters are selected for their ability to recapitulate endogenous expression levels and regulation , as well as for the lack of transactivation potential both in vitro and in vivo . these considerations are particularly important for treating was based on the following findings : sub - endogenous levels of wasp expression may hinder the reconstitution of murine b cell , t cell , and myeloid subsets and platelets ; insufficient wasp expression in b cells compared to t cells can drive acquisition of autoimmunity;21 , 22 , 23 and patients with was are predisposed to malignancies and clonal expansion.1 , 3 , 4 current clinical trials for was utilize a sin - lv with an internal promoter consisting of the proximal 1.6 kb of the endogenous was promoter ( ws1.6 ) to drive human wasp ( hwasp ) expression.10 , 12 patients treated with this sin - lv showed improvements in immunity to infections , resolved eczema , and protection from bleeding , without evidence of clonal expansion of cells10 , 12 or loss of self - tolerance.24 , 25 however , clinical improvement required relatively high levels of viral marking and alleviation of the was phenotype was incomplete with , most notably , limited or no improvement in platelet counts . in previous mouse gene therapy experiments , we found that the ws1.6 promoter did not effectively rescue wasp expression in all lineages including b cells and resulted in the acquisition of features of humoral autoimmunity . in contrast , an sin - lv using a synthetic promoter derived from a -retrovirus called mnd ( mpsv ltr , ncr deleted , dl587 pbs ) as an internal promoter rescued wasp expression in all affected lineages and reduced the risk of autoimmunity.20 , 27 , 28 in a clinical gene therapy trial for adrenoleukodystrophy , mnd has been used as an internal promoter for lv gene therapy without adverse effects . although strongly trans - activating promoters are associated with an enhanced risk of insertional transactivation , this risk can be diminished by including a chromatin insulator.28 , 30 , 31 chromatin insulators act as boundary elements preventing interactions between adjacent chromatin domains.32 , 33 , 34 candidate insulator constructs can function as barrier only elements ( acting as barriers to epigenetic chromatin silencing ) or enhancer - blocker only elements ( that prevent promoter transactivation between the domains that they separate ) , or they can have both activities ( as reviewed in emery et al . and raab et al . ) . one of the best characterized insulators , chicken globin hypersensitive site 4 ( chs4 ) , exhibits both barrier and enhancer - blocker activity . the insulator properties of the canonical 1.2-kb chs4 can be recapitulated by a 650-bp sequence consisting of the 250-bp chs4 core and 400 bp of sequence at its 3 end . because the 3 long terminal repeat ( ltr ) of the lv is copied and replaces the 5 ltr upon integration into the host chromatin , the insulator sequence that is cloned within the 3 ltr will result in the integrated lv being bordered with insulator sequences at both ends . in previous in vitro assays testing the safety of an insulated sin - lv incorporating an mnd internal promoter , we have shown that the 650-bp insulator significantly reduces transactivation of lmo2 when placed in close proximity to the lmo2 promoter . additionally , the insulated mnd lv did not promote a pre - leukemic block in differentiation of primary murine thymocytes following transduction and in vitro culture . our group also previously tested a series of chs4-insulated and non - insulated sin - lv constructs containing various internal promoters ( mnd , ef1 , and was 1.6-kb and 0.5-kb promoters ) driving gfp reporter gene expression in a mouse gene therapy model . hsct with lv containing the 650-bp chs4-insulated mnd - gfp ( 650.mnd.gfp ) resulted in gfp expression in all hematopoietic lineages , including platelets , that was stable over time . therefore , based on our combined efficacy and expression data , a producer cell clone capable of generating high - titer lv with the 650-bp chs4 insulator and mnd - wasp expression cassette ( 650.mnd.hwasp ) was developed for clinical use . in the current study , we utilize this clinical vector to perform an expanded efficacy and safety study . our analysis also includes a direct comparison of lv with and without the 650-bp chs4 insulator . efficacy and safety was assessed in a large cohort of was mice , including serial transplants . we also evaluated the safety profile of these sin - lv vectors in non - human primates ( nhps ) , expressing gfp instead of hwasp to improve assay sensitivity . our combined observations strongly support future use of the insulated mnd promoter for clinical was gene delivery . we evaluated the ability of the insulated sin - lv 650.mnd.hwasp ( figure 1a ) , generated from a clinical - grade producer cell line , to stably reconstitute wasp expression in the appropriate hematopoietic cell lineages using a was mouse gene therapy model . expression from this lv was compared , in parallel , to the non - insulated version , mnd.hwasp , that previously demonstrated reconstitution of was hematopoietic cell defects . lineage - depleted ( lin ) bone marrow ( bm ) cells from was ( ko ) mice were transduced with sin - lv at a moi of 50 and transplanted into lethally irradiated was mice . in parallel , mock transduced was or c57bl/6 lin bm cells were transplanted as negative ( ko mock ) and positive ( wt mock ) controls , respectively . cells from donor mice were distinguishable from recipients by surface staining for the congenic cd45.1/cd45.2 alleles . we also performed secondary transplants to more rigorously test vector safety and to verify the reconstitution of wasp in long - term repopulating hscs . at 1215 weeks post - transplant , peripheral blood ( pb ) samples were obtained for analysis of platelets ; 16 weeks post - transplant , mice were euthanized and a complete necropsy , as well as extensive phenotyping and functional assessment of hematopoietic cells within the thymus , bm , and spleen , was performed ( figure 1b ) . secondary recipients were similarly assessed ; however , thymus tissue was assessed only in the event that expansion of t cells was identified in the spleen or bm . analysis of lv - transduced donor lin bm cells demonstrated an equal viral copy number ( vcn ) and wasp expression for both sin - lv vectors at transplantation . wasp - expressing lin bm cells were 44% and 51% ( compared to 89% for wt mock ) after lv mnd.hwasp and 650.mnd.hwasp transduction , respectively ( figure 1c ) . at 16 weeks post - transplant , we identified high donor engraftment levels ( based on the percentage of cells congenic for the donor cd45 allele ) in the bm ( 49%61% ) and in splenic neutrophil ( 60%78% ) , monocyte ( 59%73% ) , b cell ( 82%92% ) , and t cell ( 78%88% ) compartments in all treatment groups ( figure 1d ) . using a wasp - specific antibody for intracellular staining , we found that mnd.hwasp and 650.mnd.hwasp experimental groups exhibited wt levels of wasp expression in bm and splenic populations , including neutrophils , monocytes , b cells , cd4 and cd8 t cells , and natural killer ( nk ) cells ( figures 2a2d , s1a , and s1b ) , with expression levels significantly increased relative to ko mock recipients . there was no statistically significant difference in the mean fluorescence intensity ( mfi ) of wasp intracellular staining among the two lv - treated groups ( figure s1c ) . myeloid cells lack a selective advantage for wasp expression ; thus , the fraction of wasp cells at this time point likely represents the fraction of early progenitors transduced with lv.39 , 40 an increase in the fraction of wasp cells in a peripheral subset over that observed in myeloid - derived neutrophils ( or monocytes ) therefore implies a selective advantage for wasp cells . in each cohort , we found that splenic b cells , cd4 and cd8 t cells , and nk cells had a significantly higher percentage of wasp cells compared to splenic neutrophils ( 17% and 27% in the insulated and non - insulated lvs , respectively ; solid lines in figures 2a2d ) . interestingly , we also observed a significant increase in splenic wasp nk1.1 cd3 cells in the 650.mnd.hwasp ( 48% ) versus the mnd.hwasp ( 35% ) experimental groups ( figure 2a ) , suggesting an increased selection for nk cells using the insulated lv . we also analyzed splenic lymphocytes by surface markers that allowed us to define b and t cell sub - populations along with intracellular wasp levels ( figures 2b2d ) . our findings mostly replicated those reported previously for a non - insulated mnd.hwasp lv . in both gene therapy cohorts , we observed higher percentages of wasp cells in each of the b and t cell subsets analyzed , compared to that in the myeloid compartment , indicating selection for was expression . those that were statistically significant for both gene therapy cohorts were post - transitional b cells , including mz precursors ( mzps ) , marginal zone ( mz ) and follicular mature ( fm ) b cells , and cd4 effector memory ( tem ) and regulatory ( treg ) subsets . a statistically significant selective advantage for cd8 t cells was observed only in central memory ( tcm ) and tem subsets for the 650.mnd.hwasp cohort . the numerical reconstitution of splenic lymphocyte subsets was consistent with dependence on wasp expression ( figures 2e2 g ) : lymphocyte sub - populations where wasp expression provided selective advantage in general had increased numbers in the lv - treated cohorts . there were no significant differences in cell numbers when comparing the insulated versus non - insulated lv . based on the above observations , we conclude that gene therapy using the 650.mnd.hwasp lv rescues development of affected hematopoietic cell lineages comparably to the non - insulated vector . a prominent finding in patients with was is bleeding due to a deficit in platelet numbers and function . an important consideration for was gene therapy then is the restoration of wasp levels in platelets.3 , 4 it should be noted that the platelet defect in was mice is mild relative to that seen in human subjects , so a change in blood platelet counts was not expected.41 , 42 we obtained platelets from pb 1215 weeks post - transplantation for analysis by flow cytometry for intracellular wasp expression ( figures 2h and 2i ) . we found that both lvs increased the percentage of wasp platelets relative to ko mock - treated animals . the mfi of wasp platelets in both gene therapy cohorts was similar to that of wild - type platelets , confirming the ability of the mnd promoter to drive efficient transgene expression in this lineage . wasp has a role in actin polymerization downstream of the t cell receptor ( tcr ) and its loss impacts tcr responses including proliferation of murine cd4 t cells.41 , 42 note that cd8 t cell tcr responses are only partially impacted by the loss of wasp . we tested the proliferation of murine splenic t cells in vitro in response to anti - cd3 and anti - cd28 stimulation ( cd3/28 ) , read as dilution of celltrace violet ( a fluorescent succinimidyl ester dye ) from stained cells by flow cytometry . as expected , ko mock cd4 t cells proliferated minimally in response to cd3/28 , while a substantial fraction of those from lv - treated or wt cohorts had proliferated by 72 hr . the percentage of cells that had proliferated at this time point was not significantly different between the two sin - lv treatments . we consistently found that was cd8 t cells proliferated in response to tcr stimulation even without gene therapy treatment , but lv treatment increased the overall proportion of cells proliferating in response to cd3 ( figures 3a and 3b ) . control cd4 and cd8 t cells from all treatment groups proliferated equally well in response to phorbol 12-myristate 13-acetate ( pma)-ionomycin ( p / i ) , which activates the tcr signaling pathway downstream of wasp . contrary to t cells , wasp deficiency renders b cells hyper - responsive to antigen receptor stimulation.21 , 43 however , wasp deficiency impacts b cell motility and homing in response to chemotactic agents , ultimately affecting b cell development and selection . to determine whether sin - lvs corrected this motility defect , we measured the number of total splenic b cells ( cd43-depleted splenocytes ) migrating across a transwell membrane into media with or without the chemokine cxcl13 . for both gene therapy cohorts , the addition of cxcl13 increased the percentage of b cells migrating across the transwell membrane ( figure 3c ) . a significantly higher fraction of b cells from the 650.mnd.hwasp lv cohort migrated relative to ko mock b cells , and this was comparable to the fraction of wt b cells that had migrated . thus , 650.mnd.hwasp lv rescues the homing defect in was b cells . in the presence of wasp - expressing t cells , wasp - deficient b cells trigger spontaneous germinal center responses that lead to autoantibody production and autoimmune disease in mice.21 , 22 , 23 these findings are likely to explain the increased rates of autoimmune disease in human patients with was with low - level donor chimerism following hsct . as quantified by elisa in this study , we found that total serum immunoglobulin g ( igg ) and igm levels were similar across treatment groups ( figure 3d ) . in contrast , there was a trend for reduced anti - dna autoantibodies present in ko mock recipients ( assessed as total igg or by the pathogenic igg2c subclass ) in mnd - lv - treated cohorts , approaching levels present in wt mock in 650.mnd.hwasp lv - treated mice ( figure 3e ) . these findings suggest that 650.mnd.hwasp lv drives wasp expression at levels that may limit the subsequent risk for autoimmune disease . secondary transplants were performed to determine whether 650.mnd.hwasp targeted long - term repopulating hscs and to test relative safety.45 , 46 bm cells from each primary recipient ( 16 weeks post - primary transplant ) were transplanted into two irradiated secondary recipients ( 10 10 cells / recipient ) . we performed extensive analysis of the secondary recipients at week 16 by flow cytometry , histology , hematology , and clinical chemistry . compared to primary donors , secondary recipients from all donors ( including wt mock controls ) had a smaller proportion of wasp cells within the myeloid , b cell , and t cell compartments in the bm and spleen ( figures 4a and 4b ) . interestingly , secondary recipients from the 650.mnd.hwasp group had higher proportions of wasp cells compared to the mnd.hwasp group in hematopoietic lineages of the bm ( figure 4a ) and spleen ( figure 4b ) . additionally , only the insulated lv improved was expression in platelets ( figure 4c ) , although for both vectors , platelets that did express wasp did so at equivalent levels ( mfi ) to those from the wt mock cohorts . we next determined the average vcn by qpcr in total splenocytes and bm from primary and secondary recipients ( 16 weeks after their respective bm transplants ; table 1 ) . in primary recipients , the average vcn per cell was increased in the splenic compartment relative to the bm , reflecting positive selection of wasp lymphocytes ( figure 4d ) , and the differences between the two lv groups were not statistically significant . the vcn within the bm of secondary recipients was lower than that of the primary recipients , but again increased in the spleen . notably , 650.mnd.hwasp lv - treated secondary recipients exhibited a significantly higher vcn within the spleen and a larger proportion of wasp , lin hscs within the bm compared to animals treated with mnd - hwasp lv ( figures 4d and 4e , respectively ) . these combined findings imply that inclusion of the insulator exerts a significant impact by allowing stable wasp transgene expression , thus providing a selective advantage to cells with the insulated wasp transgene . mice transplanted with 650.mnd.hwasp sin - lv - treated cells ( either as primary or secondary transplants ) had no statistically significant change in survival rates compared with the other gene therapy cohorts ( figures 5a and 5b ) . table 1 summarizes the mortality and morbidity events occurring in the course of this study . of 61 primary transplanted mice , three died from unknown causes within 4 weeks of their transplant , all from the larger 650.mnd.hwasp lv cohort . note that 32 of the primary recipient mice received 650.mnd.hwasp lv gene therapy ; the other 29 were divided into the remaining three cohorts . mortality events occurring within 4 weeks post - transplant are likely to be due to the conditioning regimen , and the rates they occurred in this study as a whole are lower than those historically seen in previous lv gene therapy models we have performed ( 7% ) , although the rate for the insulated lv group alone ( 9.4% ) is higher . at the 16-week study endpoint , we identified two mice with thymic lymphomas : one received 650.mnd.hwasp gene therapy - treated cells ( mouse 19 ) , and the other received wt mock cells ( mouse 36 ) . the cd4cd8 lymphoma of mouse 19 was identified both by flow cytometry and by histopathology of thymus and spleen ( figure 5c ) . using flow cytometry and antibodies specific to donor and recipient congenic markers , we determined that this lymphoma was recipient derived ( figure 5d ) ; consistent with this conclusion , there were no viral integrations ( by qpcr ) within genomic dna ( gdna ) obtained from thymic tissue of this mouse ( table 2 ) . mouse 36 from the wt mock cohort developed a thymic lymphoma identified during histopathology screening but not detected by flow cytometry analysis . we were unable to determine whether this tumor was donor or recipient derived using congenic antibodies . flow cytometry of harvested hematopoietic tissues and extensive histological analysis post - transplantation did not reveal evidence of clonal expansion within any of the other primary transplant recipients . complete blood counts ( cbcs ) and serum chemistries of pb were identical between the treatment groups , except for one of the mice with lymphoma ( mouse 19 ; figures s2 and s3 ) . the lifespans of secondary recipients were not significantly different than those of the primary recipients . five of the 105 secondary recipients died within 4 weeks of the transplant procedure from unknown causes : three of these mice were from the 650.mnd.hwasp gene therapy cohort , and two were from the ko mock cohort ( table 1 ) . at the 16-week endpoint , we identified five secondary transplanted mice with clonal expansion of t cells by flow - based phenotyping ( table 2 ) : two were from the 650.mnd.hwasp cohort and consisted of recipient - derived ( based on congenic markers and lack of vcn ) cd4cd8 ( mouse 116 ) and cd8 ( mouse 155 ) t cells ; three animals in the mnd.hwasp cohort ( mice 68 , 69 , and 128 ) developed cd4cd8 recipient - derived lymphomas ( and also had abnormal hematological and clinical chemistries ; figures s4 and s5 ) . we identified two additional probable lymphomas by histological analyses ( not detected by flow cytometry ) within the 650.mnd.hwasp gene therapy group ( mice 111 and 153 ) . because these tumors were identified only by histopathology , after tissue was fixed and embedded , vcn and congenic marker expression could not be assessed . a variety of radiation - associated lesions were identified during histological examination ( table s1 ) ; these were uniformly distributed across all groups . the prevalence of recipient - derived lymphomas in this was gene therapy model is consistent with the oncogenicity of the conditioning regimen in a genetically susceptible immunodeficient ( was ) mouse in the c57bl/6 background . overall , we found no evidence of clonal toxicity associated with integration of the 650.mnd.hwasp lv in this extensive pre - clinical study . to assess the safety and the integration profile of sin - lv with various internal promoters in a large animal model , we performed gene therapy in healthy juvenile pigtailed macaques ( macaca nemestrina ) using a cd34 autologous hsc myeloablative transplant protocol . similar to our mouse gene therapy studies , we first compared the efficacy and safety of lv containing either the human 1.6-kb was promoter ( ws1.6 ; currently in use in clinical trials)10 , 12 or the mnd promoter ( figure 6a ) . in a competitive repopulation experiment , equivalent numbers of marked cells ( 7.2 10 cd34 cells from each transduction / kg ; total of 14.4 10/kg body weight ) were infused into animal f09002 following 1,020-cgy total body irradiation . pb samples demonstrated hematopoietic recovery and engraftment of gene - modified cells within 3 weeks following transplant and stable cbcs and serum chemistries over a > 30-month follow - up period ( figures s6 and s7 ) . initial flow cytometry showed gene - modified cell levels of 30% and 15% for the mnd ( yellow fluorescent protein [ yfp ] ) and was1.6 ( gfp ) promoter - encoding lv - transduced cells among pb leukocytes , and these values were stably maintained for more than 1,000 days ( figure 6b ) . we observed stable granulocyte and lymphocyte marking up to the latest time point of follow - up ( 1,153 days ) , with 38.7% yfp and 15.6% gfp granulocytes and 27.0% yfp and 15% gfp lymphocytes ( data not shown ) . we also observed a marked increase in the mfi for yfp cells compared to gfp cells ( figures 6c and 6d ) , consistent with the robust expression expected from the mnd promoter compared to the was1.6 promoter . to assess the diversity of clonal contributions to this pool , we performed high - throughput retroviral integration site ( ris ) analysis on flow - sorted yfp and gfp pb leukocytes 502 and 1,153 days post - transplant . modified genome sequencing ( mgs)-pcr of gdna from sorted cells was used to determine genomic sites of provirus integration . we observed highly diverse integration sites in all samples assayed , with distinct clonal contributions in each transduced cell population ( figure 6e ) . in total , we identified 4,699 and 1,688 unique integration sites of the yfp and gfp lvs , respectively . the most abundant clone constituted 1.5% ( yfp ) and 3.7% ( gfp ) of the integration site sequences identified , implying a highly polyclonal expansion of progenitors . we identified a higher number of distinct clones in the yfp cell population , suggesting that increased gene marking in this population is not due to expansion of a single or small number of clones . riss were mapped on all chromosomes , and comparison of the global genomic distribution of integration sites in these samples ( figure 6f ) showed no promoter - specific changes in distribution pattern . this animal displayed no evidence for adverse events at nearly 4 years post - transplantation , and gene marking remained stable . owing to the correlation between retroviral promoter activity and insertional mutagenesis observed in -retrovirus - mediated gene therapy trials for both x - scid and was,13 , 14 , 15 we next tested whether inclusion of a chromatin insulator in the mnd promoter sin - lv could improve safety while maintaining efficient gene transfer , engraftment , and clonal diversity in this animal model . we performed a three - arm competitive repopulation assay ( figure s8a ) using cd34 cells transduced with either chs4.mnd.gfp ( using the 1.2-kb full - length chicken -globin insulator element ) , 650.mnd.mcherry , or 400.mnd.bfp ( an 400-bp insulator fragment ) . we observed only small differences in in vitro transduction efficiency of cd34 cells between the three lv vectors ( 38% gfp , 23% mcherry , and 35% blue fluorescent protein [ bfp ] ) . transduced cells were transplanted into the autologous myeloablated animal a11207 ( 3.5 , 4.3 , and 2.9 10 transduced cells / kg for chs4.mnd.gfp , 650.mnd.mcherry , and 400.mnd.bfp sin - lvs , respectively ; total of 10.7 10 cd34 cells / kg of body weight ) . the transplanted animal demonstrated hematopoietic recovery and engraftment of gene - modified cells within 1 week after transplant . all three arms engrafted equivalently , as evidenced by the levels of each fluorescent protein in pb . this sustained expression and gene marking supported stable hematopoiesis in this animal for nearly 2 years with no adverse events ( figures s6s8b ) . stable granulocyte and lymphocyte marking was observed up to the latest time point of follow - up ( 782 days ) , with 1.5% gfp , 1.4% mcherry , and 0.9% bfp granulocytes and 0.9% gfp , 0.4% mcherry , and 1.7% bfp lymphocytes ( data not shown ) . to assess the diversity of clonal contributions to this pool , we collected pb and bm at approximately the same time point after transplant ( days 221 and 223 post - transplant for pb for bm , respectively ) and we used fluorescence - activated cell sorting ( facs ) to sort pb leukocytes by fluorescent protein expression to distinguish between the three insulator elements . cd34 leukocytes from the bm were also purified to correlate clonal contributions observed in the pb fluorescent protein - sorted populations . ris analysis of gdna isolated from sorted and purified cells was then performed by mgs - pcr . highly diverse integration sites were observed in all samples assayed , with distinct clonal contributions in each transduced cell population ( figure s8c ) . we identified 265392 unique riss for sorted gfp , mcherry , and bfp pb leukocytes and 396 unique riss in total bm cd34 cells . there were no significant differences in the number of clones observed for each sample , which is consistent with the similar levels of gene marking in each cell population . we identified a total of six clones in the bm cd34 cell population that were also identified in one of the three pb fluorescent protein - sorted populations , demonstrating the contribution from a cd34 cell residing in the bm to a marked pb leukocyte . of these , three were gfp ( full - length insulator ) , two were mcherry ( 650-bp insulator ) , and one was bfp ( 400-bp insulator ) . of the cd34 bm clones identified , the two most prevalent contributors were both from the gfp lineage ( full - length insulator ) . after establishing the genomic positions of all lv integration sites for both animals ( f09002 and a11207 ) , we compared their positions to the 2010 rhemac3 refseq gene list available from the ucsc genome browser using a custom python script . for each insertion site , we determined the distance to the nearest transcription start site ( tss ) relative to the direction of transcription ( figures s9a and s9b ) and gene name . if the insertion fell within a gene , the gene name and genomic feature ( i.e. , within an intron , etc . ) were recorded . in both animals f09002 and a11207 , we observed an equivalent number of integration events within 100 kb of tss regardless of the promoter ( f09002 ) or insulator element ( a11207 ) tested . for animal a11207 , genes proximal to the integration sites identified at 96 , 221 , and 392 days after transplant were compared to the established list of oncogenes from the cosmic cancer gene database ( http://cancer.sanger.ac.uk/cosmic ) ( table s2 ) . of 21 integrations identified , all were within unique proto - oncogenes ; only one identical integration was found at two time points ( 96 and 392 days post - transplant ; fbxw7 , f - box and wd repeat domain containing 7 , e3 ubiquitin protein ligase ) . the integration was 69.4 kb downstream of the transcription start site of this tumor suppressor gene , within intron 3 . the contribution of this clone was < 0.1% of overall hematopoiesis at both time points , implying a lack of a biological impact from this integration during this time period . together , these data support the transduction efficiency , engraftment , and clonal diversity of insulated lv vector - transduced autologous cd34 hematopoietic cells in the clinically relevant non - human primate animal model , without evidence of insertional mutagenesis . here we report evidence of long - term efficacy and safety of an sin - lv with an insulated mnd internal promoter for was gene therapy in was mice and for a non - human primate model using a reporter gene . in primary recipient was mice , we found similar features of restored immune cell function in both gene therapy cohorts ( 650.mnd.hwasp and mnd.hwasp ) , including stable engraftment of wasp - expressing cells in all hematopoietic lineages examined ; progressive selection for wasp cells among t , nk , and b cell lineages ; correction of t and b cell development and function ; and restoration of wild - type levels of wasp expression in platelets . these findings expand upon those of a previous murine gene therapy study using a non - insulated mnd.hwasp sin - lv that used a different lv backbone than used here . in the current study , mice receiving the insulated 650.mnd.hwasp gene therapy also exhibited a trend for lower levels of autoimmune - associated anti - dna autoantibodies of the igg and igg2c subtypes compared to animals treated with the non - insulated lv construct . in our murine gene therapy experiments , serial bm transfers into secondary was recipients were performed to verify long - term hematopoietic stem cell ( lt - hsc ) transduction and long - term efficacy of the viral vectors , and to increase the sensitivity of detecting vector - induced transformation events in vivo . we found that the 650.mnd.hwasp gene therapy vector resulted in superior immune cell reconstitution in the secondary recipients compared with the non - insulated mnd.hwasp lv , including significantly higher percentages of wasp immune cells in bm , spleen , and pb . additionally , although the number of viral integrations per cell in bm and splenic cells of primary recipients were similar for both lv gene therapy cohorts , both the number of viral integrations and wasp expression in hscs and progeny was significantly higher in 650.mnd.hwasp-treated secondary recipients by the 16-week experimental endpoint . these striking observations are consistent with the interpretation that the insulated vector more effectively prevents epigenetic silencing in transduced long - term repopulating hscs . in this scenario , higher - level wasp expression in donor hscs ( derived from primary recipients ) is predicted to facilitate increased competitive repopulation in secondary recipients , an outcome that would also promote the observed selective expansion of wasp differentiated lymphoid subsets in 650.mnd.hwasp secondary recipients . this idea is supported by previous work showing that migration defects in wasp - deficient hscs hamper long - term hematopoietic reconstitution in a competitive repopulation setting . together , these findings strongly imply that inclusion of the 650-insulator element is likely to improve sustained expression in lt - hscs in a clinical application . there were no statistically significant differences in survival rates between treatment groups in this study . despite large cohorts receiving gene therapy - treated hscs ( 42 primary and 70 secondary transplant recipients ) , six incidences of clonally expanded cells were identified by flow cytometry at autopsy ( one primary and five secondary recipients ) ; all originated from recipient progenitor cells , with no viral integrations detected by pcr . in addition , three cases of thymic lymphoma , including two in the secondary 650.mnd.hwasp lv and one in the primary wt mock treatment groups , were identified based only on detailed examination of histological sections . while we were unable to definitively determine whether these latter tumors were donor or recipient derived , this rate of lymphoma development is consistent with our previous baseline observations in was - deficient recipient mice . compared with mice , large animal gene therapy trials more closely model the number of transduced lt - hscs that must be transplanted into a human subject , as well as their higher proliferative demands over a longer time span . because of the close genetic relationship between humans and nhps , nhp hscs respond to many of the recombinant human cytokines and other reagents used for clinical mobilization , selection , culture , transduction , and myeloablative total body irradiation ( tbi).52 , 53 in order to directly compare expression from two different internal promoters in the same animal , we used fluorescent reporter genes ( gfp and yfp ) transferred using sin - lv gene therapy of bm from a pigtailed macaque , followed by autologous transplantation . here we found that the proportion of cells in the periphery expressing either fluorescent protein was stable over 3 years . however , expression from the mnd promoter was evident in a higher proportion and at a higher mfi in pb cells of all cell lineages compared with that of the ws1.6 promoter . using a similar strategy in a second pigtailed macaque gene therapy experiment , we were able to simultaneously test three different fragments of the canonical 1.2-kb chs4 insulator ( upstream of the mnd - fluorescent protein cassette ) for their ability to prevent clonal dominance . as suggested by in vitro assays , insertional mutagenesis was not detected even after 2 years , and engraftment remained polyclonal with no evidence of clonal dominance ( i.e. , no individual clonal contribution of > 20% to the total pool identified ) . these data imply that increased expression elicited by the viral mnd promoter over the ws1.6 promoter in the context of a sin lentivirus vector does not compromise transduction efficiency or clonal diversity of autologous cd34 hematopoietic cells in the clinically relevant non - human primate animal model . although there was no change in the level of engrafted gene - modified cells as a function of the different insulator elements used , inclusion of an insulator may reduce overall engraftment levels when the number of gene - modified pb leukocytes is compared between animals f09002 and a11207 . finally , although the non - insulated mnd promoter showed insertional transactivation when inserted proximal to the lmo2 locus in jurkat t cells by cre - mediated cassette exchange and in a previous murine study,20 , 28 we found no evidence for toxicity in our murine or nhp studies , suggesting that these animal models , as has been previously noted,46 , 54 may lack the sensitivity to distinguish between lv vectors with low rates of genotoxic events . together , these studies suggest that the use of an insulated sin - lv vector incorporating an mnd internal promoter may strike a necessary balance for was gene therapy : driving sufficient expression of wasp to stably restore the immune cell deficits of was and wt levels of wasp expression in platelets while minimizing the potential of integrated proviral cassettes to create transactivational mutagenesis . furthermore , our observations regarding the capacity of this vector to reconstitute both lymphocyte function and wasp expression in platelets strongly support consideration of the 650.mnd.hwasp sin - lv for clinical gene therapy trials for the treatment of was . finally , our data suggest that use of insulated sin - lv vectors harboring mnd internal promoter expression cassettes may be considered for other hematopoietic disorders in which sustained high - level transgene expression in multiple lineages is required to achieve developmental and/or functional reconstitution . the sin - lv backbone pcl20cw has been described previously.55 , 56 plasmid pcl.hs4 has the full 1.2-kb hs4 insulator element from the chicken -globin locus inserted into the 3 u3 region of the viral ltr of pcl20cw . pcl400 contains 400 bp of the insulator sequence from the 3 end of the 1.2-kb insulator fragment , while pcl650 has 650 bp of the insulator ( comprising the 250-bp core and 400 bp from the 3 end of the 1.2-kb insulator fragment ) integrated into the 3 ltr . we used the mnd promoter as the internal promoter driving transgene expression in all vectors except pcl.ws1.6.gfp , which instead uses the 1.6-kb human was promoter to drive gfp expression . cdna encoding human wasp was inserted downstream of the mnd promoter in a pcl650 backbone to generate pcl650.mnd.wasp , and wasp was replaced by gfp to make pcl650.mnd.gfp . pcl.400,mnd.mcherry had the coding region for mcherry in a pcl400 backbone and pcl.mnd.yfp was created by insertion of an mnd.yfp cassette into the pcl backbone without any insulator element . third - generation lv stocks were generated by transient transfection of hek293 t cells with the vector plasmid , pcagkgp1.1r ( packaging ) , pcag4-rtr2 ( rev ) , and pcag4 vsvg ( envelope ) plasmids.58 , 59 forty - eight hours post - transfection , the viral supernatant was filtered through a 0.22-m filter and centrifuged at 8,500 g overnight at 4c . the virus was suspended in hank s balanced salt solution to achieve a 100-fold concentration , then stored at 80c . lv stocks for pcl.650.mnd.wasp were generated from a tet - regulated version of the same ( tl- cl.650.mnd.wasp ) by transfection of ligated vector concatamers into gprt - g helper cells as described previously . lv titers were determined by infection of nalm-6 cells with serial dilutions of viral stocks and detection of proviral sequences by qpcr . viral titers were calculated as proviral integrations using a standard curve generated from gdna of a nalm-6 clone with a single copy of integrated provirus . strains of mice used were c57bl/6 ( wt ) , was ( b6:129-was crossed into the c57bl/6 background for nine generations ) , and wt and was strains that were cross - bred with b6.sjl-ptprc pepc / boyj ( jackson laboratories ) for congenic marking with the cd45.1 ( ptprc ) allele ( c57bl/6 mice have the cd45.2/ptprc allele ) . mice were maintained in a specific pathogen - free facility at the seattle children s research institute ( scri ) . all studies were performed according to the association for assessment and accreditation of laboratory animal care ( aaalac ) standards and were approved by the scri institutional animal care and use committee ( iacuc ) . methods for collection and lv transduction of murine lin bm , and transplantation of lv - treated cells into irradiated recipients , were similar to those previously described.20 , 60 bm cells isolated from euthanized 6- to 8-week - old donor mice ( wt or was ; congenically marked either cd45.1 or cd45.2 ) were enriched for lineage - negative cells using the easysep mouse hematopoietic progenitor cell enrichment kit ( stemcell technologies ) . lin cells ( 4 10 cells / ml ) in hsc media ( stemspan sfem ; stemcell technologies ) with murine stem cell factor ( scf ) and thrombopoietin ( 50 and 20 ng / ml , respectively ) were transduced without ( mock ) or with lv at a moi of 50 for 10 hr ( viral dose was administered twice , 5 hr apart ) . the lv used was either clinical - grade 650.mnd.hwasp ( titer : 2 10 viral particles [ vps]/ml ) or non - insulated mnd.hwasp ( titer : 4.8 10 vp / ml ) . after 10-hr viral transduction , donor cells were washed with pbs and 2 10 cells were transferred by intravenous ( i.v . ) injection into irradiated 6- to 8-week - old was recipients ( the irradiation regimen was two 450-cgy doses administered 4 hr apart , the last one immediately prior to transplant ) . all was recipients had congenically distinct cd45 alleles from that of the donor cells . in parallel , 0.5 10 mock or lv - transduced lin bm cells were cultured in vitro in hsc media for 7 days to compare wasp expression and viral copy number of input cells using the two lentiviral constructs . the health status of all ( primary and secondary ) recipient animals post - transplant was evaluated daily by assessment of body condition . mice that reached predefined humane endpoints were euthanized and analyzed for evidence of clonal expansion of donor or recipient immune cells , histopathology , and serum chemistry . all other recipients were euthanized 16 weeks post - transplant , and tissues were analyzed as described . bm collected from primary recipients at the 16-week time point was also serially transplanted into secondary recipients as described below . pb samples were collected in lithium - coated tubes for cbc and serum chemistry analyses ( mouse profile no . 892 ; phoenix central laboratories ) or in heparin for flow cytometry - based immunophenotyping ( below ) . a full necropsy was performed , which included gross visual inspection of all organs and documentation of organomegaly . major organs were weighed and then fixed in formalin ; for the spleen and thymus , sections were removed for formalin fixation , and the remaining spleen and thymus were then re - weighed and processed for immunophenotyping and functional assays described below . paraffin embedding , sectioning , and hematoxylin and eosin staining were performed by the histology and imaging core at the university of washington s department of comparative medicine . organs examined included the heart , skeletal muscle , tongue , thyroid glands , lungs , esophagus , liver , kidneys , adrenal glands , thymus , spleen , salivary glands , stomach , small intestine , cecum , colon , pancreas , reproductive organs , skin , brain ( cerebellum and cerebrum ) , pituitary glands , eyes , harderian glands , ears , and bm . thymus and spleen were processed for immunophenotyping and functional assays ( below ) as follows . single cell suspensions were obtained by dissociating tissues with frosted glass slides , followed by erythrocytes lysis with ammonium - chloride - potassium buffer ( ack ; 0.15 m ammonium chloride , 10.0 mm potassium bicarbonate , and 0.1 mm edta ) and filtration through a 40-m mesh . serum from heparinized pb and bm collected in pbs were also subjected to erythrocyte lysis in ack and filtration using a 40-m mesh . cells were stained with fluorochrome - labeled monoclonal antibodies against cell surface markers , as well as a polyclonal wasp antibody for intracellular wasp expression , using the cytofix / cytoperm kit ( bd biosciences ) as described . stained cells were analyzed on an lsr ii flow cytometer ( bd biosciences ) and facs data were analyzed with flowjo software ( tree star ) . the absolute numbers of splenic and thymic cells were calculated using accucount rainbow fluorescent beads ( spherotech ) , extrapolating from the percentage of each tissue processed . platelets were isolated from whole peripheral blood and stained for intracellular wasp and platelet surface marker cd41 expression , as described previously . total bm from each experimental and control gene therapy primary recipient mouse was transplanted into two secondary recipient mice ( 10 10 cells per mouse ) . secondary recipients received the same conditioning regimen as described above and had the same congenic cd45 allele as the primary recipient . primary recipients with less than 5% wasp expression in myeloid cells , or with less than 20% engraftment of donor myeloid cells , were excluded from secondary bm transfer experiments . mice were monitored and analyzed at 16 weeks post - transplantation as described above for the primary recipient mice . gdna was extracted from total spleen or bm cells using the qiamp dna blood mini kit ( qiagen ) . proviral integrations were detected using the following gag - specific primers and probe : forward primer ( 5-ggagctagaacgattcgcagtta-3 ) , reverse primer ( 5-ggttgtagctgtcccagtatttgtc-3 ) , and probe ( 5-[6fam]acagccttctgatgtttctaacaggccagg[bhq1]-3 ) . dna content was normalized using the taqman gene expression array for murine -actin ( life technologies ) . maxima probe / rox qpcr master mix ( fermentas ) was used for amplification with 100 ng input dna and qpcrs were run on abi 7500 real - time pcr system ( applied biosystems ) . proviral integrations were quantified using a standard curve established using gdna extracted from a murine b cell line harboring six lv integrations per diploid genome . splenic cells were cultured in lymphocyte media ( rpmi with 55 m 2-mercaptoethanol and 10% fetal calf serum ) , and incubations were at 37c with 5% co2 . for testing t cell proliferation , total splenic cells were labeled with celltrace violet ( thermo fisher ) and stimulated with 1 g / ml plate - bound anti - cd3 and 0.25 g / ml soluble cd28 ( clones okt-3 and 9.3 , respectively ; university of california , san francisco monoclonal antibody core ) , followed by a 72-hr incubation and then flow cytometry for wasp expression and celltrace violet dilution . as a positive control , some cells were stimulated with 2 ng / ml pma and 500 ng / ml ionomycin . the b cell migration assays were performed using cd43-depleted splenocytes ( cd43 microbeads ; miltenyi biotec ) . 10/ml ) was seeded in duplicate upper wells of a 5-m transwell plate ( corning costar ) . the lower wells had 600 l lymphocyte media and 300 nm recombinant murine cxcl13 ( r&d systems ) . following a 1-hr incubation , the numbers of cells migrating to the lower well were counted , and the percentage of cells migrating was calculated based on the input cell number . serum anti - dna igg and igg2c antibodies levels were quantified by elisa as described . healthy juvenile pigtailed macaques ( m. nemestrina ) weighing between 3 and 8 kg were housed at the washington regional primate research center under conditions approved by aaalac . all studies and procedures were reviewed and approved by the university of washington iacuc . animals were given a 4-day ( days 7 to 4 ) regimen of recombinant human ( rh ) cytokines including granulocyte colony - stimulating factor ( rhg - csf ; 100 g / kg ; amgen ) and stem cell factor ( rhscf ; 50 g / kg ; amgen ) as subcutaneous ( s.c . ) injections . one day later ( day 3 ) , marrow volumes totaling 10% body weight in kilograms were aspirated from the femora and/or humeri and collected in preservative - free heparin . briefly , cells were incubated with immunoglobulin m monoclonal antibody ( clone 12.8 ) at 4c for 30 min , washed , and then incubated with rat monoclonal anti - mouse immunoglobulin m microbeads for an additional 30 min at 4c , followed by immunomagnetic column separation ( miltenyi biotec ) per the manufacturer s instructions . resulting cd34 cells were incubated at 37c and 5% co2 in a humidified incubator in stemspan sfem media containing 100 ng / ml each of rhscf , flt3-l , and thrombopoietin ( tpo ) for a period of 1618 hr . following this pre - stimulation , cells were then divided into equivalent aliquots and were transferred into non - tissue culture - treated vessels pre - coated with 2 g / cm ch-296 ( retronectin ) . cells were exposed to virus at an moi of 5 infectious units ( iu ) per cell for 8 hr . an additional volume of virus equivalent to 5 iu / cell was added to each culture and cells were cultured overnight for a final moi of 10 iu / cell . in preparation for transplantation , animals received myeloablative tbi ( 1,020 cgy ) administered from a linear accelerator at 7 cgy / min in four equally divided fractions over 2 days ( days 2 and 1 ) . the following day , cells were harvested and washed and a sample was collected to determine colony - forming capacity and transduction efficiency prior to infusion ( day 0 ) through a central vein catheter . following transplantation , supportive treatments of rhg - csf ( 100 g / kg ) were administered daily i.v . until a neutrophil count of > 500/l leukocytes were collected from heparinized pb and bm after ammonium chloride lysis at multiple time points after transplant . expression of fluorescent proteins ( gfp , yfp , mcherry , or bfp ) was analyzed by flow cytometry on either a facs canto or lsr ii cell analyzer ( both from bd biosciences ) . fluorochrome expression in lineage - specific populations was determined by gating based on forward and right - angle light scatter characteristics or by expression of lineage - specific cd markers using flowjo software ( version 9 or 10 ; tree star ) . gdna was extracted from leukocytes collected at various time points from either pb or bm or fluorochrome or cd marker - sorted populations by a qiagen blood dna mini kit per the manufacturer s instructions . resulting sequence libraries were subjected to ion torrent semiconductor sequencing and resulting sequence reads were analyzed using the vector integration site analysis ( visa ) server ( grant trobridge , washington state university ) . genomic sequences were mapped to the rhesus macaque genome ( rhemac3 ) using a stand - alone version of blat available from the ucsc genome browser ( http://genome.ucsc.edu/ ) . sequences corresponding to the same integration locus were grouped together to determine the total number of unique integration sites ( clones ) identified in the sample . relative contributions of each clone were determined by the number of integration site ( is)-associated sequence reads corresponding to that clone . a quality control check was performed to reveal clones over - represented by pcr bias by comparing the number of is - associated sequence reads with the number of different fragment lengths observed for each genomic locus . tests of statistical significance were performed using the unpaired two - tailed student s t test ; p values less than 0.05 were considered significant . d.j.r . conceived , designed , and directed the overall project ; h .- p.k . , j.a . , and a.n .

wiskott - aldrich syndrome ( was ) is a life - threatening immunodeficiency caused by mutations within the was gene . viral gene therapy to restore was protein ( wasp ) expression in hematopoietic cells of patients with was has the potential to improve outcomes relative to the current standard of care , allogeneic bone marrow transplantation . however , the development of viral vectors that are both safe and effective has been problematic . while use of viral transcriptional promoters may increase the risk of insertional mutagenesis , cellular promoters may not achieve wasp expression levels necessary for optimal therapeutic effect . here we evaluate a self - inactivating ( sin ) lentiviral vector combining a chromatin insulator upstream of a viral mnd ( mpsv ltr , ncr deleted , dl587 pbs ) promoter driving wasp expression . used as a gene therapeutic in was/ mice , this vector resulted in stable wasp+ cells in all hematopoietic lineages and rescue of t and b cell defects with a low number of viral integrations per cell , without evidence of insertional mutagenesis in serial bone marrow transplants . in a gene transfer experiment in non - human primates , the insulated mnd promoter ( driving gfp expression ) demonstrated long - term polyclonal engraftment of gfp+ cells . these observations demonstrate that the insulated mnd promoter safely and efficiently reconstitutes clinically effective wasp expression and should be considered for future was therapy .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

wiskott - aldrich syndrome ( was ) is an x - linked primary immunodeficiency disease resulting from mutations within the was gene that result in the loss of was protein ( wasp ) or function . subjects with was suffer from combined immunodeficiency , thrombocytopenia , and eczema and also frequently develop autoimmunity and lymphoid malignancies.1 , 2 , 3 , 4 wasp is a scaffold protein involved in signal transduction pathways that activate the actin cytoskeleton downstream of multiple cell surface receptors , including the b and t cell antigen receptors.5 , 6 , 7 although the disease phenotype can be alleviated with hematopoietic stem cell transplantation ( hsct ) , the success of this therapy is variable , depending on factors such as the patient s age , donor compatibility , conditioning regimen , and the extent of reconstitution . in the absence of a histocompatibility leukocyte antigen ( hla)-matched donor , transplantation with a mismatched donor has a reduced survival rate.3 , 8 , 9 since the phenotype of was deficiency impacts only hematopoietic cells , gene therapy is a possible alternative . previous and ongoing clinical trials have demonstrated the efficacy of gene therapy for alleviating the pathologies of was.10 , 11 , 12 importantly , following development of t cell leukemia due to insertional mutagenesis in -retroviral gene therapy trials for both severe combined immunodeficiency ( scid ) and was,13 , 14 , 15 much research has focused on strategies for eliminating this risk . the use of self - inactivating ( sin ) lentiviruses ( lvs ) for gene transfer is one critical improvement , combining a safer integration profile ( less affinity for insertions near promoters than -retroviruses16 , 17 , 18 ) with the ability to select internal promoters that optimize transgene expression and safety . these considerations are particularly important for treating was based on the following findings : sub - endogenous levels of wasp expression may hinder the reconstitution of murine b cell , t cell , and myeloid subsets and platelets ; insufficient wasp expression in b cells compared to t cells can drive acquisition of autoimmunity;21 , 22 , 23 and patients with was are predisposed to malignancies and clonal expansion.1 , 3 , 4 current clinical trials for was utilize a sin - lv with an internal promoter consisting of the proximal 1.6 kb of the endogenous was promoter ( ws1.6 ) to drive human wasp ( hwasp ) expression.10 , 12 patients treated with this sin - lv showed improvements in immunity to infections , resolved eczema , and protection from bleeding , without evidence of clonal expansion of cells10 , 12 or loss of self - tolerance.24 , 25 however , clinical improvement required relatively high levels of viral marking and alleviation of the was phenotype was incomplete with , most notably , limited or no improvement in platelet counts . in previous mouse gene therapy experiments , we found that the ws1.6 promoter did not effectively rescue wasp expression in all lineages including b cells and resulted in the acquisition of features of humoral autoimmunity . in contrast , an sin - lv using a synthetic promoter derived from a -retrovirus called mnd ( mpsv ltr , ncr deleted , dl587 pbs ) as an internal promoter rescued wasp expression in all affected lineages and reduced the risk of autoimmunity.20 , 27 , 28 in a clinical gene therapy trial for adrenoleukodystrophy , mnd has been used as an internal promoter for lv gene therapy without adverse effects . although strongly trans - activating promoters are associated with an enhanced risk of insertional transactivation , this risk can be diminished by including a chromatin insulator.28 , 30 , 31 chromatin insulators act as boundary elements preventing interactions between adjacent chromatin domains.32 , 33 , 34 candidate insulator constructs can function as barrier only elements ( acting as barriers to epigenetic chromatin silencing ) or enhancer - blocker only elements ( that prevent promoter transactivation between the domains that they separate ) , or they can have both activities ( as reviewed in emery et al . additionally , the insulated mnd lv did not promote a pre - leukemic block in differentiation of primary murine thymocytes following transduction and in vitro culture . our group also previously tested a series of chs4-insulated and non - insulated sin - lv constructs containing various internal promoters ( mnd , ef1 , and was 1.6-kb and 0.5-kb promoters ) driving gfp reporter gene expression in a mouse gene therapy model . hsct with lv containing the 650-bp chs4-insulated mnd - gfp ( 650.mnd.gfp ) resulted in gfp expression in all hematopoietic lineages , including platelets , that was stable over time . we also evaluated the safety profile of these sin - lv vectors in non - human primates ( nhps ) , expressing gfp instead of hwasp to improve assay sensitivity . our combined observations strongly support future use of the insulated mnd promoter for clinical was gene delivery . we evaluated the ability of the insulated sin - lv 650.mnd.hwasp ( figure 1a ) , generated from a clinical - grade producer cell line , to stably reconstitute wasp expression in the appropriate hematopoietic cell lineages using a was mouse gene therapy model . expression from this lv was compared , in parallel , to the non - insulated version , mnd.hwasp , that previously demonstrated reconstitution of was hematopoietic cell defects . at 1215 weeks post - transplant , peripheral blood ( pb ) samples were obtained for analysis of platelets ; 16 weeks post - transplant , mice were euthanized and a complete necropsy , as well as extensive phenotyping and functional assessment of hematopoietic cells within the thymus , bm , and spleen , was performed ( figure 1b ) . secondary recipients were similarly assessed ; however , thymus tissue was assessed only in the event that expansion of t cells was identified in the spleen or bm . using a wasp - specific antibody for intracellular staining , we found that mnd.hwasp and 650.mnd.hwasp experimental groups exhibited wt levels of wasp expression in bm and splenic populations , including neutrophils , monocytes , b cells , cd4 and cd8 t cells , and natural killer ( nk ) cells ( figures 2a2d , s1a , and s1b ) , with expression levels significantly increased relative to ko mock recipients . myeloid cells lack a selective advantage for wasp expression ; thus , the fraction of wasp cells at this time point likely represents the fraction of early progenitors transduced with lv.39 , 40 an increase in the fraction of wasp cells in a peripheral subset over that observed in myeloid - derived neutrophils ( or monocytes ) therefore implies a selective advantage for wasp cells . in each cohort , we found that splenic b cells , cd4 and cd8 t cells , and nk cells had a significantly higher percentage of wasp cells compared to splenic neutrophils ( 17% and 27% in the insulated and non - insulated lvs , respectively ; solid lines in figures 2a2d ) . interestingly , we also observed a significant increase in splenic wasp nk1.1 cd3 cells in the 650.mnd.hwasp ( 48% ) versus the mnd.hwasp ( 35% ) experimental groups ( figure 2a ) , suggesting an increased selection for nk cells using the insulated lv . our findings mostly replicated those reported previously for a non - insulated mnd.hwasp lv . there were no significant differences in cell numbers when comparing the insulated versus non - insulated lv . based on the above observations , we conclude that gene therapy using the 650.mnd.hwasp lv rescues development of affected hematopoietic cell lineages comparably to the non - insulated vector . a prominent finding in patients with was is bleeding due to a deficit in platelet numbers and function . an important consideration for was gene therapy then is the restoration of wasp levels in platelets.3 , 4 it should be noted that the platelet defect in was mice is mild relative to that seen in human subjects , so a change in blood platelet counts was not expected.41 , 42 we obtained platelets from pb 1215 weeks post - transplantation for analysis by flow cytometry for intracellular wasp expression ( figures 2h and 2i ) . the mfi of wasp platelets in both gene therapy cohorts was similar to that of wild - type platelets , confirming the ability of the mnd promoter to drive efficient transgene expression in this lineage . for both gene therapy cohorts , the addition of cxcl13 increased the percentage of b cells migrating across the transwell membrane ( figure 3c ) . in the presence of wasp - expressing t cells , wasp - deficient b cells trigger spontaneous germinal center responses that lead to autoantibody production and autoimmune disease in mice.21 , 22 , 23 these findings are likely to explain the increased rates of autoimmune disease in human patients with was with low - level donor chimerism following hsct . compared to primary donors , secondary recipients from all donors ( including wt mock controls ) had a smaller proportion of wasp cells within the myeloid , b cell , and t cell compartments in the bm and spleen ( figures 4a and 4b ) . interestingly , secondary recipients from the 650.mnd.hwasp group had higher proportions of wasp cells compared to the mnd.hwasp group in hematopoietic lineages of the bm ( figure 4a ) and spleen ( figure 4b ) . in primary recipients , the average vcn per cell was increased in the splenic compartment relative to the bm , reflecting positive selection of wasp lymphocytes ( figure 4d ) , and the differences between the two lv groups were not statistically significant . mortality events occurring within 4 weeks post - transplant are likely to be due to the conditioning regimen , and the rates they occurred in this study as a whole are lower than those historically seen in previous lv gene therapy models we have performed ( 7% ) , although the rate for the insulated lv group alone ( 9.4% ) is higher . we identified two additional probable lymphomas by histological analyses ( not detected by flow cytometry ) within the 650.mnd.hwasp gene therapy group ( mice 111 and 153 ) . the prevalence of recipient - derived lymphomas in this was gene therapy model is consistent with the oncogenicity of the conditioning regimen in a genetically susceptible immunodeficient ( was ) mouse in the c57bl/6 background . similar to our mouse gene therapy studies , we first compared the efficacy and safety of lv containing either the human 1.6-kb was promoter ( ws1.6 ; currently in use in clinical trials)10 , 12 or the mnd promoter ( figure 6a ) . owing to the correlation between retroviral promoter activity and insertional mutagenesis observed in -retrovirus - mediated gene therapy trials for both x - scid and was,13 , 14 , 15 we next tested whether inclusion of a chromatin insulator in the mnd promoter sin - lv could improve safety while maintaining efficient gene transfer , engraftment , and clonal diversity in this animal model . together , these data support the transduction efficiency , engraftment , and clonal diversity of insulated lv vector - transduced autologous cd34 hematopoietic cells in the clinically relevant non - human primate animal model , without evidence of insertional mutagenesis . here we report evidence of long - term efficacy and safety of an sin - lv with an insulated mnd internal promoter for was gene therapy in was mice and for a non - human primate model using a reporter gene . in primary recipient was mice , we found similar features of restored immune cell function in both gene therapy cohorts ( 650.mnd.hwasp and mnd.hwasp ) , including stable engraftment of wasp - expressing cells in all hematopoietic lineages examined ; progressive selection for wasp cells among t , nk , and b cell lineages ; correction of t and b cell development and function ; and restoration of wild - type levels of wasp expression in platelets . these findings expand upon those of a previous murine gene therapy study using a non - insulated mnd.hwasp sin - lv that used a different lv backbone than used here . in the current study , mice receiving the insulated 650.mnd.hwasp gene therapy also exhibited a trend for lower levels of autoimmune - associated anti - dna autoantibodies of the igg and igg2c subtypes compared to animals treated with the non - insulated lv construct . in our murine gene therapy experiments , serial bm transfers into secondary was recipients were performed to verify long - term hematopoietic stem cell ( lt - hsc ) transduction and long - term efficacy of the viral vectors , and to increase the sensitivity of detecting vector - induced transformation events in vivo . we found that the 650.mnd.hwasp gene therapy vector resulted in superior immune cell reconstitution in the secondary recipients compared with the non - insulated mnd.hwasp lv , including significantly higher percentages of wasp immune cells in bm , spleen , and pb . additionally , although the number of viral integrations per cell in bm and splenic cells of primary recipients were similar for both lv gene therapy cohorts , both the number of viral integrations and wasp expression in hscs and progeny was significantly higher in 650.mnd.hwasp-treated secondary recipients by the 16-week experimental endpoint . these striking observations are consistent with the interpretation that the insulated vector more effectively prevents epigenetic silencing in transduced long - term repopulating hscs . in this scenario , higher - level wasp expression in donor hscs ( derived from primary recipients ) is predicted to facilitate increased competitive repopulation in secondary recipients , an outcome that would also promote the observed selective expansion of wasp differentiated lymphoid subsets in 650.mnd.hwasp secondary recipients . this idea is supported by previous work showing that migration defects in wasp - deficient hscs hamper long - term hematopoietic reconstitution in a competitive repopulation setting . together , these findings strongly imply that inclusion of the 650-insulator element is likely to improve sustained expression in lt - hscs in a clinical application . despite large cohorts receiving gene therapy - treated hscs ( 42 primary and 70 secondary transplant recipients ) , six incidences of clonally expanded cells were identified by flow cytometry at autopsy ( one primary and five secondary recipients ) ; all originated from recipient progenitor cells , with no viral integrations detected by pcr . compared with mice , large animal gene therapy trials more closely model the number of transduced lt - hscs that must be transplanted into a human subject , as well as their higher proliferative demands over a longer time span . here we found that the proportion of cells in the periphery expressing either fluorescent protein was stable over 3 years . however , expression from the mnd promoter was evident in a higher proportion and at a higher mfi in pb cells of all cell lineages compared with that of the ws1.6 promoter . using a similar strategy in a second pigtailed macaque gene therapy experiment , we were able to simultaneously test three different fragments of the canonical 1.2-kb chs4 insulator ( upstream of the mnd - fluorescent protein cassette ) for their ability to prevent clonal dominance . as suggested by in vitro assays , insertional mutagenesis was not detected even after 2 years , and engraftment remained polyclonal with no evidence of clonal dominance ( i.e. these data imply that increased expression elicited by the viral mnd promoter over the ws1.6 promoter in the context of a sin lentivirus vector does not compromise transduction efficiency or clonal diversity of autologous cd34 hematopoietic cells in the clinically relevant non - human primate animal model . finally , although the non - insulated mnd promoter showed insertional transactivation when inserted proximal to the lmo2 locus in jurkat t cells by cre - mediated cassette exchange and in a previous murine study,20 , 28 we found no evidence for toxicity in our murine or nhp studies , suggesting that these animal models , as has been previously noted,46 , 54 may lack the sensitivity to distinguish between lv vectors with low rates of genotoxic events . together , these studies suggest that the use of an insulated sin - lv vector incorporating an mnd internal promoter may strike a necessary balance for was gene therapy : driving sufficient expression of wasp to stably restore the immune cell deficits of was and wt levels of wasp expression in platelets while minimizing the potential of integrated proviral cassettes to create transactivational mutagenesis . furthermore , our observations regarding the capacity of this vector to reconstitute both lymphocyte function and wasp expression in platelets strongly support consideration of the 650.mnd.hwasp sin - lv for clinical gene therapy trials for the treatment of was . finally , our data suggest that use of insulated sin - lv vectors harboring mnd internal promoter expression cassettes may be considered for other hematopoietic disorders in which sustained high - level transgene expression in multiple lineages is required to achieve developmental and/or functional reconstitution . we used the mnd promoter as the internal promoter driving transgene expression in all vectors except pcl.ws1.6.gfp , which instead uses the 1.6-kb human was promoter to drive gfp expression . viral titers were calculated as proviral integrations using a standard curve generated from gdna of a nalm-6 clone with a single copy of integrated provirus . in parallel , 0.5 10 mock or lv - transduced lin bm cells were cultured in vitro in hsc media for 7 days to compare wasp expression and viral copy number of input cells using the two lentiviral constructs . cells were stained with fluorochrome - labeled monoclonal antibodies against cell surface markers , as well as a polyclonal wasp antibody for intracellular wasp expression , using the cytofix / cytoperm kit ( bd biosciences ) as described . primary recipients with less than 5% wasp expression in myeloid cells , or with less than 20% engraftment of donor myeloid cells , were excluded from secondary bm transfer experiments . following a 1-hr incubation , the numbers of cells migrating to the lower well were counted , and the percentage of cells migrating was calculated based on the input cell number . sequences corresponding to the same integration locus were grouped together to determine the total number of unique integration sites ( clones ) identified in the sample .

lung carcinoma is the most common cause of cancer mortality world - wide . because patients often present at a late stage , average 5-year survival is approximately 15% . non - small cell lung carcinoma ( nsclc ) pathology predominates , representing approximately 80% of all lung carcinomas . recently , adenocarcinoma surpassed squamous cell carcinoma as the most common form of nsclc in the united states . because of differences in therapy , distinguishing adenocarcinoma from squamous cell carcinoma by morphology and/or immunohistochemistry furthermore , the introduction of tyrosine kinase inhibitors ( tkis ) heralds entry into an era of personalized medicine , where genotype identification and not solely phenotype is critical to optimizing therapy . identification of mutations in adenocarcinomas , including in epidermal growth factor receptor ( egfr ) or kirsten rat sarcoma ( kras ) virus oncogene homolog , among others , determines eligibility for treatment with targeted therapies , clinical trials , and/or ruling out tki - therapy . evaluating mutation status before initiating therapy is important because tki administration to patients with unknown mutation status is , overall , detrimental rather than beneficial . introduction of target - specific therapy for lung adenocarcinoma has coincided with the performance of greater numbers of minimally invasive procedures , including computed tomography - guided ( ct - guided ) fine - needle aspiration ( fna ) biopsy , endobronchial ultrasound - guided ( ebus ) fna biopsy , and navigational bronchoscopic fna biopsy for diagnosis and/or staging of pulmonary and mediastinal lesions or serial testing to monitor therapeutic response and/or drug resistance . because resection of locally advanced or metastatic disease as first line therapy is discouraged , fewer than 30% of patients are surgical candidates at diagnosis . in contrast to histopathologic resection specimens , cytologic specimens , including fna biopsies and exfoliative specimens , have relatively lower cellular volume and may represent the only tissue available for immunohistochemical and/or molecular testing . there is concern by some as to whether cytology specimens are acceptable for molecular testing . routine use of procedures that provide higher quantities of tissue has been proposed because low tumor volume or proportion may theoretically cause mutant alleles to fall below the level of detection and be masked by non - neoplastic cells resulting in reporting of false negative results . alternatively , a potential concern is that cytology specimens may not be representative since carcinomas demonstrate intratumoral histologic and genetic heterogeneity , and a mutated clone may be missed . according to recently published guidelines from the college of american pathologists , international association for the study of lung cancer ( iaslc ) , and association for molecular pathology , cytologic samples are suitable for egfr testing , and cell blocks ( cbs ) are the preferred medium . however , while aspirate cytology - derived cb material is more convenient than and generally preferred to direct smears for mutation status evaluation , direct comparison of specific mutations between matched histology and cytology specimens has been predominantly reported for analysis of smear derived material . in this study , molecular testing results of fna cytology cb specimens are evaluated and compared with corresponding histologic specimens to validate the expert consensus guidelines . patients with available histology and cytology samples that both contained lung adenocarcinoma were identified retrospectively over a period of 122 months . patient age at the time of diagnosis , sex of the patient , interval between acquisition of the cytology and histology specimens and their respective locations were recorded . targets of histopathologic ( biopsy and resection ) specimens and cytologic fna biopsies included lung lesions , lymph nodes ( lns ) , and distant metastases . histology specimens were collected by ct - guided , transthoracic , spring - loaded , core needle ( 20-gauge ) biopsy ( n = 3 ) , endoscopic , transbronchial biopsy ( n = 3 ) , video - assisted , thoracic surgical resection ( n = 8) or open surgical resections ( n = 16 , from 15 patients ) . one punch biopsy of a metastasis to skin was also collected . cytology specimens were collected by ct - guided - fna ( 22-gauge ) biopsy ( n = 8) or ebus - fna ( 21-gauge ) biopsy ( n = 18 ) with rapid on - site evaluation performed by a cytopathologist and/or cytotechnologist . cbs were prepared by allowing the specimen to clot and placing it directly into 10% neutral buffered formalin and/or fixing it in the needle rinse placed in cytolyt ( hologic ) . following centrifugation of the specimen in a 50 ml tube for 5 min , the supernatant was removed . well - formed clots were placed directly in bio - wrap ( leica biosystems , buffalo grove , il ) and fixed in 10% neutral buffered paraffin before paraffin embedding . for the remainder , histogel ( thermo fisher scientific , waltham , ma ) was added to the pellet and solidified in the refrigerator at 4c . solidified pellets were then placed in bio - wrap , fixed in 10% neutral buffered formalin , and embedded in paraffin . histologic and cytologic specimens were evaluated using the 2004 world health organization classification for lung tumors and the small biopsy and cytology classification proposed by the iaslc , american thoracic society and european respiratory society . at our institution , diagnosis of lung adenocarcinoma or adenosquamous carcinoma prompts reflex mutational analysis of egfr and kras . initially , the reflex testing was for kras , subsequently it was for both egfr and kras . depending on the available deoxyribonucleic acid ( dna ) and/or the test result , one or both tests were performed . ( fluorescence in situ hybridization to detect rearrangement of the anaplastic lymphoma kinase ( alk ) gene is also performed and mutational analysis of braf is subsequently undertaken if sufficient material remains ) . except when a specimen has no or few isolated cells on each slide , there are no strict criteria for the minimum number of cells for molecular testing . multiple serial sections of the block are utilized and stained with cresyl violet to identify and collect the neoplastic cells . when necessary , the carcinoma is microdissected manually or with laser capture , depending on the tumor content and its relationship to the surrounding non - neoplastic cells , to enrich the sample for molecular analysis . polymerase chain reactions ( pcr ) with flanking intronic primers were performed to amplify regions of interest and identify all mutations in egfr exons 18 - 21 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . raw sequence data were analyzed and aligned using seqscape ( life technologies , grand island , ny ) software . common kras mutations were detected with the kras codon 12/13 amplification - refractory mutation system - scorpions assay ( qiagen ) per the manufacturer 's instructions . briefly , real - time pcr with allele - specific primers covalently linked to fluorophores with signal quenchers was performed to amplify regions potentially containing seven common kras mutations ( listed in the supplementary data ) . the fluorophores and quenchers separate upon binding to amplified sequences , resulting in increased fluorescence in the reaction tubes . the number of cycles necessary to detect fluorescent signal above background indicated presence or absence of mutation . beginning in 2012 , a pcr - based method for identifying kras mutations was employed . briefly , pcr with flanking intronic primers were performed to amplify regions of interest in kras exon 2 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . patients with available histology and cytology samples that both contained lung adenocarcinoma were identified retrospectively over a period of 122 months . patient age at the time of diagnosis , sex of the patient , interval between acquisition of the cytology and histology specimens and their respective locations were recorded . targets of histopathologic ( biopsy and resection ) specimens and cytologic fna biopsies included lung lesions , lymph nodes ( lns ) , and distant metastases . histology specimens were collected by ct - guided , transthoracic , spring - loaded , core needle ( 20-gauge ) biopsy ( n = 3 ) , endoscopic , transbronchial biopsy ( n = 3 ) , video - assisted , thoracic surgical resection ( n = 8) or open surgical resections ( n = 16 , from 15 patients ) . one punch biopsy of a metastasis to skin was also collected . cytology specimens were collected by ct - guided - fna ( 22-gauge ) biopsy ( n = 8) or ebus - fna ( 21-gauge ) biopsy ( n = 18 ) with rapid on - site evaluation performed by a cytopathologist and/or cytotechnologist . cbs were prepared by allowing the specimen to clot and placing it directly into 10% neutral buffered formalin and/or fixing it in the needle rinse placed in cytolyt ( hologic ) . following centrifugation of the specimen in a 50 ml tube for 5 min , the supernatant was removed . well - formed clots were placed directly in bio - wrap ( leica biosystems , buffalo grove , il ) and fixed in 10% neutral buffered paraffin before paraffin embedding . for the remainder , histogel ( thermo fisher scientific , waltham , ma ) was added to the pellet and solidified in the refrigerator at 4c . solidified pellets were then placed in bio - wrap , fixed in 10% neutral buffered formalin , and embedded in paraffin . histologic and cytologic specimens were evaluated using the 2004 world health organization classification for lung tumors and the small biopsy and cytology classification proposed by the iaslc , american thoracic society and european respiratory society . at our institution , diagnosis of lung adenocarcinoma or adenosquamous carcinoma prompts reflex mutational analysis of egfr and kras . initially , the reflex testing was for kras , subsequently it was for both egfr and kras . depending on the available deoxyribonucleic acid ( dna ) and/or the test result , one or both tests were performed . ( fluorescence in situ hybridization to detect rearrangement of the anaplastic lymphoma kinase ( alk ) gene is also performed and mutational analysis of braf is subsequently undertaken if sufficient material remains ) . except when a specimen has no or few isolated cells on each slide , there are no strict criteria for the minimum number of cells for molecular testing . multiple serial sections of the block are utilized and stained with cresyl violet to identify and collect the neoplastic cells . when necessary , the carcinoma is microdissected manually or with laser capture , depending on the tumor content and its relationship to the surrounding non - neoplastic cells , to enrich the sample for molecular analysis . polymerase chain reactions ( pcr ) with flanking intronic primers were performed to amplify regions of interest and identify all mutations in egfr exons 18 - 21 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . raw sequence data were analyzed and aligned using seqscape ( life technologies , grand island , ny ) software . common kras mutations were detected with the kras codon 12/13 amplification - refractory mutation system - scorpions assay ( qiagen ) per the manufacturer 's instructions . briefly , real - time pcr with allele - specific primers covalently linked to fluorophores with signal quenchers was performed to amplify regions potentially containing seven common kras mutations ( listed in the supplementary data ) . the fluorophores and quenchers separate upon binding to amplified sequences , resulting in increased fluorescence in the reaction tubes . the number of cycles necessary to detect fluorescent signal above background indicated presence or absence of mutation . beginning in 2012 , a pcr - based method for identifying kras mutations briefly , pcr with flanking intronic primers were performed to amplify regions of interest in kras exon 2 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . egfr and/or kras testing were performed on histology and cytology samples from 26 patients , 18 female and 8 male ( mean age = 69 years , median age = 72 years ) . of the 26 samples , egfr and kras testing was performed on 13 , egfr only on 9 , and kras only on 4 . egfr mutation testing was performed on both histology and cytology specimens from 22 separate patients , including 5 men and 17 women with median age 73.5 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 15 negative for egfr mutation . five of the 15 pairs negative for egfr mutation were subsequently shown to harbor kras mutation ( patients 5 , 9 , 13 , 17 and 22 ) . eleven concordant sample pairs consisted of a primary tumor and its ln or distant metastasis ( patients 1 , 4 , 5 , 6 , 9 , 12 , 16 , 21 , 23 , 24 , and 26 ) . egfr mutation was identified in 5 of these 11 cancers ( patients 4 , 6 , 12 , 23 , and 24 ) . concordant results of molecular testing on cytology and histology specimens the histology specimen in the majority of patients consisted of a tumor resection . however , the histology specimen from 5 patients consisted of a small ( endoscopic snare or transthoracic ct - guided core needle ) biopsy ( patients 4 , 9 , 24 , 25 , and 26 ) . egfr mutation was identified in 2 of these 5 cancers ( patients 4 and 24 ) . in two additional patients ( 6 and 12 ) for whom egfr was evaluated on more than one histology specimen , one of the histology specimens consisted of a small biopsy . kras mutation testing was performed on both histology and cytology specimens from 17 separate patients , including 6 men and 11 women with median age 76 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 10 negative for kras mutation . two of the 10 pairs negative for kras mutation had been previously shown to harbor egfr mutation . seven sample pairs consisted of a primary tumor and its metastasis ( patients 5 , 8 , 9 , 19 , 21 , 23 , and 26 ) . kras mutation was identified in 4 of these 7 cancers ( patients 5 , 8 , 9 , and 19 ) . the histology specimen from 3 patients consisted of a small biopsy ( patients 9 , 25 , and 26 ) . egfr mutation testing was performed on both histology and cytology specimens from 22 separate patients , including 5 men and 17 women with median age 73.5 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 15 negative for egfr mutation . interestingly five of the 15 pairs negative for egfr mutation were subsequently shown to harbor kras mutation ( patients 5 , 9 , 13 , 17 and 22 ) . eleven concordant sample pairs consisted of a primary tumor and its ln or distant metastasis ( patients 1 , 4 , 5 , 6 , 9 , 12 , 16 , 21 , 23 , 24 , and 26 ) . egfr mutation was identified in 5 of these 11 cancers ( patients 4 , 6 , 12 , 23 , and 24 ) . concordant results of molecular testing on cytology and histology specimens the histology specimen in the majority of patients consisted of a tumor resection . however , the histology specimen from 5 patients consisted of a small ( endoscopic snare or transthoracic ct - guided core needle ) biopsy ( patients 4 , 9 , 24 , 25 , and 26 ) . egfr mutation was identified in 2 of these 5 cancers ( patients 4 and 24 ) . in two additional patients ( 6 and 12 ) for whom egfr was evaluated on more than one histology specimen , one of the histology specimens consisted of a small biopsy . kras mutation testing was performed on both histology and cytology specimens from 17 separate patients , including 6 men and 11 women with median age 76 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 10 negative for kras mutation . two of the 10 pairs negative for kras mutation had been previously shown to harbor egfr mutation . seven sample pairs consisted of a primary tumor and its metastasis ( patients 5 , 8 , 9 , 19 , 21 , 23 , and 26 ) . kras mutation was identified in 4 of these 7 cancers ( patients 5 , 8 , 9 , and 19 ) . the histology specimen from 3 patients consisted of a small biopsy ( patients 9 , 25 , and 26 ) . in recent years , a significant proportion of lung carcinomas have been diagnosed on cytology specimens obtained with minimally invasive techniques . recent trends demonstrate that use of minimally invasive procedures such as fnas for diagnosis of lung carcinoma is increasing . however , adequacy of cytology specimens for molecular testing is controversial because of contamination by non - neoplastic cells , lack of standardized cytological preparations , and relatively small size . the possibility of false negative results , particularly in specimens with scant tumor , may contribute to the reluctance to the use of cytology specimens for molecular testing . accordingly , in one study , < 5% of 239 samples tested for egfr mutation were obtained by fna . the current study demonstrates that cbs prepared from ebus and ct - guided fnas provide sufficient material for egfr and/or kras mutation analysis . in fact , fukui et al . achieved better results with cytological preparations than histological ones regardless of tumor volume . smouse et al . also demonstrated that mutation detection in cytological specimens is equally sensitive to that in histological specimens , and no significant difference exists between the frequency of inconclusive results observed in cytology and surgical pathology specimens . the results of the current study also illustrate that egfr and kras molecular testing results of cytology specimens are concordant with those of corresponding histology specimens in 39/39 tests performed - 22/22 egfr tests ( including 7/7 positive results ) and 17/17 kras tests ( including 7/7 positive results ) . previous studies have directly compared the results of mutational analysis predominantly between matched histology specimens and direct cytology smears ; we compare both egfr and kras results on matched histology specimens and aspirate cytology - derived cbs . though the published guidelines recommend prioritizing testing for egfr and alk , we test for kras mutation as part of a reflex protocol when sufficient tissue is available . this is by far the largest such study of lung adenocarcinoma , and the first to include comparison between both egfr and kras on cytological aspirate cb material and corresponding surgical pathology material . the concordant results indicate that cbs provide sufficient tissue for analysis beyond the recommended egfr molecular testing . khode et al . , performed only egfr analysis on 37 pairs of cytology smears and surgical pathology specimens of lung adenocarcinoma / nsclc . of those 37 pairs , only one showed discrepant egfr mutation results between the cytology smear and the biopsy specimen ( 97% concordance ) . aisner et al . have demonstrated the presence of the same egfr mutation in three cases for which resection and cytology evaluation had been performed on the same site in the same patient and a cytology cb was available . similarly , van eijk et al . in their study have reported complete concordance of the results of mutation analysis in a series of 17 cbs from nsclc patients with corresponding histology specimens . however , only four of their study patients received a diagnosis of adenocarcinoma , and two of them were found to harbor a kras mutation . sun et al . demonstrated an overall egfr concordance of 91.7% between histologic and cytologic specimens ; however , the cytologic specimens were not all obtained from the primary site from which the histologic specimens were obtained . they also performed no kras and only egfr analysis on both cbs and scrapings from smears . molecular testing results were concordant among all 13 sample pairs consisting of a primary tumor and its metastasis . of these , six sample pairs were evaluated for the presence of egfr mutation alone , and two tested positive . two pairs were evaluated for the presence of kras mutation alone , and both tested positive . of the five pairs in which both genes were evaluated , egfr mutation was identified in one pair and kras mutation was identified in two pairs . such perfect concordance is interesting as lung tumors are often heterogeneous , and phenotypic and genotypic differences between primary tumors and their metastases have been described . indeed , kalikaki et al . described discordance in 28% ( 7/25 ) and 24% ( 6/25 ) of primary and metastatic sites for egfr and kras , respectively . the 23 patients with matched pairs of cytology smears and surgical specimens from different anatomic sites showed a concordance rate of 82% . the cytological specimens from their study included pleural fluids , bronchial washings , and bronchial brushings in addition to aspirate material . however , for cases in which a diagnostic sample is available from only one site , primary or metastatic , performance of molecular testing on that lone sample may suffice to appropriately guide treatment . despite the small size of cytology samples , they provide material for molecular testing of equal or possibly greater quality than that of larger histology samples . the nsclc working group recommends using tissue blocks rich in neoplastic cells for molecular testing , and effective methods to enrich cytology samples for tumor currently exist . these include use of fixative containing a hemolytic agent , rapid on - site evaluation , or inclusion of a beacon or marker to visualize the level at which cells are concentrated . either formalin or alcohol is suitable for egfr testing , but other fixatives , including heavy metal fixatives ( e.g. , b5 , acid zinc formalin , zenker ) and acidic solutions ( e.g. , decalcifying solution , bouin ) , should be avoided because they interfere with testing . a high ratio of neoplastic to non - neoplastic cells predicts the ability of molecular testing to detect mutations . though reliable results with as few as 10 - 20% neoplastic cells have been reported , > 40 - 50% neoplastic cells are considered optimal for evaluation . the rarity of mutated cells in a specimen might exceed the sensitivity of the assay . macro - or micro - dissection can enrich the sample by increasing the ratio of neoplastic to non - neoplastic cells , and these techniques can be performed on cytology preparations . results of direct sequencing mutation detection with laser capture microdissection may increase yield from 21% to 40% . demonstrated that 50 tumor cells microdissected with laser capture from cytology specimens were sufficient for egfr and kras mutation detection . adequate results from other preparations , including liquid - based cytology preparations such as thinprep and scrapings from archival slides stained with romanowsky or papaniocolaou stains , have been reported . however , yields from cells in cytolyt are lower when compared to those obtained with microdissection of cells on a slide . cytology specimens pose challenges for pathologists , who must perform ancillary tests , including immunophenotyping and/or molecular testing , and for oncologists , who rely on results for selection of therapy . the data from the current study indicate high concordance between results of molecular testing performed on cytology cbs and histology specimens and between primary and metastatic sites . therefore , it is likely that results obtained from molecular testing of cytology specimens in lung adenocarcinoma are valid , and fna - derived cbs provide a valuable medium . each author has participated sufficiently in the work and takes public responsibility for appropriate portions of the content of this article . this study was conducted with approval from institutional review board ( irb ) of the institution associated with this study ( columbia university medical center ) . to ensure the integrity and highest quality of cytojournal publications , the review process of this manuscript was conducted under a double blind model ( authors are blinded for reviewers and vice versa ) through automatic online system .

background : lung cancer is a leading cause of mortality , and patients often present at a late stage . more recently , advances in screening , diagnosing , and treating lung cancer have been made . for instance , greater numbers of minimally invasive procedures are being performed , and identification of lung adenocarcinoma driver mutations has led to the implementation of targeted therapies . advances in molecular techniques enable use of scant tissue , including cytology specimens . in addition , per recently published consensus guidelines , cytology - derived cell blocks ( cbs ) are preferred over direct smears . yet , limited comparison of molecular testing of fine - needle aspiration ( fna ) cbs and corresponding histology specimens has been performed . this study aimed to establish concordance of epidermal growth factor receptor ( egfr ) and kirsten rat sarcoma ( kras ) virus homolog testing between fna cbs and histology samples from the same patients.materials and methods : patients for whom molecular testing for egfr or kras was performed on both fna cbs and histology samples containing lung adenocarcinoma were identified retrospectively . following microdissection , when necessary , concordance of egfr and kras molecular testing results between fna cbs and histology samples was evaluated.results:egfr and/or kras testing was performed on samples obtained from 26 patients . concordant results were obtained for all egfr ( 22/22 ) and kras ( 17/17 ) mutation analyses performed.conclusions:identification of mutations in lung adenocarcinomas affects clinical decision - making , and it is important that results from small samples be accurate . this study demonstrates that molecular testing on cytology cbs is as sensitive and specific as that on histology .

INTRODUCTION MATERIALS AND METHODS Patient specimens Histologic and cytologic interpretation EGFR and KRAS mutation status RESULTS Evaluation of EGFR mutation status Evaluation of KRAS mutation status DISCUSSION CONCLUSIONS COMPETING INTERESTS STATEMENT BY ALL AUTHORS AUTHORSHIP STATEMENT BY ALL AUTHORS ETHICS STATEMENT BY ALL AUTHORS EDITORIAL/PEER-REVIEW STATEMENT

lung carcinoma is the most common cause of cancer mortality world - wide . because patients often present at a late stage , average 5-year survival is approximately 15% . recently , adenocarcinoma surpassed squamous cell carcinoma as the most common form of nsclc in the united states . identification of mutations in adenocarcinomas , including in epidermal growth factor receptor ( egfr ) or kirsten rat sarcoma ( kras ) virus oncogene homolog , among others , determines eligibility for treatment with targeted therapies , clinical trials , and/or ruling out tki - therapy . evaluating mutation status before initiating therapy is important because tki administration to patients with unknown mutation status is , overall , detrimental rather than beneficial . introduction of target - specific therapy for lung adenocarcinoma has coincided with the performance of greater numbers of minimally invasive procedures , including computed tomography - guided ( ct - guided ) fine - needle aspiration ( fna ) biopsy , endobronchial ultrasound - guided ( ebus ) fna biopsy , and navigational bronchoscopic fna biopsy for diagnosis and/or staging of pulmonary and mediastinal lesions or serial testing to monitor therapeutic response and/or drug resistance . in contrast to histopathologic resection specimens , cytologic specimens , including fna biopsies and exfoliative specimens , have relatively lower cellular volume and may represent the only tissue available for immunohistochemical and/or molecular testing . there is concern by some as to whether cytology specimens are acceptable for molecular testing . routine use of procedures that provide higher quantities of tissue has been proposed because low tumor volume or proportion may theoretically cause mutant alleles to fall below the level of detection and be masked by non - neoplastic cells resulting in reporting of false negative results . alternatively , a potential concern is that cytology specimens may not be representative since carcinomas demonstrate intratumoral histologic and genetic heterogeneity , and a mutated clone may be missed . according to recently published guidelines from the college of american pathologists , international association for the study of lung cancer ( iaslc ) , and association for molecular pathology , cytologic samples are suitable for egfr testing , and cell blocks ( cbs ) are the preferred medium . however , while aspirate cytology - derived cb material is more convenient than and generally preferred to direct smears for mutation status evaluation , direct comparison of specific mutations between matched histology and cytology specimens has been predominantly reported for analysis of smear derived material . in this study , molecular testing results of fna cytology cb specimens are evaluated and compared with corresponding histologic specimens to validate the expert consensus guidelines . patients with available histology and cytology samples that both contained lung adenocarcinoma were identified retrospectively over a period of 122 months . patient age at the time of diagnosis , sex of the patient , interval between acquisition of the cytology and histology specimens and their respective locations were recorded . targets of histopathologic ( biopsy and resection ) specimens and cytologic fna biopsies included lung lesions , lymph nodes ( lns ) , and distant metastases . histology specimens were collected by ct - guided , transthoracic , spring - loaded , core needle ( 20-gauge ) biopsy ( n = 3 ) , endoscopic , transbronchial biopsy ( n = 3 ) , video - assisted , thoracic surgical resection ( n = 8) or open surgical resections ( n = 16 , from 15 patients ) . cytology specimens were collected by ct - guided - fna ( 22-gauge ) biopsy ( n = 8) or ebus - fna ( 21-gauge ) biopsy ( n = 18 ) with rapid on - site evaluation performed by a cytopathologist and/or cytotechnologist . well - formed clots were placed directly in bio - wrap ( leica biosystems , buffalo grove , il ) and fixed in 10% neutral buffered paraffin before paraffin embedding . for the remainder , histogel ( thermo fisher scientific , waltham , ma ) was added to the pellet and solidified in the refrigerator at 4c . solidified pellets were then placed in bio - wrap , fixed in 10% neutral buffered formalin , and embedded in paraffin . at our institution , diagnosis of lung adenocarcinoma or adenosquamous carcinoma prompts reflex mutational analysis of egfr and kras . initially , the reflex testing was for kras , subsequently it was for both egfr and kras . except when a specimen has no or few isolated cells on each slide , there are no strict criteria for the minimum number of cells for molecular testing . when necessary , the carcinoma is microdissected manually or with laser capture , depending on the tumor content and its relationship to the surrounding non - neoplastic cells , to enrich the sample for molecular analysis . polymerase chain reactions ( pcr ) with flanking intronic primers were performed to amplify regions of interest and identify all mutations in egfr exons 18 - 21 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . briefly , real - time pcr with allele - specific primers covalently linked to fluorophores with signal quenchers was performed to amplify regions potentially containing seven common kras mutations ( listed in the supplementary data ) . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . patients with available histology and cytology samples that both contained lung adenocarcinoma were identified retrospectively over a period of 122 months . patient age at the time of diagnosis , sex of the patient , interval between acquisition of the cytology and histology specimens and their respective locations were recorded . targets of histopathologic ( biopsy and resection ) specimens and cytologic fna biopsies included lung lesions , lymph nodes ( lns ) , and distant metastases . histology specimens were collected by ct - guided , transthoracic , spring - loaded , core needle ( 20-gauge ) biopsy ( n = 3 ) , endoscopic , transbronchial biopsy ( n = 3 ) , video - assisted , thoracic surgical resection ( n = 8) or open surgical resections ( n = 16 , from 15 patients ) . cytology specimens were collected by ct - guided - fna ( 22-gauge ) biopsy ( n = 8) or ebus - fna ( 21-gauge ) biopsy ( n = 18 ) with rapid on - site evaluation performed by a cytopathologist and/or cytotechnologist . well - formed clots were placed directly in bio - wrap ( leica biosystems , buffalo grove , il ) and fixed in 10% neutral buffered paraffin before paraffin embedding . for the remainder , histogel ( thermo fisher scientific , waltham , ma ) was added to the pellet and solidified in the refrigerator at 4c . solidified pellets were then placed in bio - wrap , fixed in 10% neutral buffered formalin , and embedded in paraffin . at our institution , diagnosis of lung adenocarcinoma or adenosquamous carcinoma prompts reflex mutational analysis of egfr and kras . initially , the reflex testing was for kras , subsequently it was for both egfr and kras . except when a specimen has no or few isolated cells on each slide , there are no strict criteria for the minimum number of cells for molecular testing . when necessary , the carcinoma is microdissected manually or with laser capture , depending on the tumor content and its relationship to the surrounding non - neoplastic cells , to enrich the sample for molecular analysis . polymerase chain reactions ( pcr ) with flanking intronic primers were performed to amplify regions of interest and identify all mutations in egfr exons 18 - 21 . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . briefly , real - time pcr with allele - specific primers covalently linked to fluorophores with signal quenchers was performed to amplify regions potentially containing seven common kras mutations ( listed in the supplementary data ) . dna was extracted from paraffin - embedded histology specimens and cbs using qiamp ( qiagen , inc . , cycle dideoxy terminator sequencing of the pcr amplicons was performed using the abi bigdye terminator ( applied biosystems , carlsbad , ca ) kit v1.1 per the manufacturer 's instructions . egfr and/or kras testing were performed on histology and cytology samples from 26 patients , 18 female and 8 male ( mean age = 69 years , median age = 72 years ) . of the 26 samples , egfr and kras testing was performed on 13 , egfr only on 9 , and kras only on 4 . egfr mutation testing was performed on both histology and cytology specimens from 22 separate patients , including 5 men and 17 women with median age 73.5 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 15 negative for egfr mutation . five of the 15 pairs negative for egfr mutation were subsequently shown to harbor kras mutation ( patients 5 , 9 , 13 , 17 and 22 ) . egfr mutation was identified in 5 of these 11 cancers ( patients 4 , 6 , 12 , 23 , and 24 ) . concordant results of molecular testing on cytology and histology specimens the histology specimen in the majority of patients consisted of a tumor resection . however , the histology specimen from 5 patients consisted of a small ( endoscopic snare or transthoracic ct - guided core needle ) biopsy ( patients 4 , 9 , 24 , 25 , and 26 ) . in two additional patients ( 6 and 12 ) for whom egfr was evaluated on more than one histology specimen , one of the histology specimens consisted of a small biopsy . kras mutation testing was performed on both histology and cytology specimens from 17 separate patients , including 6 men and 11 women with median age 76 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 10 negative for kras mutation . seven sample pairs consisted of a primary tumor and its metastasis ( patients 5 , 8 , 9 , 19 , 21 , 23 , and 26 ) . kras mutation was identified in 4 of these 7 cancers ( patients 5 , 8 , 9 , and 19 ) . the histology specimen from 3 patients consisted of a small biopsy ( patients 9 , 25 , and 26 ) . egfr mutation testing was performed on both histology and cytology specimens from 22 separate patients , including 5 men and 17 women with median age 73.5 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 15 negative for egfr mutation . interestingly five of the 15 pairs negative for egfr mutation were subsequently shown to harbor kras mutation ( patients 5 , 9 , 13 , 17 and 22 ) . eleven concordant sample pairs consisted of a primary tumor and its ln or distant metastasis ( patients 1 , 4 , 5 , 6 , 9 , 12 , 16 , 21 , 23 , 24 , and 26 ) . egfr mutation was identified in 5 of these 11 cancers ( patients 4 , 6 , 12 , 23 , and 24 ) . concordant results of molecular testing on cytology and histology specimens the histology specimen in the majority of patients consisted of a tumor resection . in two additional patients ( 6 and 12 ) for whom egfr was evaluated on more than one histology specimen , one of the histology specimens consisted of a small biopsy . kras mutation testing was performed on both histology and cytology specimens from 17 separate patients , including 6 men and 11 women with median age 76 [ table 1 ] . evaluation of all sample pairs yielded concordant results - 7 positive and 10 negative for kras mutation . seven sample pairs consisted of a primary tumor and its metastasis ( patients 5 , 8 , 9 , 19 , 21 , 23 , and 26 ) . kras mutation was identified in 4 of these 7 cancers ( patients 5 , 8 , 9 , and 19 ) . the histology specimen from 3 patients consisted of a small biopsy ( patients 9 , 25 , and 26 ) . in recent years , a significant proportion of lung carcinomas have been diagnosed on cytology specimens obtained with minimally invasive techniques . recent trends demonstrate that use of minimally invasive procedures such as fnas for diagnosis of lung carcinoma is increasing . however , adequacy of cytology specimens for molecular testing is controversial because of contamination by non - neoplastic cells , lack of standardized cytological preparations , and relatively small size . the possibility of false negative results , particularly in specimens with scant tumor , may contribute to the reluctance to the use of cytology specimens for molecular testing . accordingly , in one study , < 5% of 239 samples tested for egfr mutation were obtained by fna . the current study demonstrates that cbs prepared from ebus and ct - guided fnas provide sufficient material for egfr and/or kras mutation analysis . also demonstrated that mutation detection in cytological specimens is equally sensitive to that in histological specimens , and no significant difference exists between the frequency of inconclusive results observed in cytology and surgical pathology specimens . the results of the current study also illustrate that egfr and kras molecular testing results of cytology specimens are concordant with those of corresponding histology specimens in 39/39 tests performed - 22/22 egfr tests ( including 7/7 positive results ) and 17/17 kras tests ( including 7/7 positive results ) . previous studies have directly compared the results of mutational analysis predominantly between matched histology specimens and direct cytology smears ; we compare both egfr and kras results on matched histology specimens and aspirate cytology - derived cbs . though the published guidelines recommend prioritizing testing for egfr and alk , we test for kras mutation as part of a reflex protocol when sufficient tissue is available . this is by far the largest such study of lung adenocarcinoma , and the first to include comparison between both egfr and kras on cytological aspirate cb material and corresponding surgical pathology material . the concordant results indicate that cbs provide sufficient tissue for analysis beyond the recommended egfr molecular testing . , performed only egfr analysis on 37 pairs of cytology smears and surgical pathology specimens of lung adenocarcinoma / nsclc . have demonstrated the presence of the same egfr mutation in three cases for which resection and cytology evaluation had been performed on the same site in the same patient and a cytology cb was available . in their study have reported complete concordance of the results of mutation analysis in a series of 17 cbs from nsclc patients with corresponding histology specimens . however , only four of their study patients received a diagnosis of adenocarcinoma , and two of them were found to harbor a kras mutation . demonstrated an overall egfr concordance of 91.7% between histologic and cytologic specimens ; however , the cytologic specimens were not all obtained from the primary site from which the histologic specimens were obtained . they also performed no kras and only egfr analysis on both cbs and scrapings from smears . molecular testing results were concordant among all 13 sample pairs consisting of a primary tumor and its metastasis . of these , six sample pairs were evaluated for the presence of egfr mutation alone , and two tested positive . two pairs were evaluated for the presence of kras mutation alone , and both tested positive . of the five pairs in which both genes were evaluated , egfr mutation was identified in one pair and kras mutation was identified in two pairs . such perfect concordance is interesting as lung tumors are often heterogeneous , and phenotypic and genotypic differences between primary tumors and their metastases have been described . described discordance in 28% ( 7/25 ) and 24% ( 6/25 ) of primary and metastatic sites for egfr and kras , respectively . the cytological specimens from their study included pleural fluids , bronchial washings , and bronchial brushings in addition to aspirate material . however , for cases in which a diagnostic sample is available from only one site , primary or metastatic , performance of molecular testing on that lone sample may suffice to appropriately guide treatment . despite the small size of cytology samples , they provide material for molecular testing of equal or possibly greater quality than that of larger histology samples . the nsclc working group recommends using tissue blocks rich in neoplastic cells for molecular testing , and effective methods to enrich cytology samples for tumor currently exist . these include use of fixative containing a hemolytic agent , rapid on - site evaluation , or inclusion of a beacon or marker to visualize the level at which cells are concentrated . either formalin or alcohol is suitable for egfr testing , but other fixatives , including heavy metal fixatives ( e.g. , b5 , acid zinc formalin , zenker ) and acidic solutions ( e.g. a high ratio of neoplastic to non - neoplastic cells predicts the ability of molecular testing to detect mutations . though reliable results with as few as 10 - 20% neoplastic cells have been reported , > 40 - 50% neoplastic cells are considered optimal for evaluation . macro - or micro - dissection can enrich the sample by increasing the ratio of neoplastic to non - neoplastic cells , and these techniques can be performed on cytology preparations . demonstrated that 50 tumor cells microdissected with laser capture from cytology specimens were sufficient for egfr and kras mutation detection . adequate results from other preparations , including liquid - based cytology preparations such as thinprep and scrapings from archival slides stained with romanowsky or papaniocolaou stains , have been reported . cytology specimens pose challenges for pathologists , who must perform ancillary tests , including immunophenotyping and/or molecular testing , and for oncologists , who rely on results for selection of therapy . the data from the current study indicate high concordance between results of molecular testing performed on cytology cbs and histology specimens and between primary and metastatic sites . therefore , it is likely that results obtained from molecular testing of cytology specimens in lung adenocarcinoma are valid , and fna - derived cbs provide a valuable medium . this study was conducted with approval from institutional review board ( irb ) of the institution associated with this study ( columbia university medical center ) .

colorectal cancer is the fourth most common cause of cancer death in the world ( 0.69 million , 8.5% ) . early detection and treatment are essential for reducing high mortality from colorectal cancer , given that adenomas and early colorectal cancers are usually small and asymptomatic . however , participation in colorectal cancer screening shows a wide variation across different socioeconomic or regional conditions , e.g. , from 53% in oklahoma to 72% in delaware in the united states during 2006 - 2010 , from 33% in the most deprived area to 67% in the least deprived area in england for the year 2008 ( organized screening ) , and from 22% in gyeongnam ( a rural province ) to 28% in daejeon ( a metropolitan province ) in south korea ( korea hereafter ) for the year 2012 ( organized screening ) . this disparity in colorectal cancer screening might exacerbate disparity in health status , making more contribution to those with higher participation in this intervention . however , the current cost effectiveness analysis of colorectal cancer screening focuses on improving population health and ignores health disparity generated by this intervention . but recent studies show that the purposes of maximizing population health and minimizing health disparity often contradict with each other : the promotion of a targeted reminder designed to increase participation in organized colorectal cancer screening among deprived populations with ethnic diversity minimizes health disparity , whereas the promotion of a universal reminder designed to increase participation in organized colorectal cancer screening among the entire population maximizes population health in the united kingdom . for this reason , several researchers started to develop distributional cost effectiveness analysis ( dcea ) of healthcare interventions , a combination of cost effectiveness analysis and health disparity examination . socioeconomic disparity in organized colorectal cancer screening has slightly decreased in korea since 2005 , e.g. , 10.3% vs. 16.9% for medicaid vs. insured in 2006 , then 22.2% vs. 26.1% in 2012 . however , regional disparity in this intervention still persists in the nation , i.e. , from 22% in gyeongnam ( a rural province ) to 28% in daejeon ( a metropolitan province ) for 2012 . indeed , korea has much lower national participation in organized colorectal cancer screening than does the united kingdom , i.e. , 26% in 2012 vs. 52% in 2008 . in this context , this study evaluated the cost effectiveness of organized colorectal cancer screening interventions ( i.e. , screening with various types of reminders ) , with their impacts on regional health disparity being considered . markov cohort simulation was conducted for hypothetical cohorts aged 50 years as of the year 2013 in each of 16 korean provinces . the interventions until the age of 80 were ( 1 ) annual fecal occult blood test ( fobt ) ( standard screening ) , ( 2 ) annual fobt with basic reminder letters for eight provinces with higher mortalities from either all causes or colorectal cancer than the national average ( i.e. , busan [ metropolis ] , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam [ rural areas ] ) ( targeted reminder ) , ( 3 ) annual fobt with basic reminder letters for all provinces ( universal reminder 1 ) , and ( 4 ) annual fobt with enhanced reminder letters ( i.e. , personal reminder letters with tailored information packages ) for all provinces ( universal reminder 2 ) . the markov states were ( 1 ) healthy , ( 2 ) polyps not detected by screening ( p1 ) , ( 3 ) polyps detected by screening ( p2 ) , ( 4 ) symptom - free early colorectal cancer not detected by screening ( ecc1 ) , ( 5 ) symptom - free early colorectal cancer detected by screening ( ecc2 ) , ( 6 ) symptomatic early colorectal cancer ( ecc3 ) , ( 7 ) symptom - free advanced colorectal cancer not detected by screening ( acc1 ) , ( 8) symptom - free advanced colorectal cancer detected by screening ( acc2 ) , ( 9 ) symptomatic advanced colorectal cancer ( acc3 ) , ( 10 ) death from colorectal cancer , and ( 11 ) death from other causes ( the true positive goes through colorectal cancer treatment without further colorectal cancer screening ) . the length of the cycle was 1 year and the length of the duration was 40 years between the ages of 50 and 90 . atkinson incremental cost effectiveness ratio ( atkinson icer ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index ( to be elaborated below ) was used to evaluate the cost effectiveness of the four interventions with their impacts on health disparity being considered . 1 shows a simplified version of the markov model for cohort simulation . in each cycle , the healthy can stay healthy , develops polys ( p1/2 ) or dies from causes other than colorectal cancer . without treatment ( polypectomy ) , one with polyps ( 1 ) stays as he or she is , ( 2 ) develops early colorectal cancer with the annual polyp - ecc transition rate of 0.005 , i.e. , p1 to ecc1 , p2 to ecc2 , or ( 3 ) dies from causes other than colorectal cancer . without treatment , one with early colorectal cancer ( 1 ) stays as he or she is , ( 2 ) develops advanced colorectal cancer with the mean ecc dwelling time of 2 years , i.e. , ecc1 to acc1 , ecc2 to acc2 , ecc3 to acc3 , or ( 3 ) dies from causes other than colorectal cancer . also , without treatment , the mean sojourn time from symptom - free to symptomatic colorectal cancer ( i.e. , from ecc1/2 to ecc3 , from acc1/2 to acc3 ) is 5 years . a new incidence of polyps is detected by a screening , while a new incidence of colorectal cancer is detected by either a screening or a symptom . the incidence rate of polyps or colorectal cancer is the same between screened and unscreened groups . suspicious lesions detected by colonoscopy are biopsied and those detected by fobt receive colonoscopy ( and biopsy if applicable ) . patients who receive treatment either stay as cancer patients or die , and those who survived for 5 years after treatment have the same mortality rates with healthy people in the same age group . utilities for the 11 markov states came from a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . values on screening - related variables and the natural history of colorectal cancer were obtained from statistics korea , previous studies on cancer registry data ( for korea and for germany ) , existing literature on the cost effectiveness of colorectal cancer screening , and bayesian calibration for the natural history of colorectal cancer . data sources on survival after treatment were randomized trials through 13 and 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . the cost of a basic / enhanced reminder letter , fobt , colonoscopy and biopsy per participant in korea for 2013 were derived from randomized trials for the cost effectiveness of reminder letters for colorectal cancer screening in the united states and uk for 2005 and 2009 , respectively , the korea ministry of health and welfare notifications and the korea health insurance review and assessment service guidelines on health insurance medical cost including drug components and materials for medical treatment . based on the randomized trials , the basic ( or enhanced ) reminder letters are expected to increase the participation rate in colorectal cancer screening by 6% ( or 12% ) ( the cost of the reminder letter in the united states / united kingdom was adjusted by the ratio of korea s per - capita health expenditure to its united states / united kingdom counterpart for 2013 ) . for instance , screening participation was assumed to be higher by 6% in the targeted - reminder intervention than in the standard - screening intervention in busan , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam , e.g. , 27% , 35% , and 25% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in busan from supplementary table 1 , respectively . the participation gap between the universal - reminder-1 and standard - screening interventions was presumed to be 6% in every area , e.g. , 31% , 41% , and 29% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in seoul from supplementary table 1 , respectively . likewise , the participation difference between the universal - reminder-2 and standard - screening interventions was supposed to be 12% in every area , e.g. , 37% , 47% , and 35% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in seoul from supplementary table 1 , respectively . for the calculation of treatment cost per patient for colorectal cancer by the phase of care in korea for the year 2013 , treatment cost per patient for colorectal cancer from the korea national health insurance corporation was adjusted by the share of direct cost for cancer care in the united states for the year 2010 by the phase of care . the cost in korean won was converted to us dollars with the exchange rate of $ 1=1,055.4 won as of the year 2013 . the annual inflation rate adjusted by the annual discount rate for cost was assumed to be 1 . the crux of dcea is to adjust total effectiveness outcome ( the un - weighted mean or sum of individuals effectiveness outcomes in the conventional cost effectiveness analysis ) by an inequality index so that interventions with greater health disparity lead to smaller total effectiveness outcomes . ( 1 ) , a function of inequality aversion , i.e. , public aversion to health disparity . usually , inequality aversion on income distribution is measured as public opinion on the ideal rate of exchange between the incomes of those with highest income and those with the lowest income ( leaky bucket questionnaire ) . likewise , inequality aversion on health distribution can be measured as public opinion on the ideal rate of exchange between the health of those with the best health and those with the worst health . once the atkinson inequality index is calculated for every intervention , the atkinson icer in eq . ( 2 ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index , can be used to compare both cost effectiveness and health disparity from the interventions . ( 2 ) , is different from the conventional icer in the following manner : as inequality aversion increases , individuals with smaller qalys / worse health have more weights in total effectiveness outcome than do those with greater qalys / better health . the atkinson inequality index becomes 0 and the atkinson icer becomes the conventional icer when inequality aversion becomes 0 . in this vein , the atkinson inequality index and the atkinson icer were calculated to compare both cost effectiveness and health disparity from the standard screening , the targeted reminder and , the universal reminder 1 and 2 in korea . per - capita qalys and percapita cost were computed for each intervention for each area based on the markov cohort simulation described above ( 2014 , treeage software inc . , then , the atkinson inequality index and the atkinson icer for the nation were calculated based on the equations below . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion markov cohort simulation was conducted for hypothetical cohorts aged 50 years as of the year 2013 in each of 16 korean provinces . the interventions until the age of 80 were ( 1 ) annual fecal occult blood test ( fobt ) ( standard screening ) , ( 2 ) annual fobt with basic reminder letters for eight provinces with higher mortalities from either all causes or colorectal cancer than the national average ( i.e. , busan [ metropolis ] , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam [ rural areas ] ) ( targeted reminder ) , ( 3 ) annual fobt with basic reminder letters for all provinces ( universal reminder 1 ) , and ( 4 ) annual fobt with enhanced reminder letters ( i.e. , personal reminder letters with tailored information packages ) for all provinces ( universal reminder 2 ) . the markov states were ( 1 ) healthy , ( 2 ) polyps not detected by screening ( p1 ) , ( 3 ) polyps detected by screening ( p2 ) , ( 4 ) symptom - free early colorectal cancer not detected by screening ( ecc1 ) , ( 5 ) symptom - free early colorectal cancer detected by screening ( ecc2 ) , ( 6 ) symptomatic early colorectal cancer ( ecc3 ) , ( 7 ) symptom - free advanced colorectal cancer not detected by screening ( acc1 ) , ( 8) symptom - free advanced colorectal cancer detected by screening ( acc2 ) , ( 9 ) symptomatic advanced colorectal cancer ( acc3 ) , ( 10 ) death from colorectal cancer , and ( 11 ) death from other causes ( the true positive goes through colorectal cancer treatment without further colorectal cancer screening ) . the length of the cycle was 1 year and the length of the duration was 40 years between the ages of 50 and 90 . atkinson incremental cost effectiveness ratio ( atkinson icer ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index ( to be elaborated below ) was used to evaluate the cost effectiveness of the four interventions with their impacts on health disparity being considered . 1 shows a simplified version of the markov model for cohort simulation . in each cycle , the healthy can stay healthy , develops polys ( p1/2 ) or dies from causes other than colorectal cancer . without treatment ( polypectomy ) , one with polyps ( 1 ) stays as he or she is , ( 2 ) develops early colorectal cancer with the annual polyp - ecc transition rate of 0.005 , i.e. , p1 to ecc1 , p2 to ecc2 , or ( 3 ) dies from causes other than colorectal cancer . without treatment , one with early colorectal cancer ( 1 ) stays as he or she is , ( 2 ) develops advanced colorectal cancer with the mean ecc dwelling time of 2 years , i.e. , ecc1 to acc1 , ecc2 to acc2 , ecc3 to acc3 , or ( 3 ) dies from causes other than colorectal cancer . also , without treatment , the mean sojourn time from symptom - free to symptomatic colorectal cancer ( i.e. , from ecc1/2 to ecc3 , from acc1/2 to acc3 ) is 5 years . a new incidence of polyps is detected by a screening , while a new incidence of colorectal cancer is detected by either a screening or a symptom . the incidence rate of polyps or colorectal cancer is the same between screened and unscreened groups . suspicious lesions detected by colonoscopy are biopsied and those detected by fobt receive colonoscopy ( and biopsy if applicable ) . patients who receive treatment either stay as cancer patients or die , and those who survived for 5 years after treatment have the same mortality rates with healthy people in the same age group . utilities for the 11 markov states came from a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . values on screening - related variables and the natural history of colorectal cancer were obtained from statistics korea , previous studies on cancer registry data ( for korea and for germany ) , existing literature on the cost effectiveness of colorectal cancer screening , and bayesian calibration for the natural history of colorectal cancer . data sources on survival after treatment were randomized trials through 13 and 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . the cost of a basic / enhanced reminder letter , fobt , colonoscopy and biopsy per participant in korea for 2013 were derived from randomized trials for the cost effectiveness of reminder letters for colorectal cancer screening in the united states and uk for 2005 and 2009 , respectively , the korea ministry of health and welfare notifications and the korea health insurance review and assessment service guidelines on health insurance medical cost including drug components and materials for medical treatment . based on the randomized trials , the basic ( or enhanced ) reminder letters are expected to increase the participation rate in colorectal cancer screening by 6% ( or 12% ) ( the cost of the reminder letter in the united states / united kingdom was adjusted by the ratio of korea s per - capita health expenditure to its united states / united kingdom counterpart for 2013 ) . for instance , screening participation was assumed to be higher by 6% in the targeted - reminder intervention than in the standard - screening intervention in busan , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam , e.g. , 27% , 35% , and 25% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in busan from supplementary table 1 , respectively . the participation gap between the universal - reminder-1 and standard - screening interventions was presumed to be 6% in every area , e.g. , 31% , 41% , and 29% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in seoul from supplementary table 1 , respectively . likewise , the participation difference between the universal - reminder-2 and standard - screening interventions was supposed to be 12% in every area , e.g. , 37% , 47% , and 35% for those aged 51 - 60 , 61 - 70 , and 71 - 80 in seoul from supplementary table 1 , respectively . for the calculation of treatment cost per patient for colorectal cancer by the phase of care in korea for the year 2013 , treatment cost per patient for colorectal cancer from the korea national health insurance corporation was adjusted by the share of direct cost for cancer care in the united states for the year 2010 by the phase of care . the cost in korean won was converted to us dollars with the exchange rate of $ 1=1,055.4 won as of the year 2013 . the annual inflation rate adjusted by the annual discount rate for cost was assumed to be 1 . the crux of dcea is to adjust total effectiveness outcome ( the un - weighted mean or sum of individuals effectiveness outcomes in the conventional cost effectiveness analysis ) by an inequality index so that interventions with greater health disparity lead to smaller total effectiveness outcomes . ( 1 ) , a function of inequality aversion , i.e. , public aversion to health disparity . usually , inequality aversion on income distribution is measured as public opinion on the ideal rate of exchange between the incomes of those with highest income and those with the lowest income ( leaky bucket questionnaire ) . likewise , inequality aversion on health distribution can be measured as public opinion on the ideal rate of exchange between the health of those with the best health and those with the worst health . once the atkinson inequality index is calculated for every intervention , the atkinson icer in eq . ( 2 ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index , can be used to compare both cost effectiveness and health disparity from the interventions . ( 2 ) , is different from the conventional icer in the following manner : as inequality aversion increases , individuals with smaller qalys / worse health have more weights in total effectiveness outcome than do those with greater qalys / better health . the atkinson inequality index becomes 0 and the atkinson icer becomes the conventional icer when inequality aversion becomes 0 . in this vein , the atkinson inequality index and the atkinson icer were calculated to compare both cost effectiveness and health disparity from the standard screening , the targeted reminder and , the universal reminder 1 and 2 in korea . per - capita qalys and percapita cost were computed for each intervention for each area based on the markov cohort simulation described above ( 2014 , treeage software inc . , then , the atkinson inequality index and the atkinson icer for the nation were calculated based on the equations below . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion table 2 shows per - capita qalys and cost of interventions and the comparison by province in korea . on a national level , non - screening was dominated by the standard screening , the targeted reminder and , the universal reminder 1 and 2 : on a national level , non - screening resulted in smaller percapita qalys and greater per - capita cost than did the standard screening , the targeted reminder , and the universal reminder 1 and 2 . only four provinces had higher per - capita qalys than the national mean across interventions and the comparison , i.e. , seoul ( the capital of the nation ) , gyeonggi ( the province surrounding seoul ) , daejeon ( the province specialized in research and development ) , and jeju ( a southern island ) . the pattern was similar for the per - capita cost of non - screening , the standard screening , and the universal reminder 1 and 2 . as expected , however , eight provinces with basic reminder letters , i.e. , busan ( metropolis ) , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam ( rural areas ) , had the higher per - capita cost of the targeted - reminder intervention than the national mean . figs . 2 or 3 presents per - capita qaly gains of the targeted reminder , and the universal reminder 1 and 2 compared to the standard screening by qaly group ( or by province ) . 2 , q1 is 25,000 participants with the smallest qalys and q2 is 25,000 with qalys greater than q1 s and smaller than the median qaly of the cohort . likewise , q4 is 25,000 participants with the greatest qalys and q3 is 25,000 with qalys smaller than q4 s and greater than the median qaly of the cohort . the per - capita qaly gain of the targeted - reminder intervention compared to the standard screening was greatest for q1 ( 0.0125 ) , followed by q2 ( 0.0056 ) , q3 ( 0.0006 ) , and q4 ( 0.0000 ) . a similar pattern was found for the universal reminder 1 and 2 albeit by a smaller gap between q1 and another qaly group in terms of percapita qaly gain . table 3 displays the atkinson inequality indexes and the atkinson icers of interventions and the comparison across different values of inequality aversion . health disparity , measured by the atkinson inequality index , was smallest ( or greatest ) in non - screening ( or the standard screening ) . across inequality aversion , indeed , the targeted reminder had smaller health disparity , and greater cost effectiveness with respect to the standard screening , than did the universal reminder 1 and 2 , e.g. , $ 6,140 vs. $ 6,868 and $ 8,726 ( or $ 5,744 vs. $ 6,831 and $ 8,835 ) for the atkinson icer with the inequality aversion of 0 ( or 7 ) . moreover , as inequality aversion increases , ( 1 ) the atkinson icer of the targeted reminder becomes smaller , ( 2 ) the atkinson icer of the universal reminder 2 becomes greater , and ( 3 ) a gap between the targeted reminder and the universal reminder 1 ( or 2 ) in terms of the atkinson icer expands ( fig . if public aversion to health disparity becomes greater , these results suggest , the gap between the cost effectiveness of the targeted reminder ( minimizing health disparity ) and the universal reminder 1 and 2 ( maximizing population health ) will become greater . this study might be the first dcea of cancer screening interventions in east asia , evaluating the cost effectiveness of organized colorectal cancer screening interventions with various types of reminders , with their impacts on regional health disparity being considered . based on the results of this study , health disparity was smallest ( or greatest ) in non - screening ( or the standard screening ) across inequality aversion . across inequality aversion , in addition , the targeted reminder had smaller health disparity , and greater cost effectiveness with respect to the standard screening , than did the universal reminder 1 and 2 . moreover , if public aversion to health disparity increases , a difference between the cost effectiveness of the targeted reminder ( minimizing health disparity ) and the universal reminder 1 and 2 ( maximizing population health ) will increase . these findings are largely consistent with those on the distributional cost effectiveness of organized colorectal cancer screening interventions in the united kingdom ( i.e. , the standard screening , the targeted reminder and the universal reminder 1 ) . as in korea , across all or most values of inequality aversion , health disparity was smallest in non - screening and the targeted reminder had smaller health disparity and greater cost effectiveness ( with respect to the standard screening ) than did the universal reminder 1 in the united kingdom . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion for model validation , simulated results for seoul and gyeonggi ( covering 45% of korea s population ) were compared with actual data from randomized trials through 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . simulation outcomes were comparable to actual data regarding the relative risk of 30-year mortality with annual screening for a cohort aged 60 at the baseline , i.e. , 0.62 for seoul and gyeonggi vs. 0.68 for the united states . a major policy implication of this study and dcea in general is that public aversion to health disparity can be an important factor for the cost effectiveness of a healthcare intervention especially in a region with significant health disparity : if inequality aversion increases , healthcare interventions designed to minimize health disparity ( e.g. , the targeted reminder ) will become relatively more cost effective than those designed to maximize population health ( e.g. , the universal reminder 1 and 2 ) . based on a global analysis of inequality aversion on income distribution for 1999 , the range of inequality aversion was 0.42 - 1.88 in the united states , 0.67 - 3.35 in the united kingdom , 0.68 - 3.67 in spain , and 0.72 - 3.83 in italy with population sizes and per - capita gdps of the latter three nations comparable to korea s for the year 2013 . first , sensitivity analysis for different values of utility for polyps and colorectal cancer might present additional insights on this line of research , even though these values in this study came from well - established existing literature including a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . second , this study did not consider whether various types of reminders are economically feasible . in some cases , new interventions , which were proven to be cost effective , examining the economic feasibility of these reminders is expected to expand the boundary of knowledge on this topic . third , this study focused on a dynamic interplay between cost effectiveness and regional health disparity from colorectal cancer screening interventions . using micro - simulation to highlight disparity in patient - level variables such as family history , socioeconomic status and social support might be another promising direction of research on this important issue . fourth , a significant part of data for this study came from existing literature for other nations with different healthcare systems . as promotional effort for colorectal cancer screening , the targeted reminder might be more cost effective than its universal - reminder counterpart with their effects on health disparity being considered . this study helps to develop promotional effort for colorectal cancer screening with the greatest cost effectiveness and the smallest health disparity at the same time .

purposethe purpose of this study was to evaluate the cost effectiveness of colorectal cancer screening interventions with their effects on health disparity being considered.materials and methodsmarkov cohort simulation was conducted with the cycle / duration of 1/40 year(s ) . data came from the results of randomized trials and others . participants were hypothetical cohorts aged 50 years as of year 2013 in 16 korean provinces . the interventions until the age of 80 were annual organized fecal occult blood test ( fobt ) ( standard screening ) , annual fobt with basic reminders for provinces with higher mortalities than the national average ( targeted reminder ) and annual fobt with basic / enhanced reminders for all provinces ( universal reminder 1 and 2 ) . the comparison was non - screening , the outcome was quality - adjusted life years , and only medical costs for screening and treatment were considered from a societal perspective . the atkinson incremental cost effectiveness ratio ( atkinson icer ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index , was used to evaluate the cost effectiveness of the four interventions with their impacts on regional health disparity being considered.resultshealth disparity was smallest ( or greatest ) in non - screening ( or the standard screening ) . the targeted reminder had smaller health disparity , and smaller atkinson icer with respect to standard screening , than did the universal reminder 1 and 2.conclusionthe targeted reminder might be more cost effective than the universal reminders with their effects on health disparity being considered . this study helps to develop promotional effort for colorectal cancer screening with both the greatest cost effectiveness and the smallest health disparity

Introduction Materials and Methods 1. Participant, intervention, comparison, and outcome 2. Model structure 3. Utility, screening, natural history, and survival after treatment and cost 4. Social welfare analysis Results Discussion Conclusion Electronic Supplementary Material

however , the current cost effectiveness analysis of colorectal cancer screening focuses on improving population health and ignores health disparity generated by this intervention . but recent studies show that the purposes of maximizing population health and minimizing health disparity often contradict with each other : the promotion of a targeted reminder designed to increase participation in organized colorectal cancer screening among deprived populations with ethnic diversity minimizes health disparity , whereas the promotion of a universal reminder designed to increase participation in organized colorectal cancer screening among the entire population maximizes population health in the united kingdom . in this context , this study evaluated the cost effectiveness of organized colorectal cancer screening interventions ( i.e. , screening with various types of reminders ) , with their impacts on regional health disparity being considered . markov cohort simulation was conducted for hypothetical cohorts aged 50 years as of the year 2013 in each of 16 korean provinces . the interventions until the age of 80 were ( 1 ) annual fecal occult blood test ( fobt ) ( standard screening ) , ( 2 ) annual fobt with basic reminder letters for eight provinces with higher mortalities from either all causes or colorectal cancer than the national average ( i.e. , busan [ metropolis ] , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam [ rural areas ] ) ( targeted reminder ) , ( 3 ) annual fobt with basic reminder letters for all provinces ( universal reminder 1 ) , and ( 4 ) annual fobt with enhanced reminder letters ( i.e. , personal reminder letters with tailored information packages ) for all provinces ( universal reminder 2 ) . the markov states were ( 1 ) healthy , ( 2 ) polyps not detected by screening ( p1 ) , ( 3 ) polyps detected by screening ( p2 ) , ( 4 ) symptom - free early colorectal cancer not detected by screening ( ecc1 ) , ( 5 ) symptom - free early colorectal cancer detected by screening ( ecc2 ) , ( 6 ) symptomatic early colorectal cancer ( ecc3 ) , ( 7 ) symptom - free advanced colorectal cancer not detected by screening ( acc1 ) , ( 8) symptom - free advanced colorectal cancer detected by screening ( acc2 ) , ( 9 ) symptomatic advanced colorectal cancer ( acc3 ) , ( 10 ) death from colorectal cancer , and ( 11 ) death from other causes ( the true positive goes through colorectal cancer treatment without further colorectal cancer screening ) . atkinson incremental cost effectiveness ratio ( atkinson icer ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index ( to be elaborated below ) was used to evaluate the cost effectiveness of the four interventions with their impacts on health disparity being considered . without treatment , one with early colorectal cancer ( 1 ) stays as he or she is , ( 2 ) develops advanced colorectal cancer with the mean ecc dwelling time of 2 years , i.e. utilities for the 11 markov states came from a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . values on screening - related variables and the natural history of colorectal cancer were obtained from statistics korea , previous studies on cancer registry data ( for korea and for germany ) , existing literature on the cost effectiveness of colorectal cancer screening , and bayesian calibration for the natural history of colorectal cancer . data sources on survival after treatment were randomized trials through 13 and 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . the cost of a basic / enhanced reminder letter , fobt , colonoscopy and biopsy per participant in korea for 2013 were derived from randomized trials for the cost effectiveness of reminder letters for colorectal cancer screening in the united states and uk for 2005 and 2009 , respectively , the korea ministry of health and welfare notifications and the korea health insurance review and assessment service guidelines on health insurance medical cost including drug components and materials for medical treatment . based on the randomized trials , the basic ( or enhanced ) reminder letters are expected to increase the participation rate in colorectal cancer screening by 6% ( or 12% ) ( the cost of the reminder letter in the united states / united kingdom was adjusted by the ratio of korea s per - capita health expenditure to its united states / united kingdom counterpart for 2013 ) . for instance , screening participation was assumed to be higher by 6% in the targeted - reminder intervention than in the standard - screening intervention in busan , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam , e.g. likewise , the participation difference between the universal - reminder-2 and standard - screening interventions was supposed to be 12% in every area , e.g. for the calculation of treatment cost per patient for colorectal cancer by the phase of care in korea for the year 2013 , treatment cost per patient for colorectal cancer from the korea national health insurance corporation was adjusted by the share of direct cost for cancer care in the united states for the year 2010 by the phase of care . the cost in korean won was converted to us dollars with the exchange rate of $ 1=1,055.4 won as of the year 2013 . the crux of dcea is to adjust total effectiveness outcome ( the un - weighted mean or sum of individuals effectiveness outcomes in the conventional cost effectiveness analysis ) by an inequality index so that interventions with greater health disparity lead to smaller total effectiveness outcomes . once the atkinson inequality index is calculated for every intervention , the atkinson icer in eq . ( 2 ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index , can be used to compare both cost effectiveness and health disparity from the interventions . the atkinson inequality index becomes 0 and the atkinson icer becomes the conventional icer when inequality aversion becomes 0 . in this vein , the atkinson inequality index and the atkinson icer were calculated to compare both cost effectiveness and health disparity from the standard screening , the targeted reminder and , the universal reminder 1 and 2 in korea . , then , the atkinson inequality index and the atkinson icer for the nation were calculated based on the equations below . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion markov cohort simulation was conducted for hypothetical cohorts aged 50 years as of the year 2013 in each of 16 korean provinces . the interventions until the age of 80 were ( 1 ) annual fecal occult blood test ( fobt ) ( standard screening ) , ( 2 ) annual fobt with basic reminder letters for eight provinces with higher mortalities from either all causes or colorectal cancer than the national average ( i.e. , busan [ metropolis ] , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam [ rural areas ] ) ( targeted reminder ) , ( 3 ) annual fobt with basic reminder letters for all provinces ( universal reminder 1 ) , and ( 4 ) annual fobt with enhanced reminder letters ( i.e. , personal reminder letters with tailored information packages ) for all provinces ( universal reminder 2 ) . the markov states were ( 1 ) healthy , ( 2 ) polyps not detected by screening ( p1 ) , ( 3 ) polyps detected by screening ( p2 ) , ( 4 ) symptom - free early colorectal cancer not detected by screening ( ecc1 ) , ( 5 ) symptom - free early colorectal cancer detected by screening ( ecc2 ) , ( 6 ) symptomatic early colorectal cancer ( ecc3 ) , ( 7 ) symptom - free advanced colorectal cancer not detected by screening ( acc1 ) , ( 8) symptom - free advanced colorectal cancer detected by screening ( acc2 ) , ( 9 ) symptomatic advanced colorectal cancer ( acc3 ) , ( 10 ) death from colorectal cancer , and ( 11 ) death from other causes ( the true positive goes through colorectal cancer treatment without further colorectal cancer screening ) . atkinson incremental cost effectiveness ratio ( atkinson icer ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index ( to be elaborated below ) was used to evaluate the cost effectiveness of the four interventions with their impacts on health disparity being considered . without treatment ( polypectomy ) , one with polyps ( 1 ) stays as he or she is , ( 2 ) develops early colorectal cancer with the annual polyp - ecc transition rate of 0.005 , i.e. without treatment , one with early colorectal cancer ( 1 ) stays as he or she is , ( 2 ) develops advanced colorectal cancer with the mean ecc dwelling time of 2 years , i.e. utilities for the 11 markov states came from a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . values on screening - related variables and the natural history of colorectal cancer were obtained from statistics korea , previous studies on cancer registry data ( for korea and for germany ) , existing literature on the cost effectiveness of colorectal cancer screening , and bayesian calibration for the natural history of colorectal cancer . data sources on survival after treatment were randomized trials through 13 and 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . the cost of a basic / enhanced reminder letter , fobt , colonoscopy and biopsy per participant in korea for 2013 were derived from randomized trials for the cost effectiveness of reminder letters for colorectal cancer screening in the united states and uk for 2005 and 2009 , respectively , the korea ministry of health and welfare notifications and the korea health insurance review and assessment service guidelines on health insurance medical cost including drug components and materials for medical treatment . based on the randomized trials , the basic ( or enhanced ) reminder letters are expected to increase the participation rate in colorectal cancer screening by 6% ( or 12% ) ( the cost of the reminder letter in the united states / united kingdom was adjusted by the ratio of korea s per - capita health expenditure to its united states / united kingdom counterpart for 2013 ) . for instance , screening participation was assumed to be higher by 6% in the targeted - reminder intervention than in the standard - screening intervention in busan , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam , e.g. likewise , the participation difference between the universal - reminder-2 and standard - screening interventions was supposed to be 12% in every area , e.g. for the calculation of treatment cost per patient for colorectal cancer by the phase of care in korea for the year 2013 , treatment cost per patient for colorectal cancer from the korea national health insurance corporation was adjusted by the share of direct cost for cancer care in the united states for the year 2010 by the phase of care . the cost in korean won was converted to us dollars with the exchange rate of $ 1=1,055.4 won as of the year 2013 . the crux of dcea is to adjust total effectiveness outcome ( the un - weighted mean or sum of individuals effectiveness outcomes in the conventional cost effectiveness analysis ) by an inequality index so that interventions with greater health disparity lead to smaller total effectiveness outcomes . once the atkinson inequality index is calculated for every intervention , the atkinson icer in eq . ( 2 ) , the incremental cost effectiveness ratio adjusted by the atkinson inequality index , can be used to compare both cost effectiveness and health disparity from the interventions . the atkinson inequality index becomes 0 and the atkinson icer becomes the conventional icer when inequality aversion becomes 0 . in this vein , the atkinson inequality index and the atkinson icer were calculated to compare both cost effectiveness and health disparity from the standard screening , the targeted reminder and , the universal reminder 1 and 2 in korea . , then , the atkinson inequality index and the atkinson icer for the nation were calculated based on the equations below . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion table 2 shows per - capita qalys and cost of interventions and the comparison by province in korea . on a national level , non - screening was dominated by the standard screening , the targeted reminder and , the universal reminder 1 and 2 : on a national level , non - screening resulted in smaller percapita qalys and greater per - capita cost than did the standard screening , the targeted reminder , and the universal reminder 1 and 2 . only four provinces had higher per - capita qalys than the national mean across interventions and the comparison , i.e. the pattern was similar for the per - capita cost of non - screening , the standard screening , and the universal reminder 1 and 2 . , busan ( metropolis ) , gangwon , chungbuk , chungnam , jeonbuk , jeonnam , gyeongbuk , and gyeongnam ( rural areas ) , had the higher per - capita cost of the targeted - reminder intervention than the national mean . 2 or 3 presents per - capita qaly gains of the targeted reminder , and the universal reminder 1 and 2 compared to the standard screening by qaly group ( or by province ) . 2 , q1 is 25,000 participants with the smallest qalys and q2 is 25,000 with qalys greater than q1 s and smaller than the median qaly of the cohort . likewise , q4 is 25,000 participants with the greatest qalys and q3 is 25,000 with qalys smaller than q4 s and greater than the median qaly of the cohort . the per - capita qaly gain of the targeted - reminder intervention compared to the standard screening was greatest for q1 ( 0.0125 ) , followed by q2 ( 0.0056 ) , q3 ( 0.0006 ) , and q4 ( 0.0000 ) . a similar pattern was found for the universal reminder 1 and 2 albeit by a smaller gap between q1 and another qaly group in terms of percapita qaly gain . health disparity , measured by the atkinson inequality index , was smallest ( or greatest ) in non - screening ( or the standard screening ) . across inequality aversion , indeed , the targeted reminder had smaller health disparity , and greater cost effectiveness with respect to the standard screening , than did the universal reminder 1 and 2 , e.g. , $ 6,140 vs. $ 6,868 and $ 8,726 ( or $ 5,744 vs. $ 6,831 and $ 8,835 ) for the atkinson icer with the inequality aversion of 0 ( or 7 ) . moreover , as inequality aversion increases , ( 1 ) the atkinson icer of the targeted reminder becomes smaller , ( 2 ) the atkinson icer of the universal reminder 2 becomes greater , and ( 3 ) a gap between the targeted reminder and the universal reminder 1 ( or 2 ) in terms of the atkinson icer expands ( fig . if public aversion to health disparity becomes greater , these results suggest , the gap between the cost effectiveness of the targeted reminder ( minimizing health disparity ) and the universal reminder 1 and 2 ( maximizing population health ) will become greater . this study might be the first dcea of cancer screening interventions in east asia , evaluating the cost effectiveness of organized colorectal cancer screening interventions with various types of reminders , with their impacts on regional health disparity being considered . based on the results of this study , health disparity was smallest ( or greatest ) in non - screening ( or the standard screening ) across inequality aversion . across inequality aversion , in addition , the targeted reminder had smaller health disparity , and greater cost effectiveness with respect to the standard screening , than did the universal reminder 1 and 2 . moreover , if public aversion to health disparity increases , a difference between the cost effectiveness of the targeted reminder ( minimizing health disparity ) and the universal reminder 1 and 2 ( maximizing population health ) will increase . these findings are largely consistent with those on the distributional cost effectiveness of organized colorectal cancer screening interventions in the united kingdom ( i.e. , the standard screening , the targeted reminder and the universal reminder 1 ) . as in korea , across all or most values of inequality aversion , health disparity was smallest in non - screening and the targeted reminder had smaller health disparity and greater cost effectiveness ( with respect to the standard screening ) than did the universal reminder 1 in the united kingdom . a atkinson inequality index for qi qaly , individual i q qaly , per - capita ( mean ) inequality aversion c intervention 2/1 s per - capita cost a intervention 2/1 s atkinson index q intervention 2/1 s per - capita qaly inequality aversion for model validation , simulated results for seoul and gyeonggi ( covering 45% of korea s population ) were compared with actual data from randomized trials through 30 years of follow - up for the cost effectiveness of colorectal cancer screening in the united states . a major policy implication of this study and dcea in general is that public aversion to health disparity can be an important factor for the cost effectiveness of a healthcare intervention especially in a region with significant health disparity : if inequality aversion increases , healthcare interventions designed to minimize health disparity ( e.g. , the targeted reminder ) will become relatively more cost effective than those designed to maximize population health ( e.g. , the universal reminder 1 and 2 ) . based on a global analysis of inequality aversion on income distribution for 1999 , the range of inequality aversion was 0.42 - 1.88 in the united states , 0.67 - 3.35 in the united kingdom , 0.68 - 3.67 in spain , and 0.72 - 3.83 in italy with population sizes and per - capita gdps of the latter three nations comparable to korea s for the year 2013 . first , sensitivity analysis for different values of utility for polyps and colorectal cancer might present additional insights on this line of research , even though these values in this study came from well - established existing literature including a systematic review of colorectal cancer utilities and a cost effectiveness analysis of colorectal cancer screening with blood - based biomarkers . third , this study focused on a dynamic interplay between cost effectiveness and regional health disparity from colorectal cancer screening interventions . as promotional effort for colorectal cancer screening , the targeted reminder might be more cost effective than its universal - reminder counterpart with their effects on health disparity being considered . this study helps to develop promotional effort for colorectal cancer screening with the greatest cost effectiveness and the smallest health disparity at the same time .

bernardo alberto houssay in his lecture in 1947 the blood sugar and the production and consumption of glucose are kept within normal bounds , therefore there is an equilibrium between the glands of internal secretions which reduce the blood sugar ( pancreas ) and those which raise it ( anterohypophysis , adrenals , thyroid , etc . ) . deficit or excess of thyroid hormones can break this equilibrium leading to alterations of carbohydrate metabolism . overt hyperthyroidism has been related to glucose intolerance and even ketoacidosis . with regards to hypothyroidism , cases of hypoglycemia have been reported in the literature despite the fact that peripheral insulin resistance may be present . in the century that has elapsed , since the first observations of uncontrolled glucose metabolism in thyrotoxic diabetic patients , new pathways involved in the regulation of glucose homeostasis by thyroid hormones have been unveiled . novel findings include the stimulation of hepatic glucose production by thyroid hormones acting via a sympathetic pathway from the hypothalamus and the discovery of transcriptional regulators of metabolic and mitochondrial genes that , influenced by intracellular t3 levels , may contribute to the development of insulin resistance . the calorigenic - thermogenic activity of t3 long ascribed solely to uncoupling of mitochondrial oxidative phosphorylation has recently been related to t3-induced gating of mitochondrial permeability transition pore ( ptp ) of the inner mitochondrial membrane where the whole t3 transduction pathway integrates genomic and nongenomic activities of t3 in regulating mitochondrial energetics . in this paper , we summarize the effects of thyroid hormones in glucose metabolism and its alterations when thyroid dysfunction is present . thyroid receptor - mediated effects on gene transcription and translation are key in the regulation of glucose metabolism . according to the results of studies with complementary dna ( cdna ) microarray analysis in mouse liver , several genes involved in gluconeogenesis , glycogen metabolism , and insulin signaling that are regulated by thyroid hormones have been identified . in the study by feng et al . , rna from hypothyroid mice treated with t3 was prepared , labeled with fluorescent dye , and hybridized with the cdna microarray . this enzyme hydrolyzes glucose-6-phosphate and completes the final step in gluconeogenesis and glycogenolysis , therefore playing an important role in the homeostatic regulation of blood glucose levels . another finding was a decrease in mrna expression of akt2 ( protein kinase b ) , a serine / threonine kinase that is an essential molecule in the insulin signaling pathway . akt2 has been shown to promote glycogen synthesis in liver by inactivating glycogen synthase kinase 3 . thus , a decrease in akt2 activity would decrease glycogen synthesis explaining the antagonistic insulin effect of thyroid hormones at the liver . moreover , an induction of 2-adrenergic receptor mrna and repression of inhibitory g protein ( gi ) rna of the adenylate cyclase cascade by t3 were also reported . all these results are in favour of a permissive influence of t3 in the glycogenolytic and gluconeogenic effects of epinephrine and glucagon . other hepatic gluconeogenic enzymes that have been found to be positively regulated by thyroid hormones include phosphoenolpyruvate carboxykinase ( pepck ) , the enzyme that catalyzes the rate - controlling step of gluconeogenesis and pyruvate carboxylase , involved in the synthesis of oxaloacetate from pyruvate . the catalytic activity of pyruvate carboxylase has been found increased approximately 2-fold in hyperthyroid rats compared with untreated or treated euthyroid controls . another mechanism , whereby thyroid hormones are known to increase hepatic glucose output , is through increased hepatic expression of the glucose transporter glut2 as previously shown in a rat model where glut2 protein concentration in crude liver membranes was twice as high in chronically hyperthyroid versus hypothyroid animals . it has been previously reported that , despite an expected resistance towards the insulin inhibitory effect on gluconeogenesis , the transcription of several enzymes involved in lipid synthesis or lipid metabolism is increased in hyperinsulinemic , insulin - resistant mice . furthermore , t3 induction of lipogenesis through the transcriptional activation of malic enzyme , involved in fatty acid synthesis , has been previously reported . therefore , it is possible that by the induction of lipogenic enzymes , t3 could be further aggravating the dysregulation of liver glucose and lipid metabolism characteristic of insulin resistance . as a result of the long time quest for thyroid analogs that possess the favourable actions on metabolism without the unwanted thyroid cardiac effects , the differential distribution of thyroid receptor ( tr ) isoforms in the tissues has been key for the development of these analogs . with regards to lipogenesis , carbohydrate - response element - binding protein ( chrebp ) is a major transcription factor controlling the activation of glucose - induced lipogenesis in liver and is a direct target of thyroid hormones in liver and white adipose tissue ( wat ) , the two main lipogenic tissues in mice . chrebp is shown to be specifically regulated by trbeta but not by tralpha in vivo , in liver where trbeta represents 80% of the thyroid hormone bound tr , but also in wat where both tr isoforms are expressed . although the area of thyroid analogs is beyond the scope of this paper , it is to be mentioned that some thyromimetic analogs , such as 3,5-l - diiodothyronine ( t(2 ) ) , exert their beneficial action on metabolism , without inducing a thyrotoxic state , through a mechanism that does not involve binding to thyroid hormone receptors . in rats fed a high - fat diet , the addition of t(2 ) to lipid - overloaded cells resulted in reduction in lipid content ; downregulation of peroxisome proliferator - activated receptors ( ppar) , ppar , and alternative oxidase ( aox ) expression ; increase in ppar expression ; and stimulation of mitochondrial uncoupling thus preventing and reversing hepatic steatosis in this animal model . surprisingly , in this study , these lipid - lowering actions not mediated by tr were also observed with t3 . to summarize , all these findings have helped to understand that thyroid hormones have insulin antagonistic effects at the liver that lead to an increased glucose hepatic output , via an enhanced rate of gluconeogenesis and glycogenolysis . with regards to lipid metabolism , however , in the context of insulin resistance , the conversion of glucose into fatty acids together with nonsuppressed gluconeogenesis is simply perpetuating the hyperinsulinemic state . furthermore , nutritional influences , such as those of high - fat diets , should also be taken into consideration as modifiers of the effects of thyroid hormones on insulin sensitivity . opposite to what occurs at the liver level , at peripheral tissues , thyroid hormones have been shown to exert some of their actions synergically with insulin . the upregulation of the expression of genes such as glut-4 or phosphoglycerate kinase ( pgk ) , involved in glucose transport and glycolysis respectively , is a good proof of concept . in skeletal muscle , the main site of insulin - mediated glucose disposal , glucose transporter glut4 , is induced by t3 , revealing that it can increase basal and insulin - stimulated glucose transport in this tissue . another t3 target in skeletal muscle is mitochondrial uncoupling protein 3 ( ucp3 ) . unveiling this association may be important since progressive reduction of ucp3 levels results in insulin resistance accompanied by decreased fatty acid oxidation and a less intense akt / pkb and 5 adenosine monophosphate - activated protein kinase ( ampk ) signaling . although discrepancies between the regulation by t3 of ucp3 expression in rats , humans , and mice have been observed , the rat model has shed some light into t3 actions in this tissue . t3 intravenous ( i.v . ) administration in hypothyroid rats showed a rise in serum fatty acid levels concomitant with a rapid increase in ucp3 expression in gastrocnemius muscle . these findings point to ucp as a possible molecular determinant of the action of t3 on energy metabolism . , t2 actions have been also explored in skeletal muscle . in a model of high - fat diet - induced insulin resistance in rat , the administration of t2 , on the gastrocnemius muscle , induced remarkable changes on the metabolic / structural phenotype and insulin signaling . t2 increased insulin - stimulated akt phosphorylation levels , the muscle contents of fast / glycolytic fibers and sarcolemmal glut4 . moreover , the result of cdna microarray analysis on skeletal muscle of a group of healthy men receiving 75 g / d of t3 for 14 days has shown that not only genes with agonistic insulin effects but several others with antagonistic insulin effects are upregulated by treatment with t3 , underlying the pleiotropic effect of thyroid hormone in energy metabolism . cdna array data have also provided a molecular basis to the effect of t3 on adipose tissue . in human adipocytes , t3 increases the mrna levels of the lipolytic 2-ar , favouring catecolamine - induced lipolysis and it also downregulates sterol regulatory element binding protein ( srebp1c ) , involved in lipogenesis , which may constitute a link between hyperthyroidism and insulin resistance . skin fibroblasts have been also used to study thyroid hormone - responsive genes involved in metabolism in human cells . although they are not as metabolically active as hepatic cells , they are easily obtained and also , thyroid hormone - responsive . in cultured human fibroblasts , moeller et al . observed that , opposite to a posttranscriptional regulation as reported for other growth factors and hormones , the mrna of the transcription factor hif-1 ( hypoxia - inducible factor 1 ) , a key mediator of glycolysis , increased in response to t3 . as the glucose transporter glut1 , several enzymes of glycolysis , and the lactate exporter slc16a3 were all also found induced by t3 and are target genes of the transcription factor hif-1 , the authors postulated that the effect of thyroid hormones on the induction of these genes most probably was indirect and hif-1 mediated . furthermore , a new mechanism of thyroid action was unraveled by this group of researchers . it was shown that t3 bound to trbeta , in lieu of initiating gene transcription in the nucleus , activates the phosphatidylinositol 3-kinase ( pi3k ) signaling pathway in the cytosol in order to activate hif-1 gene expression . at the cellular level , thyroid hormones can also increase mitochondrial biogenesis , fatty acid oxidation , and tca cycle activity . these findings are quite relevant since the role of mitochondrial dysfunction , leading to cellular lipid excess and impaired oxidative metabolism , has been clearly demonstrated in the pathogenesis of type 2 diabetes [ 2325 ] . furthermore , it has been described that in skeletal muscle , the lack of thyroid hormones might dysregulate mitochondrial gene expression . ppar gamma coactivator-1 alpha ( pgc-1 alpha ) , a key transcriptional regulator of mitochondrial content and function , fatty acid oxidation , and gluconeogenesis , has been involved in the process whereby thyroid hormones regulate mitochondrial function . it has been shown that pgc-1 alpha gene expression is increased by t3 , as much as 13-fold 6 hours after t3 treatment . the regulation pattern of t3 on pgc-1 alpha is complex and may occur through nongenomic activation of kinases to induce the expression of pgc-1 alpha or through transcriptional upregulation via the presence of a thyroid responsive element ( tre ) in the pgc-1 alpha promoter or by genomic upregulation of a transcription factor ( via a tre ) , which then binds to the pgc-1 alpha promoter and increases pgc-1 alpha transcription . it is hypothesized that pgc-1 alpha can be dysregulated by reduced t3 levels , thus contributing to insulin resistance . not only low circulating but also , intracellular t3 levels , could count for this matter . a lower expression and activity of type 2 iodothyronine - deiodinase ( d2 ) , the enzyme that is key for the conversion of t4 into t3 in muscle and thus , amplifies thyroid hormone signaling in individual cells , has been found linked to insulin resistance [ 29 , 30 ] . if pgc-1 alpha effects on mitochondrial gene expression may indeed be regulated by thyroid hormone , normal activity of deiodinase type 2 is very relevant . bile acids are potent stimulators of the enzyme and may play an important role in the relationship between thyroid action and glucose metabolism . on the other hand , the natural occurrence of polymorphisms of deiodinase type 2 such as thr92ala , with a lower activity , has also been implicated with increased risk for diabetes type 2 . it has been shown that the hypothalamus can modulate endogenous glucose production by using functionally reciprocal sympathetic and parasympathetic autonomic outputs to the liver . moreover , a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver has been proposed as a central pathway for modulation of hepatic glucose metabolism by thyroid hormone . klieveric et al . demonstrated that upon selective administration to the paraventricular nucleus ( pvn ) , t3 increases endogenous glucose production and plasma glucose , and that these hypothalamic t3 effects are mediated via sympathetic projections to the liver . in order to arrive to such remarkable findings first they administered an intracerebroventricular ( i.c.v . ) t3 or vehicle ( veh ) infusion , and there was a significant increase in plasma glucose compared with veh - treated rats . this meant that central t3 infusion could reproduce the characteristic increase in hepatic glucose output of thyrotoxicosis . to further identify the neuroanatomic region responsible for these changes , the authors infused t3 within the hypothalamus at the pvn . a similar response was obtained , that was independent of plasma t3 , insulin , and corticosterone concentrations . they repeated the experiment in surgically hepatic sympathectomized animals ( hsx ) and sham - denervated animals . the principal finding of this study is the description of a neural ( autonomic ) modulation of hepatic glucose metabolism by t3 at the hypothalamus that takes place independently of plasma glucoregulatory hormone concentrations . thyrotoxic subjects frequently show impaired glucose tolerance . this is a result of increased glucose turnover with increased glucose absorption through the gastrointestinal tract , postabsorptive hyperglycemia , elevated hepatic glucose output , with elevated fasting and/or postprandial insulin and proinsulin levels , elevated free fatty acid concentrations and elevated peripheral glucose transport and utilization . the literature about this topic is vast and has been previously comprehensively reviewed by dimitriadis and raptis . although ketoacidosis may result per se from the insulin resistance present in thyrotoxicosis , the stimulatory action of thyroid hormones in excess on lipolysis and free fatty acids availability can also contribute to increased ketogenesis . thyrotoxicosis has been reported to increase endogenous glucose production in the liver in the basal state and to decrease hepatic insulin sensitivity in humans . the different mechanisms to explain this phenomenon include increased rates of gluconeogenesis and glycogenolysis mainly explained by the above - mentioned effects on the liver by thyroid hormones . to summarize , these effects include thyroid receptor - mediated effects on liver gene transcription , increased sympathetic action in the liver mediated by hypothalamus , and increased concentrations of the glut2 glucose transporters in the liver plasma membrane that allows for glucose efflux [ 8 , 39 ] together with increased concentration of free fatty acids in plasma . the interpretation of the effects of hyperthyroidism on glucose utilization by peripheral tissues is by far the most complex issue on this topic . on one hand , the rates of glucose uptake in peripheral tissues have been found increased by thyroid hormones , suggesting that glucose utilization is highly increased , specially in skeletal muscle [ 34 , 37 , 4144 ] . this increased utilization , as shown by indirect calorimetry during euglycemic hyperinsulinemic clamps , is mainly due to an increase in insulin - stimulated glucose oxidation rates [ 43 , 4548 ] . however , a decrease in insulin - stimulated nonoxidative glucose disposal , through reduced glycogenogenesis [ 43 , 44 , 49 ] , takes place , with intracellular glucose being redirected towards glycolysis and lactate formation . the release of lactate from peripheral tissues back to the liver is a major contributor to the cori cycle where more hepatic glucose is being produced [ 43 , 4951 ] . although glucose intolerance in hyperthyroidism can be easily explained by hepatic insulin resistance without involvement of peripheral tissues , impaired insulin - stimulated peripheral glucose uptake has also been proven in some studies . by means of the arteriovenous difference technique in the forearm muscles of hyperthyroid subjects after the consumption of a mixed meal , it has been clearly demonstrated that muscle blood flow is increased , masking a defect in insulin - stimulated glucose uptake . moreover , in disagreement with previous reports [ 41 , 45 ] , shen et al . also described decreased peripheral insulin sensitivity in hyperthyroidism . alternative explanations for peripheral insulin resistance in hyperthyroidism include an increased secretion of bioactive mediators ( adipokines ) such as interleukin 6 ( il6 ) and tumour necrosis factor a ( tnf ) from adipose tissue in hyperthyroidism . these adipokines , that exert both proinflammatory and insulin resistant effects , have been found elevated in hyperthyroid women . in hyperthyroidism , decreased , normal , or even increased levels of plasma insulin have been reported . however , a rather consistent finding has been the increased degradation of insulin in hyperthyroid subjects [ 43 , 55 ] . it has been postulated that , in the long run , severe thyrotoxicosis can lead to irreversible pancreatic damage [ 56 , 57 ] . with regards to glucagon , its secretion and metabolic clearance rates have been reported increased , explaining the normal fasting plasma levels described in hyperthyroidism . subclinical hyperthyroidism has also been associated with insulin resistance [ 5961 ] in some but not all studies . endogenous subclinical hyperthyroidism may have a larger impact on glucose metabolism due to its chronicity and higher t3 levels when compared to exogenous administration of t4 . this phenomenon can be interpreted in the light of reduced levels of gluconeogenesis leading to decreased liver glucose output [ 63 , 64 ] . on the other hand , insulin resistance has been shown to be present in peripheral tissues in hypothyroid animal models [ 65 , 66 ] . hence , a poor utilization of glucose in hypothyroidism may be offset by a reduced release to circulation maintaining a balance of the glucose metabolism . studies performed in adipocytes and skeletal muscle of rats made hypothyroid have shown that these tissues are less responsive to insulin with regards to glucose metabolism [ 63 , 6569 ] . czech et al . studied insulin responsiveness in adipocytes and skeletal muscle of mature rats rendered hypothyroid by a low iodine diet and propylthiouracil . it was observed that glucose conversion to glycogen was partially inhibited while the glycolytic flux stimulation by insulin was totally frustrated . other authors [ 66 , 67 ] showed decreased insulin - stimulated glucose transport and/or phosphorylation , as well as a lower rate of glycolysis in the isolated , incubated soleus muscle of the hypothyroid rat and also suggested that the effects of hypothyroidism in muscle were not mediated through an interference of insulin binding to its receptor . it was postulated that they rather occurred through a postreceptor mechanism that may include abnormal phosphorylation of insulin signaling proteins . insulin resistance was confirmed in another study of rats with mild hypothyroidism . in this study , insulin responsiveness was measured by an insulin tolerance test and an euglycemic - hyperinsulinemic clamp followed by measurements of tissue - specific glucose utilization indices with the labelled 2-deoxy - d-[1 - 3h]glucose ( 2-dg ) technique in muscles ( red quadriceps ) and white adipose tissue ( epididymal fat ) . several other parameters were also determined such as muscle triglyceride content , plasma leptin and nonesterified fatty acids ( nefa ) levels , and mrna expression of resistin in adipose tissue as well as adipose tissue and liver carnitine palmitoyl transferase 1 ( cpt-1 ) , and muscle carnitine palmitoyl transferase 1 ( cpt-1 ) mrna levels , explored by real - time quantitative pcr . plasma leptin levels were lower and adipose tissue mrna expression of resistin higher , in the hypothyroid state . looking for a potential role of leptin in the metabolic consequences of hypothyroidism , leptin was infused , and it was found that glucose disposal was recovered . increased expression of muscle and adipose tissue carnitine palmitoyl transferases , decreased plasma nefa levels , and reduced muscle triglyceride content after leptin infusion were interpreted as the mediators of the recovery of the insulin resistant state . therefore , one possible explanation to decreased insulin responsiveness in hypothyroidism , according to the authors of this study , includes a dysregulation of leptin action at the hypothalamus . adipocyte - myocyte crosstalk by adipokines has been reported to play a significant role in skeletal muscle insulin resistance and may partially explain insulin resistance present in hypothyroidism . however , other factors associated with insulin resistance in hypothyroidism , such as altered blood flow , impaired glut4 translocation , decreased glycogen synthesis , and decreased muscle oxidative capacity have to be also considered . compared to the number of reports about insulin resistance in hyperthyroid patients , there are relatively fewer studies in humans dealing with the effects of hypothyroidism on glucose metabolism . measured whole - body sensitivity of glucose disposal to insulin in hypothyroid patients using the euglycemic - hyperinsulinemic clamp technique . they demonstrated that hypothyroidism induced a decrease in the insulin - mediated glucose disposal that reverted upon treatment . the same clamp technique and also measuring glucose tolerance and beta - cell activity with an oral glucose tolerance tests ( ogtt ) , handisurya et al . confirmed these findings and added the knowledge that glucose - induced insulin secretion is diminished by thyroid replacement corresponding well with the observed improvement of insulin sensitivity . dimitriadis et al . explored glucose uptake in muscle and adipose tissue of hypothyroid and control subjects by means of the arteriovenous difference technique in the anterior abdominal subcutaneous adipose tissue and the forearm muscles after the consumption of a mixed meal . a decreased net extraction of glucose and blood flow after the meal in hypothyroid muscle and adipose tissue was reported . this impairment in the ability of insulin to increase blood flow rate to the hypothyroid tissues is an alternative explanation to the mechanism whereby hypothyroidism can induce lower glucose disposal . insulin tolerance test has been used to explore insulin sensitivity in acute overt hypothyroid patients . a previous study in overt hypothyroid patients based on the homeostasis model assessment ( homa - ir ) showed no association between hypothyroidism and insulin sensitivity . found unimpaired insulin - stimulated glucose disposal in the forearm of hypothyroid patients after treatment with levothyroxine . with regards to subclinical hypothyroidism , insulin resistance has been demonstrated in some [ 60 , 74 , 79 ] but not all studies , where homa levels were found comparable to a control group [ 8082 ] . however , in some of these negative studies [ 80 , 81 ] , hyperinsulinemia was reported in subclinical hypothyroid subjects and interpreted as an early sign of impairment of glucose metabolism . thyroid hormones have a large impact on glucose metabolism . a direct regulation on thyroid responsive genes at the target organ has been described and more recently an indirect effect involving hypothalamic pathways that regulate glucose metabolism via control of the sympathetic nervous system has been reported . furthermore , thyroid hormone effects can be insulin agonistic , such as demonstrated in muscle or antagonistic such as observed in the liver . in hyperthyroidism , dysregulation of this balance may end in glucose intolerance mainly due to hepatic insulin resistance . in hypothyroidism the results are less evident . however , the available data suggest that insulin resistance is present mainly at the peripheral tissues . possible explanations hypothesized to explain this phenomenon span from the dysregulation of mitochondrial oxidative metabolism to the reduction of blood flow in muscle and adipose tissue under hypothyroid conditions .

evidence for a relationship between t4 and t3 and glucose metabolism appeared over 100 years ago when the influence of thyroid hormone excess in the deterioration of glucose metabolism was first noticed . since then , it has been known that hyperthyroidism is associated with insulin resistance . more recently , hypothyroidism has also been linked to decreased insulin sensitivity . the explanation to this apparent paradox may lie in the differential effects of thyroid hormones at the liver and peripheral tissues level . the purpose of this paper is to explore the effects of thyroid hormones in glucose metabolism and analyze the mechanisms whereby alterations of thyroid hormones lead to insulin resistance .

1. Introduction 2. Effects of Thyroid Hormones on Glucose Metabolism ( 3. Insulin Resistance as a Consequence of Hyperthyroidism 4. Hypothyroidism Can Also Lead to Insulin Resistance 5. Conclusions

bernardo alberto houssay in his lecture in 1947 the blood sugar and the production and consumption of glucose are kept within normal bounds , therefore there is an equilibrium between the glands of internal secretions which reduce the blood sugar ( pancreas ) and those which raise it ( anterohypophysis , adrenals , thyroid , etc . ) deficit or excess of thyroid hormones can break this equilibrium leading to alterations of carbohydrate metabolism . overt hyperthyroidism has been related to glucose intolerance and even ketoacidosis . with regards to hypothyroidism , cases of hypoglycemia have been reported in the literature despite the fact that peripheral insulin resistance may be present . in the century that has elapsed , since the first observations of uncontrolled glucose metabolism in thyrotoxic diabetic patients , new pathways involved in the regulation of glucose homeostasis by thyroid hormones have been unveiled . novel findings include the stimulation of hepatic glucose production by thyroid hormones acting via a sympathetic pathway from the hypothalamus and the discovery of transcriptional regulators of metabolic and mitochondrial genes that , influenced by intracellular t3 levels , may contribute to the development of insulin resistance . in this paper , we summarize the effects of thyroid hormones in glucose metabolism and its alterations when thyroid dysfunction is present . thyroid receptor - mediated effects on gene transcription and translation are key in the regulation of glucose metabolism . according to the results of studies with complementary dna ( cdna ) microarray analysis in mouse liver , several genes involved in gluconeogenesis , glycogen metabolism , and insulin signaling that are regulated by thyroid hormones have been identified . in the study by feng et al . , rna from hypothyroid mice treated with t3 was prepared , labeled with fluorescent dye , and hybridized with the cdna microarray . this enzyme hydrolyzes glucose-6-phosphate and completes the final step in gluconeogenesis and glycogenolysis , therefore playing an important role in the homeostatic regulation of blood glucose levels . another finding was a decrease in mrna expression of akt2 ( protein kinase b ) , a serine / threonine kinase that is an essential molecule in the insulin signaling pathway . akt2 has been shown to promote glycogen synthesis in liver by inactivating glycogen synthase kinase 3 . thus , a decrease in akt2 activity would decrease glycogen synthesis explaining the antagonistic insulin effect of thyroid hormones at the liver . all these results are in favour of a permissive influence of t3 in the glycogenolytic and gluconeogenic effects of epinephrine and glucagon . other hepatic gluconeogenic enzymes that have been found to be positively regulated by thyroid hormones include phosphoenolpyruvate carboxykinase ( pepck ) , the enzyme that catalyzes the rate - controlling step of gluconeogenesis and pyruvate carboxylase , involved in the synthesis of oxaloacetate from pyruvate . the catalytic activity of pyruvate carboxylase has been found increased approximately 2-fold in hyperthyroid rats compared with untreated or treated euthyroid controls . another mechanism , whereby thyroid hormones are known to increase hepatic glucose output , is through increased hepatic expression of the glucose transporter glut2 as previously shown in a rat model where glut2 protein concentration in crude liver membranes was twice as high in chronically hyperthyroid versus hypothyroid animals . it has been previously reported that , despite an expected resistance towards the insulin inhibitory effect on gluconeogenesis , the transcription of several enzymes involved in lipid synthesis or lipid metabolism is increased in hyperinsulinemic , insulin - resistant mice . furthermore , t3 induction of lipogenesis through the transcriptional activation of malic enzyme , involved in fatty acid synthesis , has been previously reported . therefore , it is possible that by the induction of lipogenic enzymes , t3 could be further aggravating the dysregulation of liver glucose and lipid metabolism characteristic of insulin resistance . as a result of the long time quest for thyroid analogs that possess the favourable actions on metabolism without the unwanted thyroid cardiac effects , the differential distribution of thyroid receptor ( tr ) isoforms in the tissues has been key for the development of these analogs . with regards to lipogenesis , carbohydrate - response element - binding protein ( chrebp ) is a major transcription factor controlling the activation of glucose - induced lipogenesis in liver and is a direct target of thyroid hormones in liver and white adipose tissue ( wat ) , the two main lipogenic tissues in mice . chrebp is shown to be specifically regulated by trbeta but not by tralpha in vivo , in liver where trbeta represents 80% of the thyroid hormone bound tr , but also in wat where both tr isoforms are expressed . although the area of thyroid analogs is beyond the scope of this paper , it is to be mentioned that some thyromimetic analogs , such as 3,5-l - diiodothyronine ( t(2 ) ) , exert their beneficial action on metabolism , without inducing a thyrotoxic state , through a mechanism that does not involve binding to thyroid hormone receptors . surprisingly , in this study , these lipid - lowering actions not mediated by tr were also observed with t3 . to summarize , all these findings have helped to understand that thyroid hormones have insulin antagonistic effects at the liver that lead to an increased glucose hepatic output , via an enhanced rate of gluconeogenesis and glycogenolysis . with regards to lipid metabolism , however , in the context of insulin resistance , the conversion of glucose into fatty acids together with nonsuppressed gluconeogenesis is simply perpetuating the hyperinsulinemic state . furthermore , nutritional influences , such as those of high - fat diets , should also be taken into consideration as modifiers of the effects of thyroid hormones on insulin sensitivity . opposite to what occurs at the liver level , at peripheral tissues , thyroid hormones have been shown to exert some of their actions synergically with insulin . the upregulation of the expression of genes such as glut-4 or phosphoglycerate kinase ( pgk ) , involved in glucose transport and glycolysis respectively , is a good proof of concept . unveiling this association may be important since progressive reduction of ucp3 levels results in insulin resistance accompanied by decreased fatty acid oxidation and a less intense akt / pkb and 5 adenosine monophosphate - activated protein kinase ( ampk ) signaling . in a model of high - fat diet - induced insulin resistance in rat , the administration of t2 , on the gastrocnemius muscle , induced remarkable changes on the metabolic / structural phenotype and insulin signaling . moreover , the result of cdna microarray analysis on skeletal muscle of a group of healthy men receiving 75 g / d of t3 for 14 days has shown that not only genes with agonistic insulin effects but several others with antagonistic insulin effects are upregulated by treatment with t3 , underlying the pleiotropic effect of thyroid hormone in energy metabolism . in human adipocytes , t3 increases the mrna levels of the lipolytic 2-ar , favouring catecolamine - induced lipolysis and it also downregulates sterol regulatory element binding protein ( srebp1c ) , involved in lipogenesis , which may constitute a link between hyperthyroidism and insulin resistance . skin fibroblasts have been also used to study thyroid hormone - responsive genes involved in metabolism in human cells . although they are not as metabolically active as hepatic cells , they are easily obtained and also , thyroid hormone - responsive . observed that , opposite to a posttranscriptional regulation as reported for other growth factors and hormones , the mrna of the transcription factor hif-1 ( hypoxia - inducible factor 1 ) , a key mediator of glycolysis , increased in response to t3 . as the glucose transporter glut1 , several enzymes of glycolysis , and the lactate exporter slc16a3 were all also found induced by t3 and are target genes of the transcription factor hif-1 , the authors postulated that the effect of thyroid hormones on the induction of these genes most probably was indirect and hif-1 mediated . furthermore , a new mechanism of thyroid action was unraveled by this group of researchers . it was shown that t3 bound to trbeta , in lieu of initiating gene transcription in the nucleus , activates the phosphatidylinositol 3-kinase ( pi3k ) signaling pathway in the cytosol in order to activate hif-1 gene expression . at the cellular level , thyroid hormones can also increase mitochondrial biogenesis , fatty acid oxidation , and tca cycle activity . these findings are quite relevant since the role of mitochondrial dysfunction , leading to cellular lipid excess and impaired oxidative metabolism , has been clearly demonstrated in the pathogenesis of type 2 diabetes [ 2325 ] . furthermore , it has been described that in skeletal muscle , the lack of thyroid hormones might dysregulate mitochondrial gene expression . ppar gamma coactivator-1 alpha ( pgc-1 alpha ) , a key transcriptional regulator of mitochondrial content and function , fatty acid oxidation , and gluconeogenesis , has been involved in the process whereby thyroid hormones regulate mitochondrial function . it has been shown that pgc-1 alpha gene expression is increased by t3 , as much as 13-fold 6 hours after t3 treatment . the regulation pattern of t3 on pgc-1 alpha is complex and may occur through nongenomic activation of kinases to induce the expression of pgc-1 alpha or through transcriptional upregulation via the presence of a thyroid responsive element ( tre ) in the pgc-1 alpha promoter or by genomic upregulation of a transcription factor ( via a tre ) , which then binds to the pgc-1 alpha promoter and increases pgc-1 alpha transcription . it is hypothesized that pgc-1 alpha can be dysregulated by reduced t3 levels , thus contributing to insulin resistance . a lower expression and activity of type 2 iodothyronine - deiodinase ( d2 ) , the enzyme that is key for the conversion of t4 into t3 in muscle and thus , amplifies thyroid hormone signaling in individual cells , has been found linked to insulin resistance [ 29 , 30 ] . if pgc-1 alpha effects on mitochondrial gene expression may indeed be regulated by thyroid hormone , normal activity of deiodinase type 2 is very relevant . bile acids are potent stimulators of the enzyme and may play an important role in the relationship between thyroid action and glucose metabolism . on the other hand , the natural occurrence of polymorphisms of deiodinase type 2 such as thr92ala , with a lower activity , has also been implicated with increased risk for diabetes type 2 . it has been shown that the hypothalamus can modulate endogenous glucose production by using functionally reciprocal sympathetic and parasympathetic autonomic outputs to the liver . moreover , a sympathetic pathway from the hypothalamic paraventricular nucleus to the liver has been proposed as a central pathway for modulation of hepatic glucose metabolism by thyroid hormone . demonstrated that upon selective administration to the paraventricular nucleus ( pvn ) , t3 increases endogenous glucose production and plasma glucose , and that these hypothalamic t3 effects are mediated via sympathetic projections to the liver . in order to arrive to such remarkable findings first they administered an intracerebroventricular ( i.c.v . ) t3 or vehicle ( veh ) infusion , and there was a significant increase in plasma glucose compared with veh - treated rats . to further identify the neuroanatomic region responsible for these changes , the authors infused t3 within the hypothalamus at the pvn . the principal finding of this study is the description of a neural ( autonomic ) modulation of hepatic glucose metabolism by t3 at the hypothalamus that takes place independently of plasma glucoregulatory hormone concentrations . the literature about this topic is vast and has been previously comprehensively reviewed by dimitriadis and raptis . although ketoacidosis may result per se from the insulin resistance present in thyrotoxicosis , the stimulatory action of thyroid hormones in excess on lipolysis and free fatty acids availability can also contribute to increased ketogenesis . thyrotoxicosis has been reported to increase endogenous glucose production in the liver in the basal state and to decrease hepatic insulin sensitivity in humans . the different mechanisms to explain this phenomenon include increased rates of gluconeogenesis and glycogenolysis mainly explained by the above - mentioned effects on the liver by thyroid hormones . to summarize , these effects include thyroid receptor - mediated effects on liver gene transcription , increased sympathetic action in the liver mediated by hypothalamus , and increased concentrations of the glut2 glucose transporters in the liver plasma membrane that allows for glucose efflux [ 8 , 39 ] together with increased concentration of free fatty acids in plasma . the interpretation of the effects of hyperthyroidism on glucose utilization by peripheral tissues is by far the most complex issue on this topic . on one hand , the rates of glucose uptake in peripheral tissues have been found increased by thyroid hormones , suggesting that glucose utilization is highly increased , specially in skeletal muscle [ 34 , 37 , 4144 ] . however , a decrease in insulin - stimulated nonoxidative glucose disposal , through reduced glycogenogenesis [ 43 , 44 , 49 ] , takes place , with intracellular glucose being redirected towards glycolysis and lactate formation . the release of lactate from peripheral tissues back to the liver is a major contributor to the cori cycle where more hepatic glucose is being produced [ 43 , 4951 ] . although glucose intolerance in hyperthyroidism can be easily explained by hepatic insulin resistance without involvement of peripheral tissues , impaired insulin - stimulated peripheral glucose uptake has also been proven in some studies . by means of the arteriovenous difference technique in the forearm muscles of hyperthyroid subjects after the consumption of a mixed meal , it has been clearly demonstrated that muscle blood flow is increased , masking a defect in insulin - stimulated glucose uptake . also described decreased peripheral insulin sensitivity in hyperthyroidism . alternative explanations for peripheral insulin resistance in hyperthyroidism include an increased secretion of bioactive mediators ( adipokines ) such as interleukin 6 ( il6 ) and tumour necrosis factor a ( tnf ) from adipose tissue in hyperthyroidism . however , a rather consistent finding has been the increased degradation of insulin in hyperthyroid subjects [ 43 , 55 ] . it has been postulated that , in the long run , severe thyrotoxicosis can lead to irreversible pancreatic damage [ 56 , 57 ] . subclinical hyperthyroidism has also been associated with insulin resistance [ 5961 ] in some but not all studies . endogenous subclinical hyperthyroidism may have a larger impact on glucose metabolism due to its chronicity and higher t3 levels when compared to exogenous administration of t4 . this phenomenon can be interpreted in the light of reduced levels of gluconeogenesis leading to decreased liver glucose output [ 63 , 64 ] . on the other hand , insulin resistance has been shown to be present in peripheral tissues in hypothyroid animal models [ 65 , 66 ] . hence , a poor utilization of glucose in hypothyroidism may be offset by a reduced release to circulation maintaining a balance of the glucose metabolism . studies performed in adipocytes and skeletal muscle of rats made hypothyroid have shown that these tissues are less responsive to insulin with regards to glucose metabolism [ 63 , 6569 ] . czech et al . other authors [ 66 , 67 ] showed decreased insulin - stimulated glucose transport and/or phosphorylation , as well as a lower rate of glycolysis in the isolated , incubated soleus muscle of the hypothyroid rat and also suggested that the effects of hypothyroidism in muscle were not mediated through an interference of insulin binding to its receptor . insulin resistance was confirmed in another study of rats with mild hypothyroidism . plasma leptin levels were lower and adipose tissue mrna expression of resistin higher , in the hypothyroid state . looking for a potential role of leptin in the metabolic consequences of hypothyroidism , leptin was infused , and it was found that glucose disposal was recovered . therefore , one possible explanation to decreased insulin responsiveness in hypothyroidism , according to the authors of this study , includes a dysregulation of leptin action at the hypothalamus . adipocyte - myocyte crosstalk by adipokines has been reported to play a significant role in skeletal muscle insulin resistance and may partially explain insulin resistance present in hypothyroidism . however , other factors associated with insulin resistance in hypothyroidism , such as altered blood flow , impaired glut4 translocation , decreased glycogen synthesis , and decreased muscle oxidative capacity have to be also considered . compared to the number of reports about insulin resistance in hyperthyroid patients , there are relatively fewer studies in humans dealing with the effects of hypothyroidism on glucose metabolism . measured whole - body sensitivity of glucose disposal to insulin in hypothyroid patients using the euglycemic - hyperinsulinemic clamp technique . they demonstrated that hypothyroidism induced a decrease in the insulin - mediated glucose disposal that reverted upon treatment . the same clamp technique and also measuring glucose tolerance and beta - cell activity with an oral glucose tolerance tests ( ogtt ) , handisurya et al . confirmed these findings and added the knowledge that glucose - induced insulin secretion is diminished by thyroid replacement corresponding well with the observed improvement of insulin sensitivity . dimitriadis et al . explored glucose uptake in muscle and adipose tissue of hypothyroid and control subjects by means of the arteriovenous difference technique in the anterior abdominal subcutaneous adipose tissue and the forearm muscles after the consumption of a mixed meal . a decreased net extraction of glucose and blood flow after the meal in hypothyroid muscle and adipose tissue was reported . this impairment in the ability of insulin to increase blood flow rate to the hypothyroid tissues is an alternative explanation to the mechanism whereby hypothyroidism can induce lower glucose disposal . insulin tolerance test has been used to explore insulin sensitivity in acute overt hypothyroid patients . a previous study in overt hypothyroid patients based on the homeostasis model assessment ( homa - ir ) showed no association between hypothyroidism and insulin sensitivity . found unimpaired insulin - stimulated glucose disposal in the forearm of hypothyroid patients after treatment with levothyroxine . with regards to subclinical hypothyroidism , insulin resistance has been demonstrated in some [ 60 , 74 , 79 ] but not all studies , where homa levels were found comparable to a control group [ 8082 ] . however , in some of these negative studies [ 80 , 81 ] , hyperinsulinemia was reported in subclinical hypothyroid subjects and interpreted as an early sign of impairment of glucose metabolism . thyroid hormones have a large impact on glucose metabolism . a direct regulation on thyroid responsive genes at the target organ has been described and more recently an indirect effect involving hypothalamic pathways that regulate glucose metabolism via control of the sympathetic nervous system has been reported . furthermore , thyroid hormone effects can be insulin agonistic , such as demonstrated in muscle or antagonistic such as observed in the liver . in hyperthyroidism , dysregulation of this balance may end in glucose intolerance mainly due to hepatic insulin resistance . however , the available data suggest that insulin resistance is present mainly at the peripheral tissues . possible explanations hypothesized to explain this phenomenon span from the dysregulation of mitochondrial oxidative metabolism to the reduction of blood flow in muscle and adipose tissue under hypothyroid conditions .

the prompt administration of coronary reperfusion therapy for patients with an evolving acute myocardial infarction ( ami ) is crucial in reducing mortality and the risk of serious clinical complications in these patients.1 during the past decade , primary percutaneous coronary intervention ( pci ) has gradually replaced thrombolysis as the main revascularization strategy for patients presenting with st - segment elevation myocardial infarction ( stemi ) , since primary pci has been found to be superior to thrombolytic therapy when performed rapidly by expert teams.2 because the effectiveness of primary pci may be limited by delays in its prompt delivery , current clinical guidelines have recommended a door - to - balloon time of 90 minutes or less for patients hospitalized with stemi who undergo a primary pci.1 to date , while a number of studies have described the timing of receipt of a pci in patients presenting to the hospital with stemi , there are little population - based data available describing contemporary trends in the magnitude of , and factors associated with , door - to - balloon times in patients experiencing stemi who receive a primary pci;36 the limited studies in this area have shown mixed results of improvement in door - to - balloon time during varying study years and an inconsistent profile of patients who fail to be treated within recommended guidelines.36 inasmuch , there is a need to examine relatively contemporary long - term trends in the extent of , and potential risk factors associated with , delays in door - to - balloon time among patients hospitalized with stemi who undergo a primary pci , particularly from the more generalizable perspective of a population - based investigation . the primary objective of our study was to describe decade - long ( 20012011 ) trends in the extent of delay from hospital emergency department ( ed ) presentation to initiation of primary pci among patients hospitalized with stemi . our secondary objective was to examine factors associated with the failure to receive a primary pci within 90 minutes after ed arrival among patients hospitalized with stemi . described elsewhere in detail,710 the worcester heart attack study is an ongoing population - based investigation examining long - term trends in the descriptive epidemiology of ami in residents of the worcester , ma , metropolitan area ( 2000 census = 478,000 ) hospitalized at all eleven medical centers in central massachusetts on an approximate biennial basis between 1975 and 2011.710 we reviewed patient s medical records on an approximate biennial basis since the inception of this study due to the availability of federal funding support and design features of this observational study . computerized printouts of patients discharged from all greater worcester hospitals with possible ami ( international classification of disease ( ninth revision ) codes : 410414 , 786.5 ) were identified . cases of possible ami were independently validated using predefined criteria for ami;710 these criteria included a suggestive clinical history , increases in several serum biomarkers ( eg , creatine kinase , creatine kinase - mb , and troponin values ) , and serial electrocardiographic findings during hospitalization consistent with the presence of ami . patients who satisfied at least two of these three criteria and who were residents of the worcester metropolitan area since this study was population - based were included . a diagnosis of stemi was made when new st - segment elevation was present at the j point in two or more contiguous leads.11 for the purposes of this study , we restricted our sample to adult residents of the worcester metropolitan area who were hospitalized with stemi and received a primary pci at a pci - capable hospital between 2001 and 2011 , which were our most recent study years and allowed us to examine decade - long trends in the end points of interest . among the eleven medical centers in the worcester metropolitan area , the vast majority ( 99% ) of pcis were performed at the two major urban teaching and community hospitals in the city of worcester . patients who received thrombolytic therapy during hospitalization were excluded since they did not meet the criteria for receiving a primary pci . door - to - balloon time was defined as the time interval from the patient s arrival at the hospital ed to inflation of the balloon to restore coronary flow . patients who were transferred from another hospital were excluded , since the clinical guidelines of door - to - balloon time within 90 minutes were recommended for those who were initially seen at a pci - capable hospital . to increase the likelihood that we were assessing patients who received a primary pci , we excluded patients with hospital delay times that exceeded 6 hours5,12 and those who did not have door - to - balloon times documented in their hospital medical records . this study used secondary data from the review of medical records of patients hospitalized with ami . this study was approved by the institutional review board at the university of massachusetts medical school . trained nurses and physicians abstracted information on patients demographic characteristics , medical history , clinical data , and treatment practices through the review of hospital medical records . information on patient s sociodemographic characteristics ( eg , age , sex , race , marital status ) , year of hospitalization , history of previously diagnosed comorbidities ( eg , stroke , diabetes , heart failure ) , prior coronary revascularization ( pci or coronary artery bypass graft [ cabg ] surgery ) , ami event order ( initial vs prior ) , hospital ed arrival day and time , mode of transportation ( car / walked - in vs ambulance ) , and door - to - balloon time was collected . to increase the available sample size , and for ease of analysis and interpretation , we aggregated the six individual study years into three 2-year strata ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) for purposes of examining trends in our principal study outcomes . door - to - balloon time was further dichotomized as 90 minutes versus > 90 minutes based on current clinical guidelines recommendations.1 differences in the distribution of patient demographic and clinical characteristics between patients hospitalized during the three aggregated time periods were examined using the analysis of variance test for continuous variables and the chi - square test for categorical variables . the cochran armitage tests and linear regression models were used to test for linear trends over time among categorical and continuous variables , respectively . delays to the receipt of a primary pci after hospital arrival were examined by calculating the mean and median door - to - balloon times and the proportion of patients who received a primary pci within 90 minutes among patients hospitalized with stemi during the years under study . due to the relatively common nature of the primary study outcome ( ie , > 10% ) , and the advantage of providing relative risk estimates , multivariable - adjusted poisson regression models with robust error variance13 were used to examine the association between the main explanatory variable of time period of hospitalization ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) and the outcome of whether patients failed to receive a primary pci within 90 minutes after hospital ed arrival ( ie , door - to - balloon time : > 90 minutes vs 90 minutes ) while adjusting for several potentially confounding variables of prognostic importance . we dummy coded this variable with the earliest study years ( 2001/2003 ) serving as the reference group . several covariates associated with delay to the receipt of a primary pci in patients hospitalized with stemi in prior studies were examined.3,4,14,15 these factors included age , sex , race ( white vs non - white ) , marital status ( married vs unmarried ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , prior coronary revascularization ( pci or cabg surgery ) , ami event order ( initial vs prior ) , hospital ed arrival time ( regular hours : 8 am6 pm , weekday vs off - hours : before 8 am or after 6 pm , weekday and weekend ) , and mode of transportation ( car / walked - in vs ambulance ) . we also repeated our multivariable - adjusted poisson regression analysis using data from the first and the last 2-year time clusters for purposes of exploring the association between various demographic and clinical factors with delays in the receipt of a primary pci . the results of our poisson regression models with robust error variance were presented as multivariable - adjusted risk ratios ( rrs ) with accompanying 95% cis . all statistical analyses were conducted using sas version 9.3 ( sas institute , inc . , cary , nc , usa ) . trained nurses and physicians abstracted information on patients demographic characteristics , medical history , clinical data , and treatment practices through the review of hospital medical records . information on patient s sociodemographic characteristics ( eg , age , sex , race , marital status ) , year of hospitalization , history of previously diagnosed comorbidities ( eg , stroke , diabetes , heart failure ) , prior coronary revascularization ( pci or coronary artery bypass graft [ cabg ] surgery ) , ami event order ( initial vs prior ) , hospital ed arrival day and time , mode of transportation ( car / walked - in vs ambulance ) , and door - to - balloon time was collected . to increase the available sample size , and for ease of analysis and interpretation , we aggregated the six individual study years into three 2-year strata ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) for purposes of examining trends in our principal study outcomes . door - to - balloon time was further dichotomized as 90 minutes versus > 90 minutes based on current clinical guidelines recommendations.1 differences in the distribution of patient demographic and clinical characteristics between patients hospitalized during the three aggregated time periods were examined using the analysis of variance test for continuous variables and the chi - square test for categorical variables . the cochran armitage tests and linear regression models were used to test for linear trends over time among categorical and continuous variables , respectively . delays to the receipt of a primary pci after hospital arrival were examined by calculating the mean and median door - to - balloon times and the proportion of patients who received a primary pci within 90 minutes among patients hospitalized with stemi during the years under study . due to the relatively common nature of the primary study outcome ( ie , > 10% ) , and the advantage of providing relative risk estimates , multivariable - adjusted poisson regression models with robust error variance13 were used to examine the association between the main explanatory variable of time period of hospitalization ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) and the outcome of whether patients failed to receive a primary pci within 90 minutes after hospital ed arrival ( ie , door - to - balloon time : > 90 minutes vs 90 minutes ) while adjusting for several potentially confounding variables of prognostic importance . we dummy coded this variable with the earliest study years ( 2001/2003 ) serving as the reference group . several covariates associated with delay to the receipt of a primary pci in patients hospitalized with stemi in prior studies were examined.3,4,14,15 these factors included age , sex , race ( white vs non - white ) , marital status ( married vs unmarried ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , prior coronary revascularization ( pci or cabg surgery ) , ami event order ( initial vs prior ) , hospital ed arrival time ( regular hours : 8 am6 pm , weekday vs off - hours : before 8 am or after 6 pm , weekday and weekend ) , and mode of transportation ( car / walked - in vs ambulance ) . we also repeated our multivariable - adjusted poisson regression analysis using data from the first and the last 2-year time clusters for purposes of exploring the association between various demographic and clinical factors with delays in the receipt of a primary pci . the results of our poisson regression models with robust error variance were presented as multivariable - adjusted risk ratios ( rrs ) with accompanying 95% cis . all statistical analyses were conducted using sas version 9.3 ( sas institute , inc . , cary , nc , usa ) . during the years under study , there were a total of 1,853 patients hospitalized with stemi at our participating sites ; among these patients , 1,224 patients received a pci . for purposes of examining patients hospitalized with stemi who received a primary pci within the guideline - recommended door - to - balloon time , we sequentially excluded patients who received thrombolytic therapy during their acute hospitalization ( n=81 ) , who were transferred from another hospital ( n=325 ) , whose door - to - balloon time exceeded 6 hours ( n=150 ) , and who did not have door - to - balloon times documented ( n=39 ) . the final study population consisted of 629 adult residents of central massachusetts who were hospitalized with stemi and received a primary pci at the two major pci - capable urban teaching and community hospitals in the worcester metropolitan area on a biennial basis between 2001 and 2011 . overall , the average age of this patient population was 61.9 years ; 30.5% were women , 91.7% were white , and 62.0% were married . in addition , 75.8% of our study sample was hospitalized for an initial ami , 75.9% were transported to the study hospitals by ambulance , and 53.1% arrived at participating eds during off - hours ( table 1 ) . during the most recent years under study , patients who were hospitalized with stemi and received a primary pci were more likely to have a history of peripheral vascular disease or have prior coronary revascularization , compared to those hospitalized with stemi during earlier study periods ( table 1 ) . the average delay time from the patient s arrival at the ed to inflation of the balloon to restore coronary flow during the years under study was 102 minutes . there was a marked decrease in the mean delay time from 2001/2003 ( 141 minutes ) to 2009/2011 ( 85 minutes ) ( figure 1 ) . the median delay time from the patient s arrival at the hospital ed to balloon inflation during the years under study was 89 minutes . there was also a significant decrease in the median duration of pci - associated delay from 2001/2003 ( 133 minutes ) to 2009/2011 ( 73 minutes ) ( figure 1 ) . among all study patients who underwent a primary pci , 50.9% of these patients received the intervention within 90 minutes of their arrival at the ed . there was a dramatic increase in the proportion of patients with stemi who received a primary pci within guideline - recommended 90 minutes between 2001/2003 ( 17.2% ) and 2009/2011 ( 70.5% ) ( p for trend < 0.001 ) ( figure 1 ) . among all study patients who received a primary pci within 90 minutes , 46.3% were treated during the first hour after arrival at the hospital ed ; this proportion significantly increased from 10.7% in 2001/2003 to 47.8% in 2009/2011 ( p<0.001 ) . in examining changing trends in the failure to receive a primary pci within 90 minutes , after adjusting for several demographic characteristics and clinical factors , there was a significant reduction in the risk of failing to receive a primary pci in 2005/2007 ( rr = 0.53 , 95% ci = 0.400.72 ) and in 2009/2011 ( rr = 0.36 , 95% ci = 0.260.50 ) compared with those hospitalized with stemi in 2001/2003 ( table 2 ) . using multivariable - adjusted regression analyses , we examined the role of several prognostic factors associated with failure to receive a primary pci within 90 minutes in all study patients ( table 2 ) . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher risk of failing to receive a primary pci within 90 minutes ( table 3 ) . in addition , we further examined the role of various demographic and clinical factors with the failure to receive a primary pci within 90 minutes using data from the first and the last 2-year time clusters ( table 4 ) . compared with the results in our overall study population , some similar and discrepant results were found in the time periods analyzed . in these subgroup analyses , arriving at the ed by car / walk - in during off - hours was no longer significantly associated with failing to receive a primary pci within 90 minutes ; however , having previously undergone cabg surgery was significantly associated with a higher risk of failing to receive a primary pci within 90 minutes in the last 2-year time period . during the years under study , there were a total of 1,853 patients hospitalized with stemi at our participating sites ; among these patients , 1,224 patients received a pci . for purposes of examining patients hospitalized with stemi who received a primary pci within the guideline - recommended door - to - balloon time , we sequentially excluded patients who received thrombolytic therapy during their acute hospitalization ( n=81 ) , who were transferred from another hospital ( n=325 ) , whose door - to - balloon time exceeded 6 hours ( n=150 ) , and who did not have door - to - balloon times documented ( n=39 ) . the final study population consisted of 629 adult residents of central massachusetts who were hospitalized with stemi and received a primary pci at the two major pci - capable urban teaching and community hospitals in the worcester metropolitan area on a biennial basis between 2001 and 2011 . overall , the average age of this patient population was 61.9 years ; 30.5% were women , 91.7% were white , and 62.0% were married . in addition , 75.8% of our study sample was hospitalized for an initial ami , 75.9% were transported to the study hospitals by ambulance , and 53.1% arrived at participating eds during off - hours ( table 1 ) . during the most recent years under study , patients who were hospitalized with stemi and received a primary pci were more likely to have a history of peripheral vascular disease or have prior coronary revascularization , compared to those hospitalized with stemi during earlier study periods ( table 1 ) . the average delay time from the patient s arrival at the ed to inflation of the balloon to restore coronary flow during the years under study was 102 minutes . there was a marked decrease in the mean delay time from 2001/2003 ( 141 minutes ) to 2009/2011 ( 85 minutes ) ( figure 1 ) . the median delay time from the patient s arrival at the hospital ed to balloon inflation during the years under study was 89 minutes . there was also a significant decrease in the median duration of pci - associated delay from 2001/2003 ( 133 minutes ) to 2009/2011 ( 73 minutes ) ( figure 1 ) . among all study patients who underwent a primary pci , 50.9% of these patients received the intervention within 90 minutes of their arrival at the ed . there was a dramatic increase in the proportion of patients with stemi who received a primary pci within guideline - recommended 90 minutes between 2001/2003 ( 17.2% ) and 2009/2011 ( 70.5% ) ( p for trend < 0.001 ) ( figure 1 ) . among all study patients who received a primary pci within 90 minutes , 46.3% were treated during the first hour after arrival at the hospital ed ; this proportion significantly increased from 10.7% in 2001/2003 to 47.8% in 2009/2011 ( p<0.001 ) . in examining changing trends in the failure to receive a primary pci within 90 minutes , after adjusting for several demographic characteristics and clinical factors , there was a significant reduction in the risk of failing to receive a primary pci in 2005/2007 ( rr = 0.53 , 95% ci = 0.400.72 ) and in 2009/2011 ( rr = 0.36 , 95% ci = 0.260.50 ) compared with those hospitalized with stemi in 2001/2003 ( table 2 ) . using multivariable - adjusted regression analyses , we examined the role of several prognostic factors associated with failure to receive a primary pci within 90 minutes in all study patients ( table 2 ) . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher risk of failing to receive a primary pci within 90 minutes ( table 3 ) . in addition , we further examined the role of various demographic and clinical factors with the failure to receive a primary pci within 90 minutes using data from the first and the last 2-year time clusters ( table 4 ) . compared with the results in our overall study population , some similar and discrepant results were found in the time periods analyzed . in these subgroup analyses , arriving at the ed by car / walk - in during off - hours was no longer significantly associated with failing to receive a primary pci within 90 minutes ; however , having previously undergone cabg surgery was significantly associated with a higher risk of failing to receive a primary pci within 90 minutes in the last 2-year time period . the results of this observational study suggest that , among greater worcester residents who were hospitalized for stemi and received a primary pci at the two major pci - capable hospitals in central massachusetts between 2001 and 2011 , there was a fourfold increase in the proportion of patients who received a primary pci within guideline - recommended 90 minutes during the years under study . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher likelihood of failing to receive a primary pci within 90 minutes at participating study hospitals . timely medical care is crucial to reducing mortality and the risk of serious clinical complications in patients experiencing signs and symptoms of an ami . this is because it can maximize the benefits of evidence - based treatments and possibly reduce the likelihood of sudden cardiac deaths and the eventual size of the infarct . although primary pci has been shown to improve outcomes in patients with stemi , its effectiveness may be limited by delays in its more timely delivery.1 since 1999 , clinical practice guidelines for the management of patients with stemi have recommended door - to - balloon times of 90 minutes or less.1,16,17 however , earlier data from the national registry of myocardial infarction , which examined 33,647 patients hospitalized with stemi who received a primary pci between 1999 and 2002 at 421 us hospitals , reported that only 35% of patients were treated within the recommended 90 minutes after arrival at the hospital ; meaningful improvements in door - to - balloon times over the study period were not observed.3 these discouraging findings led to several national efforts dedicated to reducing door - to - balloon time in patients hospitalized with stemi . the centers for medicare and medicaid services and the joint commission began using door - to - balloon time as a performance measure in 2002.18 in 2006 , the centers for medicare and medicaid services began publicly reporting hospital achievement of door - to - balloon times of 90 minutes or less . in november 2006 , the american college of cardiology , the american heart association , and several other organizations launched the door - to - balloon ( d2b ) : an alliance for quality campaign with the goal of increasing the percentage of patients with stemi who would receive a primary pci within 90 minutes of presentation at a pci - capable hospital to 75%.19 in may 2007 , the american heart association launched mission : lifeline , another national initiative designed to educate patients and providers about the importance of rapid response to stemi and to help hospitals create coordinated stemi diagnostic and treatment systems.20 several studies in the us have shown reductions in door - to - balloon times since these national efforts have been employed.46 findings from the blue cross blue shield of michigan cardiovascular consortium of 8,771 patients with stemi who underwent a primary pci at nine hospitals between 2003 and 2008 showed that the median door - to - balloon time had decreased from 113 minutes in 2003 to 76 minutes in 2008 ( p<0.001 ) . in addition , the percentage of patients who were revascularized with a door - to - balloon time of < 90 minutes significantly increased from 29% in 2003 to 67% in 2008 ( p<0.001).4 a prior study that examined data from > 300,000 medicare patients at 900 us hospitals found that door - to - balloon times declined from a median of 96 minutes in 2005 to 64 minutes in 2010 . there were corresponding increases in the percentage of patients who had door - to - balloon times < 90 minutes ( 44%91%).5 a recent study analyzed data from 96,738 admissions for patients with stemi who underwent a primary pci from july , 2005 , through june , 2009 , at 515 us hospitals participating in the cathpci registry . median door - to - balloon times declined significantly from 83 minutes in the first year to 67 minutes in the most recent study year , and the percentage of patients for whom the door - to - balloon time was 90 minutes or less increased from 60% to 83% during the years under study ( p<0.001).6 consistent with the timeline of national efforts in reducing door - to - balloon time and prior research results , our current decade - long trend study observed a substantial decrease in the median door - to - balloon time and a dramatic increase in the proportion of patients who received a primary pci within the guideline - recommended 90 minutes since 2005/2007 . indeed , two studies have been previously published from our institution , which examined the positive impact of a regional system in central massachusetts for stemi care on shortening the door - to - balloon times for these patients during the past several years.21,22 while reductions in door - to - balloon times have taken place in many us hospitals over time , some unintended consequences of these efforts merit attention . corresponding to the national effort initiated by the american college of cardiology in 2006 to reduce door - to - balloon times , several strategies and organizational factors associated with shorter door - to - balloon time have been identified and promoted.2325 these efforts include encouraging emergency medical service ( ems ) providers and ed physicians to activate the cardiac catheterization laboratory prior to consultation with a staff cardiologist , which may have achieved a significant reduction in door - to - balloon time , while increasing the rate of false activations . indeed , a prior study of all adult patients with a suspected stemi between 2007 and 2011 at the university of michigan hospital noted that the median door - to - balloon time decreased from 67 minutes in 2007 to 55 minutes in 2011 , but the false activation rates increased from 15% to 40% of all cases.26 when the cardiac catheterization laboratory is activated emergently , resources must be collected to prepare for a potential patient . during off - hours , this often requires bringing in a full team to begin preparing the cardiac catheterization laboratory . these false cardiac catheterization laboratory activations can be a drain on staff and a poor use of resources . therefore , future studies of health care system interventions to decrease the rates of false cardiac catheterization laboratory activations while maintaining short door - to - balloon times remain warranted . a recent systematic review and meta - analysis examining the association between off - hour presentation and outcomes in patients with ami has suggested that patients with stemi presenting during off - hours have longer door - to - balloon times.27 a prior study examined data from the get with the guidelines coronary artery disease databases between 2000 and 2005 and found that , among the 5,454 patients with stemi who received a primary pci , those arriving during off - hours were less likely to achieve door - to - balloon times 90 minutes compared with those arriving during regular hours.28 in the national registry of myocardial infarction study of 33,647 patients with stemi treated with primary pci from 1999 to 2002,15 54% of patients were treated during off - hours ; door - to - balloon times were substantially longer during off - hours ( 116 minutes ) than regular hours ( 95 minutes ) . longer door - to - balloon times during off - hours were primarily due to a longer interval between obtaining the electrocardiogram ( ecg ) and patient arrival at the cardiac catheterization laboratory . similarly , our study noted that patients with stemi admitted to the ed during off - hours were less likely to have received a timely primary pci compared with those admitted during regular hours . approaches to provide onsite staffing of the cardiac catheterization laboratory and rapid access to interventional cardiologists during off - hours , including consideration of the costs of providing such coverage , would be beneficial . in this investigation , we found that patients with stemi who arrived at the ed by car / walked - in were less likely to have received a primary pci within 90 minutes , compared with those who arrived by ambulance . prehospital ecgs have been recommended and are increasingly used in the management of patients with chest pain transported by emss,1 such that paramedics can rapidly diagnose and triage patients with a suspected stemi before hospital arrival . since hospitals can use the prehospital ecg results to activate the cardiac catheterization laboratory while the patient is en route to the hospital , door - to - balloon times are shorter than when activation is initiated after the patient s arrival at the ed . several studies have shown that the use of prehospital ecgs is associated with shorter door - to - balloon times.29,30 patients who arrive at the ed by private vehicle or other means might be triaged to a lower level of estimated illness severity and , as such , experience delays in the receipt of an ecg and diagnosis of stemi . similarly , patients who present with chest pain and arrive at the hospital by ems may receive more rapid and definitive care due to the importance placed on ems arrival by providers and perception that patients transported by ems may be sicker than those who arrive by other means . although our study also identified that having previously undergone cabg surgery was significantly associated with not receiving a primary pci within the guideline - recommended time frame , a prior systematic review of factors associated with door - to - balloon time in patients with stemi treated with pci has found mixed findings with regard to the strength of association of these factors between studies.31 while these differences in study results may be due to differences in study design , definitions of key covariates , patient populations under study , and sample size considerations , our study identified several patient groups at high risk for failing to be treated in a timely manner in whom further surveillance and hospital or provider educational efforts might be directed . although several studies , including ours , have suggested encouraging reductions in door - to - balloon times over the years , health care providers should continue their efforts to educate patients about the symptoms of ami and importance of calling 911 to facilitate ems triage , treatment , and transport to reduce not only in - hospital but also prehospital treatment delays . indeed , delays in patient s medical care seeking behavior following the development of acute coronary symptoms continue to remain unduly long and have improved little over time.32,33 the strengths of the present community - based study include the examination of relatively contemporary decade - long trends in , and factors associated with , door - to - balloon time among patients hospitalized with stemi . however , several limitations need to be acknowledged in the interpretation of the present findings . since our study population included only patients who had received a primary pci at the two major pci - capable urban teaching and community hospitals in central massachusetts , one needs to be careful in extrapolating our findings to those who reside in other geographic areas . because study patients were predominantly white , the generalizability of our findings to other race / ethnic groups may be limited . in addition , there remains the potential for unmeasured confounding in our observed associations since we did not have information available on several patient - associated characteristics , such as education , psychosocial factors , and treatment preference , as well as detailed health care system - level factors , which may have affected door - to - balloon times . since our study was restricted to patients who were hospitalized with stemi and received a primary pci at a pci - capable hospital in central massachusetts between 2001 and 2011 , we aggregated data from the six individual study years in order to increase the overall sample size . therefore , our subgroup analysis using data from the first and the last 2-year time clusters was limited in providing statistically stable results . timely medical care is crucial to reducing mortality and the risk of serious clinical complications in patients experiencing signs and symptoms of an ami . this is because it can maximize the benefits of evidence - based treatments and possibly reduce the likelihood of sudden cardiac deaths and the eventual size of the infarct . although primary pci has been shown to improve outcomes in patients with stemi , its effectiveness may be limited by delays in its more timely delivery.1 since 1999 , clinical practice guidelines for the management of patients with stemi have recommended door - to - balloon times of 90 minutes or less.1,16,17 however , earlier data from the national registry of myocardial infarction , which examined 33,647 patients hospitalized with stemi who received a primary pci between 1999 and 2002 at 421 us hospitals , reported that only 35% of patients were treated within the recommended 90 minutes after arrival at the hospital ; meaningful improvements in door - to - balloon times over the study period were not observed.3 these discouraging findings led to several national efforts dedicated to reducing door - to - balloon time in patients hospitalized with stemi . the centers for medicare and medicaid services and the joint commission began using door - to - balloon time as a performance measure in 2002.18 in 2006 , the centers for medicare and medicaid services began publicly reporting hospital achievement of door - to - balloon times of 90 minutes or less . in november 2006 , the american college of cardiology , the american heart association , and several other organizations launched the door - to - balloon ( d2b ) : an alliance for quality campaign with the goal of increasing the percentage of patients with stemi who would receive a primary pci within 90 minutes of presentation at a pci - capable hospital to 75%.19 in may 2007 , the american heart association launched mission : lifeline , another national initiative designed to educate patients and providers about the importance of rapid response to stemi and to help hospitals create coordinated stemi diagnostic and treatment systems.20 several studies in the us have shown reductions in door - to - balloon times since these national efforts have been employed.46 findings from the blue cross blue shield of michigan cardiovascular consortium of 8,771 patients with stemi who underwent a primary pci at nine hospitals between 2003 and 2008 showed that the median door - to - balloon time had decreased from 113 minutes in 2003 to 76 minutes in 2008 ( p<0.001 ) . in addition , the percentage of patients who were revascularized with a door - to - balloon time of < 90 minutes significantly increased from 29% in 2003 to 67% in 2008 ( p<0.001).4 a prior study that examined data from > 300,000 medicare patients at 900 us hospitals found that door - to - balloon times declined from a median of 96 minutes in 2005 to 64 minutes in 2010 . there were corresponding increases in the percentage of patients who had door - to - balloon times < 90 minutes ( 44%91%).5 a recent study analyzed data from 96,738 admissions for patients with stemi who underwent a primary pci from july , 2005 , through june , 2009 , at 515 us hospitals participating in the cathpci registry . median door - to - balloon times declined significantly from 83 minutes in the first year to 67 minutes in the most recent study year , and the percentage of patients for whom the door - to - balloon time was 90 minutes or less increased from 60% to 83% during the years under study ( p<0.001).6 consistent with the timeline of national efforts in reducing door - to - balloon time and prior research results , our current decade - long trend study observed a substantial decrease in the median door - to - balloon time and a dramatic increase in the proportion of patients who received a primary pci within the guideline - recommended 90 minutes since 2005/2007 . indeed , two studies have been previously published from our institution , which examined the positive impact of a regional system in central massachusetts for stemi care on shortening the door - to - balloon times for these patients during the past several years.21,22 while reductions in door - to - balloon times have taken place in many us hospitals over time , some unintended consequences of these efforts merit attention . corresponding to the national effort initiated by the american college of cardiology in 2006 to reduce door - to - balloon times , several strategies and organizational factors associated with shorter door - to - balloon time have been identified and promoted.2325 these efforts include encouraging emergency medical service ( ems ) providers and ed physicians to activate the cardiac catheterization laboratory prior to consultation with a staff cardiologist , which may have achieved a significant reduction in door - to - balloon time , while increasing the rate of false activations . indeed , a prior study of all adult patients with a suspected stemi between 2007 and 2011 at the university of michigan hospital noted that the median door - to - balloon time decreased from 67 minutes in 2007 to 55 minutes in 2011 , but the false activation rates increased from 15% to 40% of all cases.26 when the cardiac catheterization laboratory is activated emergently , resources must be collected to prepare for a potential patient . during off - hours , this often requires bringing in a full team to begin preparing the cardiac catheterization laboratory . these false cardiac catheterization laboratory activations can be a drain on staff and a poor use of resources . therefore , future studies of health care system interventions to decrease the rates of false cardiac catheterization laboratory activations while maintaining short door - to - balloon times remain warranted . a recent systematic review and meta - analysis examining the association between off - hour presentation and outcomes in patients with ami has suggested that patients with stemi presenting during off - hours have longer door - to - balloon times.27 a prior study examined data from the get with the guidelines coronary artery disease databases between 2000 and 2005 and found that , among the 5,454 patients with stemi who received a primary pci , those arriving during off - hours were less likely to achieve door - to - balloon times 90 minutes compared with those arriving during regular hours.28 in the national registry of myocardial infarction study of 33,647 patients with stemi treated with primary pci from 1999 to 2002,15 54% of patients were treated during off - hours ; door - to - balloon times were substantially longer during off - hours ( 116 minutes ) than regular hours ( 95 minutes ) . longer door - to - balloon times during off - hours were primarily due to a longer interval between obtaining the electrocardiogram ( ecg ) and patient arrival at the cardiac catheterization laboratory . similarly , our study noted that patients with stemi admitted to the ed during off - hours were less likely to have received a timely primary pci compared with those admitted during regular hours . approaches to provide onsite staffing of the cardiac catheterization laboratory and rapid access to interventional cardiologists during off - hours , including consideration of the costs of providing such coverage , would be beneficial . in this investigation , we found that patients with stemi who arrived at the ed by car / walked - in were less likely to have received a primary pci within 90 minutes , compared with those who arrived by ambulance . prehospital ecgs have been recommended and are increasingly used in the management of patients with chest pain transported by emss,1 such that paramedics can rapidly diagnose and triage patients with a suspected stemi before hospital arrival . since hospitals can use the prehospital ecg results to activate the cardiac catheterization laboratory while the patient is en route to the hospital , door - to - balloon times are shorter than when activation is initiated after the patient s arrival at the ed . several studies have shown that the use of prehospital ecgs is associated with shorter door - to - balloon times.29,30 patients who arrive at the ed by private vehicle or other means might be triaged to a lower level of estimated illness severity and , as such , experience delays in the receipt of an ecg and diagnosis of stemi . similarly , patients who present with chest pain and arrive at the hospital by ems may receive more rapid and definitive care due to the importance placed on ems arrival by providers and perception that patients transported by ems may be sicker than those who arrive by other means . although our study also identified that having previously undergone cabg surgery was significantly associated with not receiving a primary pci within the guideline - recommended time frame , a prior systematic review of factors associated with door - to - balloon time in patients with stemi treated with pci has found mixed findings with regard to the strength of association of these factors between studies.31 while these differences in study results may be due to differences in study design , definitions of key covariates , patient populations under study , and sample size considerations , our study identified several patient groups at high risk for failing to be treated in a timely manner in whom further surveillance and hospital or provider educational efforts might be directed . although several studies , including ours , have suggested encouraging reductions in door - to - balloon times over the years , health care providers should continue their efforts to educate patients about the symptoms of ami and importance of calling 911 to facilitate ems triage , treatment , and transport to reduce not only in - hospital but also prehospital treatment delays . indeed , delays in patient s medical care seeking behavior following the development of acute coronary symptoms continue to remain unduly long and have improved little over time.32,33 the strengths of the present community - based study include the examination of relatively contemporary decade - long trends in , and factors associated with , door - to - balloon time among patients hospitalized with stemi . however , several limitations need to be acknowledged in the interpretation of the present findings . since our study population included only patients who had received a primary pci at the two major pci - capable urban teaching and community hospitals in central massachusetts , one needs to be careful in extrapolating our findings to those who reside in other geographic areas . because study patients were predominantly white , the generalizability of our findings to other race / ethnic groups may be limited . in addition , there remains the potential for unmeasured confounding in our observed associations since we did not have information available on several patient - associated characteristics , such as education , psychosocial factors , and treatment preference , as well as detailed health care system - level factors , which may have affected door - to - balloon times . since our study was restricted to patients who were hospitalized with stemi and received a primary pci at a pci - capable hospital in central massachusetts between 2001 and 2011 , we aggregated data from the six individual study years in order to increase the overall sample size . therefore , our subgroup analysis using data from the first and the last 2-year time clusters was limited in providing statistically stable results . between 2001 and 2011 , the likelihood of receiving a primary pci within guideline - recommended times among patients who were hospitalized with stemi at the two major teaching and community hospitals in central massachusetts has increased dramatically . although most of the identified risk factors for the less than optimal timely receipt of a primary pci were not modifiable , our findings can hopefully lead to better development of innovative , patient - centered , intervention strategies , which can further reduce the door - to - balloon times of patients hospitalized with stemi and enhance their hospital and postdischarge outcomes .

objectivesthe purpose of this study was to examine decade - long trends ( 20012011 ) in , and factors associated with , door - to - balloon time within 90 minutes of hospital presentation among patients hospitalized with st - segment elevation myocardial infarction ( stemi ) who received a primary percutaneous coronary intervention ( pci).methodsresidents of central massachusetts hospitalized with stemi who received a primary pci at two major pci - capable medical centers in central massachusetts on a biennial basis between 2001 and 2011 comprised the study population ( n=629 ) . multivariable regression analyses were used to examine factors associated with failing to receive a primary pci within 90 minutes after emergency department ( ed ) arrival.resultsthe average age of this patient population was 61.9 years ; 30.5% were women , and 91.7% were white . during the years under study , 50.9% of patients received a primary pci within 90 minutes of ed arrival ; this proportion increased from 2001/2003 ( 17.2% ) to 2009/2011 ( 70.5% ) ( p<0.001 ) . having previously undergone coronary artery bypass graft surgery , arriving at the ed by car / walk - in and during off - hours were significantly associated with a higher risk of failing to receive a primary pci within 90 minutes of ed arrival.conclusionthe likelihood of receiving a timely primary pci in residents of central massachusetts hospitalized with stemi at the major teaching / community medical centers increased dramatically during the years under study . several groups were identified for purposes of heightened surveillance and intervention efforts to reduce the likelihood of failing to receive a timely primary pci among patients acutely diagnosed with stemi .

Introduction Methods Data collection Data analysis Results Study population characteristics Trends in door-to-balloon time Factors associated with failure to receive a primary PCI within 90 minutes Discussion Trends in, and magnitude of, door-to-balloon time Factors associated with failure to receive a primary PCI within 90 minutes after ED arrival Strengths and limitations of the study Conclusion

the prompt administration of coronary reperfusion therapy for patients with an evolving acute myocardial infarction ( ami ) is crucial in reducing mortality and the risk of serious clinical complications in these patients.1 during the past decade , primary percutaneous coronary intervention ( pci ) has gradually replaced thrombolysis as the main revascularization strategy for patients presenting with st - segment elevation myocardial infarction ( stemi ) , since primary pci has been found to be superior to thrombolytic therapy when performed rapidly by expert teams.2 because the effectiveness of primary pci may be limited by delays in its prompt delivery , current clinical guidelines have recommended a door - to - balloon time of 90 minutes or less for patients hospitalized with stemi who undergo a primary pci.1 to date , while a number of studies have described the timing of receipt of a pci in patients presenting to the hospital with stemi , there are little population - based data available describing contemporary trends in the magnitude of , and factors associated with , door - to - balloon times in patients experiencing stemi who receive a primary pci;36 the limited studies in this area have shown mixed results of improvement in door - to - balloon time during varying study years and an inconsistent profile of patients who fail to be treated within recommended guidelines.36 inasmuch , there is a need to examine relatively contemporary long - term trends in the extent of , and potential risk factors associated with , delays in door - to - balloon time among patients hospitalized with stemi who undergo a primary pci , particularly from the more generalizable perspective of a population - based investigation . the primary objective of our study was to describe decade - long ( 20012011 ) trends in the extent of delay from hospital emergency department ( ed ) presentation to initiation of primary pci among patients hospitalized with stemi . our secondary objective was to examine factors associated with the failure to receive a primary pci within 90 minutes after ed arrival among patients hospitalized with stemi . a diagnosis of stemi was made when new st - segment elevation was present at the j point in two or more contiguous leads.11 for the purposes of this study , we restricted our sample to adult residents of the worcester metropolitan area who were hospitalized with stemi and received a primary pci at a pci - capable hospital between 2001 and 2011 , which were our most recent study years and allowed us to examine decade - long trends in the end points of interest . delays to the receipt of a primary pci after hospital arrival were examined by calculating the mean and median door - to - balloon times and the proportion of patients who received a primary pci within 90 minutes among patients hospitalized with stemi during the years under study . due to the relatively common nature of the primary study outcome ( ie , > 10% ) , and the advantage of providing relative risk estimates , multivariable - adjusted poisson regression models with robust error variance13 were used to examine the association between the main explanatory variable of time period of hospitalization ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) and the outcome of whether patients failed to receive a primary pci within 90 minutes after hospital ed arrival ( ie , door - to - balloon time : > 90 minutes vs 90 minutes ) while adjusting for several potentially confounding variables of prognostic importance . several covariates associated with delay to the receipt of a primary pci in patients hospitalized with stemi in prior studies were examined.3,4,14,15 these factors included age , sex , race ( white vs non - white ) , marital status ( married vs unmarried ) , previously diagnosed comorbid conditions ( angina , atrial fibrillation , heart failure , hypertension , peripheral vascular disease , stroke , diabetes , chronic obstructive pulmonary disease , depression , and chronic kidney disease ) , prior coronary revascularization ( pci or cabg surgery ) , ami event order ( initial vs prior ) , hospital ed arrival time ( regular hours : 8 am6 pm , weekday vs off - hours : before 8 am or after 6 pm , weekday and weekend ) , and mode of transportation ( car / walked - in vs ambulance ) . delays to the receipt of a primary pci after hospital arrival were examined by calculating the mean and median door - to - balloon times and the proportion of patients who received a primary pci within 90 minutes among patients hospitalized with stemi during the years under study . due to the relatively common nature of the primary study outcome ( ie , > 10% ) , and the advantage of providing relative risk estimates , multivariable - adjusted poisson regression models with robust error variance13 were used to examine the association between the main explanatory variable of time period of hospitalization ( 2001/2003 , earliest ; 2005/2007 , middle ; and 2009/2011 , most recent ) and the outcome of whether patients failed to receive a primary pci within 90 minutes after hospital ed arrival ( ie , door - to - balloon time : > 90 minutes vs 90 minutes ) while adjusting for several potentially confounding variables of prognostic importance . for purposes of examining patients hospitalized with stemi who received a primary pci within the guideline - recommended door - to - balloon time , we sequentially excluded patients who received thrombolytic therapy during their acute hospitalization ( n=81 ) , who were transferred from another hospital ( n=325 ) , whose door - to - balloon time exceeded 6 hours ( n=150 ) , and who did not have door - to - balloon times documented ( n=39 ) . the final study population consisted of 629 adult residents of central massachusetts who were hospitalized with stemi and received a primary pci at the two major pci - capable urban teaching and community hospitals in the worcester metropolitan area on a biennial basis between 2001 and 2011 . overall , the average age of this patient population was 61.9 years ; 30.5% were women , 91.7% were white , and 62.0% were married . there was a dramatic increase in the proportion of patients with stemi who received a primary pci within guideline - recommended 90 minutes between 2001/2003 ( 17.2% ) and 2009/2011 ( 70.5% ) ( p for trend < 0.001 ) ( figure 1 ) . among all study patients who received a primary pci within 90 minutes , 46.3% were treated during the first hour after arrival at the hospital ed ; this proportion significantly increased from 10.7% in 2001/2003 to 47.8% in 2009/2011 ( p<0.001 ) . in examining changing trends in the failure to receive a primary pci within 90 minutes , after adjusting for several demographic characteristics and clinical factors , there was a significant reduction in the risk of failing to receive a primary pci in 2005/2007 ( rr = 0.53 , 95% ci = 0.400.72 ) and in 2009/2011 ( rr = 0.36 , 95% ci = 0.260.50 ) compared with those hospitalized with stemi in 2001/2003 ( table 2 ) . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher risk of failing to receive a primary pci within 90 minutes ( table 3 ) . in these subgroup analyses , arriving at the ed by car / walk - in during off - hours was no longer significantly associated with failing to receive a primary pci within 90 minutes ; however , having previously undergone cabg surgery was significantly associated with a higher risk of failing to receive a primary pci within 90 minutes in the last 2-year time period . for purposes of examining patients hospitalized with stemi who received a primary pci within the guideline - recommended door - to - balloon time , we sequentially excluded patients who received thrombolytic therapy during their acute hospitalization ( n=81 ) , who were transferred from another hospital ( n=325 ) , whose door - to - balloon time exceeded 6 hours ( n=150 ) , and who did not have door - to - balloon times documented ( n=39 ) . the final study population consisted of 629 adult residents of central massachusetts who were hospitalized with stemi and received a primary pci at the two major pci - capable urban teaching and community hospitals in the worcester metropolitan area on a biennial basis between 2001 and 2011 . overall , the average age of this patient population was 61.9 years ; 30.5% were women , 91.7% were white , and 62.0% were married . there was a dramatic increase in the proportion of patients with stemi who received a primary pci within guideline - recommended 90 minutes between 2001/2003 ( 17.2% ) and 2009/2011 ( 70.5% ) ( p for trend < 0.001 ) ( figure 1 ) . among all study patients who received a primary pci within 90 minutes , 46.3% were treated during the first hour after arrival at the hospital ed ; this proportion significantly increased from 10.7% in 2001/2003 to 47.8% in 2009/2011 ( p<0.001 ) . in examining changing trends in the failure to receive a primary pci within 90 minutes , after adjusting for several demographic characteristics and clinical factors , there was a significant reduction in the risk of failing to receive a primary pci in 2005/2007 ( rr = 0.53 , 95% ci = 0.400.72 ) and in 2009/2011 ( rr = 0.36 , 95% ci = 0.260.50 ) compared with those hospitalized with stemi in 2001/2003 ( table 2 ) . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher risk of failing to receive a primary pci within 90 minutes ( table 3 ) . in these subgroup analyses , arriving at the ed by car / walk - in during off - hours was no longer significantly associated with failing to receive a primary pci within 90 minutes ; however , having previously undergone cabg surgery was significantly associated with a higher risk of failing to receive a primary pci within 90 minutes in the last 2-year time period . the results of this observational study suggest that , among greater worcester residents who were hospitalized for stemi and received a primary pci at the two major pci - capable hospitals in central massachusetts between 2001 and 2011 , there was a fourfold increase in the proportion of patients who received a primary pci within guideline - recommended 90 minutes during the years under study . having previously undergone cabg surgery and arriving at the ed by car / walked - in and during off - hours were significantly associated with a higher likelihood of failing to receive a primary pci within 90 minutes at participating study hospitals . although primary pci has been shown to improve outcomes in patients with stemi , its effectiveness may be limited by delays in its more timely delivery.1 since 1999 , clinical practice guidelines for the management of patients with stemi have recommended door - to - balloon times of 90 minutes or less.1,16,17 however , earlier data from the national registry of myocardial infarction , which examined 33,647 patients hospitalized with stemi who received a primary pci between 1999 and 2002 at 421 us hospitals , reported that only 35% of patients were treated within the recommended 90 minutes after arrival at the hospital ; meaningful improvements in door - to - balloon times over the study period were not observed.3 these discouraging findings led to several national efforts dedicated to reducing door - to - balloon time in patients hospitalized with stemi . in november 2006 , the american college of cardiology , the american heart association , and several other organizations launched the door - to - balloon ( d2b ) : an alliance for quality campaign with the goal of increasing the percentage of patients with stemi who would receive a primary pci within 90 minutes of presentation at a pci - capable hospital to 75%.19 in may 2007 , the american heart association launched mission : lifeline , another national initiative designed to educate patients and providers about the importance of rapid response to stemi and to help hospitals create coordinated stemi diagnostic and treatment systems.20 several studies in the us have shown reductions in door - to - balloon times since these national efforts have been employed.46 findings from the blue cross blue shield of michigan cardiovascular consortium of 8,771 patients with stemi who underwent a primary pci at nine hospitals between 2003 and 2008 showed that the median door - to - balloon time had decreased from 113 minutes in 2003 to 76 minutes in 2008 ( p<0.001 ) . median door - to - balloon times declined significantly from 83 minutes in the first year to 67 minutes in the most recent study year , and the percentage of patients for whom the door - to - balloon time was 90 minutes or less increased from 60% to 83% during the years under study ( p<0.001).6 consistent with the timeline of national efforts in reducing door - to - balloon time and prior research results , our current decade - long trend study observed a substantial decrease in the median door - to - balloon time and a dramatic increase in the proportion of patients who received a primary pci within the guideline - recommended 90 minutes since 2005/2007 . a recent systematic review and meta - analysis examining the association between off - hour presentation and outcomes in patients with ami has suggested that patients with stemi presenting during off - hours have longer door - to - balloon times.27 a prior study examined data from the get with the guidelines coronary artery disease databases between 2000 and 2005 and found that , among the 5,454 patients with stemi who received a primary pci , those arriving during off - hours were less likely to achieve door - to - balloon times 90 minutes compared with those arriving during regular hours.28 in the national registry of myocardial infarction study of 33,647 patients with stemi treated with primary pci from 1999 to 2002,15 54% of patients were treated during off - hours ; door - to - balloon times were substantially longer during off - hours ( 116 minutes ) than regular hours ( 95 minutes ) . in this investigation , we found that patients with stemi who arrived at the ed by car / walked - in were less likely to have received a primary pci within 90 minutes , compared with those who arrived by ambulance . although our study also identified that having previously undergone cabg surgery was significantly associated with not receiving a primary pci within the guideline - recommended time frame , a prior systematic review of factors associated with door - to - balloon time in patients with stemi treated with pci has found mixed findings with regard to the strength of association of these factors between studies.31 while these differences in study results may be due to differences in study design , definitions of key covariates , patient populations under study , and sample size considerations , our study identified several patient groups at high risk for failing to be treated in a timely manner in whom further surveillance and hospital or provider educational efforts might be directed . indeed , delays in patient s medical care seeking behavior following the development of acute coronary symptoms continue to remain unduly long and have improved little over time.32,33 the strengths of the present community - based study include the examination of relatively contemporary decade - long trends in , and factors associated with , door - to - balloon time among patients hospitalized with stemi . since our study was restricted to patients who were hospitalized with stemi and received a primary pci at a pci - capable hospital in central massachusetts between 2001 and 2011 , we aggregated data from the six individual study years in order to increase the overall sample size . although primary pci has been shown to improve outcomes in patients with stemi , its effectiveness may be limited by delays in its more timely delivery.1 since 1999 , clinical practice guidelines for the management of patients with stemi have recommended door - to - balloon times of 90 minutes or less.1,16,17 however , earlier data from the national registry of myocardial infarction , which examined 33,647 patients hospitalized with stemi who received a primary pci between 1999 and 2002 at 421 us hospitals , reported that only 35% of patients were treated within the recommended 90 minutes after arrival at the hospital ; meaningful improvements in door - to - balloon times over the study period were not observed.3 these discouraging findings led to several national efforts dedicated to reducing door - to - balloon time in patients hospitalized with stemi . in november 2006 , the american college of cardiology , the american heart association , and several other organizations launched the door - to - balloon ( d2b ) : an alliance for quality campaign with the goal of increasing the percentage of patients with stemi who would receive a primary pci within 90 minutes of presentation at a pci - capable hospital to 75%.19 in may 2007 , the american heart association launched mission : lifeline , another national initiative designed to educate patients and providers about the importance of rapid response to stemi and to help hospitals create coordinated stemi diagnostic and treatment systems.20 several studies in the us have shown reductions in door - to - balloon times since these national efforts have been employed.46 findings from the blue cross blue shield of michigan cardiovascular consortium of 8,771 patients with stemi who underwent a primary pci at nine hospitals between 2003 and 2008 showed that the median door - to - balloon time had decreased from 113 minutes in 2003 to 76 minutes in 2008 ( p<0.001 ) . median door - to - balloon times declined significantly from 83 minutes in the first year to 67 minutes in the most recent study year , and the percentage of patients for whom the door - to - balloon time was 90 minutes or less increased from 60% to 83% during the years under study ( p<0.001).6 consistent with the timeline of national efforts in reducing door - to - balloon time and prior research results , our current decade - long trend study observed a substantial decrease in the median door - to - balloon time and a dramatic increase in the proportion of patients who received a primary pci within the guideline - recommended 90 minutes since 2005/2007 . a recent systematic review and meta - analysis examining the association between off - hour presentation and outcomes in patients with ami has suggested that patients with stemi presenting during off - hours have longer door - to - balloon times.27 a prior study examined data from the get with the guidelines coronary artery disease databases between 2000 and 2005 and found that , among the 5,454 patients with stemi who received a primary pci , those arriving during off - hours were less likely to achieve door - to - balloon times 90 minutes compared with those arriving during regular hours.28 in the national registry of myocardial infarction study of 33,647 patients with stemi treated with primary pci from 1999 to 2002,15 54% of patients were treated during off - hours ; door - to - balloon times were substantially longer during off - hours ( 116 minutes ) than regular hours ( 95 minutes ) . in this investigation , we found that patients with stemi who arrived at the ed by car / walked - in were less likely to have received a primary pci within 90 minutes , compared with those who arrived by ambulance . although our study also identified that having previously undergone cabg surgery was significantly associated with not receiving a primary pci within the guideline - recommended time frame , a prior systematic review of factors associated with door - to - balloon time in patients with stemi treated with pci has found mixed findings with regard to the strength of association of these factors between studies.31 while these differences in study results may be due to differences in study design , definitions of key covariates , patient populations under study , and sample size considerations , our study identified several patient groups at high risk for failing to be treated in a timely manner in whom further surveillance and hospital or provider educational efforts might be directed . indeed , delays in patient s medical care seeking behavior following the development of acute coronary symptoms continue to remain unduly long and have improved little over time.32,33 the strengths of the present community - based study include the examination of relatively contemporary decade - long trends in , and factors associated with , door - to - balloon time among patients hospitalized with stemi . since our study was restricted to patients who were hospitalized with stemi and received a primary pci at a pci - capable hospital in central massachusetts between 2001 and 2011 , we aggregated data from the six individual study years in order to increase the overall sample size . between 2001 and 2011 , the likelihood of receiving a primary pci within guideline - recommended times among patients who were hospitalized with stemi at the two major teaching and community hospitals in central massachusetts has increased dramatically .

while fept prepared as a thin film is one of the main candidates for the next generation data storage devices , its physical properties regarding ordering dynamics were mainly investigated in the bulk . in the l10 phase the magnetocrystalline uniaxial anisotropy in the range between , 7 j cm and 80 j cm is the reason for its hard ferromagnetic constitution of about 0.5 mev anisotropy per 3d - atom . fept is therefore suitable for perpendicular magnetic data storage media applications , which offer potential for increased data storage densities with respect to the currently still common in - plane or parallel magnetization media , . recent research is already dealing with practical designs for hard discs based on a new technique , . the impact on data storage devices makes a detailed knowledge of the dynamics and diffusion processes essential . only then one can selectively design alloys that guarantee an acceptable thermal stability of the permanent magnetization in very small volumes enabling scale reduction of the memory bits in the recording media . in the last years various experiments with bulk l10 fept devoted to diffusion , , and ordering dynamics studies , , have been performed . on the contrary , the results we present here deal with the ordering mechanism of the epitaxial fept thin film system . the l10 structure may grow in bulk or in thin film samples with three different structural orientations of the c - axis as shown in fig . 1 . our recent monte carlo ( mc ) simulations , , , predicted that the c - variant of a single - crystalline l10 structure with out - of - plane easy axis of thin film magnetization yielding technologically desired magnetic axis is unstable in its atomic stacking . the mono atomic planes spontaneously reorient creating a - variant domains with the easy axis of the magnetic field in the film plane . the underlying atomic - scale mechanism is as follows : some iron atoms from the surface iron layer replace platinum atoms in the layer beneath , see fig . 2 of kozlowski et al . the effect of spontaneous l10 c - variant a - variant reorientation in stoichiometric fept structure limited by free ( 001 ) iron surfaces was achieved by the monte carlo method implementing vacancy mechanism of atomic jumps , , . nearest neighbor ( nn ) and next nearest neighbor ( nnn ) pair - interaction energies addressing fept were deduced from ab - initio calculations combined with a cluster - expansion procedure , . here we present the experimental evidence of the reorientation within c - variant ordered l10 structures of fept thin films . the rearrangements of ordered domains are caused by successive annealing at temperatures between 773 k and 898 k following order / order transformations between different l10 variants in the fept thin film : c - variant ( c ) , a - variant ( a ) , and a random , disordered r - variant ( r ) . we further report on the effect which was not observed in the mc simulations , i.e. partial recovery of the c - variant in the epitaxial thin film after long annealing times . one may consider this process as partly self - healing of the fept film driven by the ordering free energy overpowering the surface free energy . both processes originate from an initial disordering generated by ( fast ) surface diffusion followed by a partial recovery of the long - range order generated by ( slow ) bulk diffusion . the experimental data were evaluated by the johnson mehl avrami ( jma ) model . the same model was applied already to describe l10-ordering in fept after sputter deposition . in our investigation we found that the ordering is driven by different kinds of dynamics for short and long time scales . the jma - model was adapted to account for these differences and to fulfill various constraints characteristic for thin films:initial and final fractions of all variants need not to be 1 or 0 ( i.e. the dynamics do not operate in the whole thin film).transformation between following variants are included in the model : c ( a , r ) , ( a , r ) c , a r , r a.different ordering dynamics account at different time scales , i.e. different avrami exponents ( ae ) are applied . initial and final fractions of all variants need not to be 1 or 0 ( i.e. the dynamics do not operate in the whole thin film ) . transformation between following variants are included in the model : c ( a , r ) , ( a , r ) c , a r , r a. different ordering dynamics account at different time scales , i.e. different avrami exponents ( ae ) are applied . the first and simplest approach contains of two different time scales , e1(t ) for long times and e2(t ) for short times , both with corresponding ae s . the model consisting only of two time scales works sufficiently well for all measured data.(1)e1(t)=exp[(t/1)n1]e2(t)=exp[(t/2)n2].here t is the time , i the characteristic time of the process and ni the corresponding ae . the time dependence of the amount of c - variant is then determined by the sum of a decaying fraction dc(t ) and a growing fraction gc(t)(2)fc(t)=gc(t)+dc(t)=c0+c1e1(t)c2e2(t).c0 is the saturation value for t . the model allows that only a fraction c2 reorients , while the recovering of the c - variant dominates only in a fraction c1 of the film , i.e. the starting volume of the transformation is limited because a part of the film remains c - variant . according to equation ( 2 ) the fractions for the a - variant and the random r - variant were determined taking into account that a and r can be formed only by fractions a1 and r1 from the amount which was rearranged from the c - variant and vice versa , thus a1 + r1 = 1 . for the a - variant and r - variant the growing fractions ga(t ) and gr(t ) and the decaying fractions da(t ) and dr(t ) can be determined including also the initial fractions of a and r , a0 and r0 , respectively . this finally gives the total fractions of a and r:(3)fa(t)=ga(t)da(t)fr(t)=gr(t)dr(t).in the simplest approach the formation and loss of a and r have the same weights a1 and r1 , respectively . further , if a constant mass flow is assumed a r or r a for all time scales , only the rate a1 changes . to make the model flexible in describing rates of the formation or annihilation of a or r , a non - constant mass flow f between a and r was introduced ( again for the simplest case of two different time scales):1.for short times a part ( 1 f1 ) of the growing a - variant is forming the r - variant with corresponding fractions expressed by(4)ga(t)=f1ga(t)gr(t)=r0+a0+c2dc(t)ga(t).2.for long times a part ( 1 f2 ) of the decaying a - variant is not forming c but r(5)da(t)=f2da(t)dr(t)=gc(t)da(t).note that the model can only describe net - flows , i.e. the result of a r for short times is equivalent to the fact that the flow c r is larger than c a. for short times a part ( 1 f1 ) of the growing a - variant is forming the r - variant with corresponding fractions expressed by for long times a part ( 1 f2 ) of the decaying a - variant is not forming c but r the structure simulated in , , was a nano - layered l10 ab - binary stoichiometric thin film of 40 cubic fcc cells with periodic boundary conditions . a single vacancy was introduced into the system by emptying one site chosen at random . glauber dynamics with a vacancy mechanism of atomic jumps have been used in the simulations . pair - interactions of fe- and pt - atoms in two co - ordination shells were evaluated on the basis of ab - initio calculations combined with the cluster - expansion technique . the simulated isothermal order order kinetics at temperatures in equilibrium tequ followed sudden changes of temperature from a starting temperature tst to tequ . among the monitored parameters were the bragg - williams - type long - range order ( lro ) parameter ( ) dedicated to particular l10 superstructure variants , specific short - range order parameters and frequencies of particular atomic jumps . the simulated system showed a discontinuous order disorder transition at a temperature tc close to the experimental one . thin layers of fept with different variants of the l10 superstructure were simulated by removing periodic boundary conditions in direction parallel ( c - variant ) or perpendicular ( a- and b - variant ) to the c - axis . the layer thickness was varied in such a way that the vacancy concentration was constant . for thin layers 0 k tequ relaxations were performed and the atoms were forced to remain in the initial box . the film surfaces were composed one of fe - atoms and one of pt - atoms , each an interface to vacuum . two processes were observed : ( i ) homogeneous disordering i.e. generation of antisites within the volume of the layer ( as found for bulk - calculations ) ; ( ii ) formation of l10-ordered a- and b - variant domains due to the jumps of surface fe - atoms on pt antisites . the resulting ordering process nucleated heterogeneously and selectively on the fe - surface and advanced discontinuously inward the layer . for the simulation of very thin films of about 10 unit cells thickness , the reaction front reached the free pt - surface which resulted in an additional energy gain from the formation of a- and b - variant domains at the pt - surface ( the energy gain was due to the transformation / reorientation of the first layer of c - variant l10 unit cells at the fe - surface ) . the thin film froze in this configuration , which then is the final saturated state in thermodynamical equilibrium . only for thick films the reaction front does not reach the free pt - surface and the propagation depth saturates depending on the simulation temperature . monte carlo and ab - initio simulations of fe pt intermetallic alloys are not numerous but two recent papers should be commented . interesting is the derivation and application of four - body potentials on the basis of a combination of ab - initio and cluster - expansion calculations . although explored is a lattice model , the way the potentials are calculated allows to account for off - lattice effects . however , as the direct - exchange mc algorithm is used , no information on ordering kinetics is provided . in addition , the simulations start from a disordered fcc structure , so observed is a formation of a system of l10 domains . in general , the paper focuses on the correctness of the order disorder transition temperature resulting from the simulations . very important results relevant for our studies are presented by dannenberg et al . . ab - initio energies of l10 fept - surfaces with diverse orientations are calculated and it is shown that the ( 100 ) surface ( i.e. a - variant ) is energetically favourable with respect to the ( 001 ) ( i.e. c - variant ) surface . experimentally this ideal structure is represented by a thin film sample of c - variant l10-ordered fept grown on mgo . the real structure can of course never provide the same results as the monte carlo simulation with two free surfaces . further , during the preparation the atoms may migrate , segregate and diffuse . due to the presence of the substrate the films show also epitaxial strain enhancing the tetragonal distortion of the unit cell close to the substrate . the best approximation of free surface was a thin film without cover . to ensure that the reorientation effect originates genuinely from formation of a - variant domains at the surface , pt covered reference samples the pressure was better than 1 10 mbar and the substrate temperature was 623 k during preparation . a first set of samples consisted of fept(500 )/mgo(001 ) and a second identical set was covered in - situ by 20 of platinum . both sets were produced simultaneously by co - evaporation of fe ( 0.041 s ) and pt ( 0.053 s ) . additionally an independent third uncapped sample set was identically grown to check the reproducibility of the result of the sample one . for more details on the quality of the sample inclusive tem pictures , the thickness of 500 is about three times larger than the forty unit cells thick layer for mc simulations . nevertheless , the thicker sample was chosen to guarantee a relaxed , flat film of well ordered l10 structure , , . the samples were characterized regarding their chemical and magnetic order using conversion electron mssbauer spectroscopy ( cems ) , x - ray diffraction ( xrd ) , and vibrating sample magnetometer ( vsm ) . all methods give clear evidence of an almost perfect c - variant l10-ordered fept thin film . the long - range order ( lro ) of the samples was measured by xrd using cu - k radiation . spectra for the as - prepared state as well as for the samples annealed at 773 k , 848 k and 898 k are shown in fig . well pronounced ( 001 ) and ( 002 ) reflections indicate a well ordered crystalline l10 structure for all thin film samples . the c - axis lattice constant of the as - prepared sample as calculated from the position of the bragg peaks is cexp = 3.723(2 ) . this corresponds to the ideal value of the c - axis in the l10 fept structure ( cid = 3.7212(3 ) ) , . from the intensities of the ( 001 ) and ( 002 ) reflections we estimate an lro parameter to be 0.77(5 ) for the as - prepared sample . magnetization curves ( not shown ) measured with the external field perpendicular to the film surface by means of vsm reproduced the values from the cems measurements quite well . it was , however , not possible to conclusively correlate the vsm data with the fraction of differently oriented variants . we performed stepwise annealing in vacuum of 2 10 mbar or better at 773 k , 808 k , 848 k and 898 k ( a different sample at each temperature ) followed by cems characterization at room temperature . to double - check the stability of the specimen during annealing procedure , xrd and high resolution xrd ( hrxrd ) were aplied . for the cems measurements we used a flowing - gas detector ( he ch4 ) and a standard mssbauer drive and electronics . the source was 50 mci co in rh - matrix and the calibration was done with a foil of high - purity natural iron . intensities of cems resonances were fitted with the software including the hyperfine field distribution ( recoil ) . intensities of mssbauer lines , especially the intensity of the second and fifth line contains information about the orientation of the incident k vector of the 14.4 kev -quanta with respect to the b vector of the magnetic hyperfine field . detailed analysis of the cems spectra allows therefore to determine the fraction of different l10 variants in the structure of the fept thin film samples : c - variant ( kb ) , a - variant ( kb ) as well as the random distribution ( with a randomly oriented magnetic field vector ) . enhanced intensities of the second and fifth mssbauer lines are an evidence of the lower c - variant content and , consequently , higher a - variant ( or random domains ) level and vice versa . the changes in the relative intensities of 2nd and 5th line are visible comparing e.g. mssbauer spectra in fig . 3(a c ) . a larger fraction of very broad lines ( the lines are broad due to large hyperfine field distribution ) in fig . we want to point out that these variants are identical crystalline l10-ordered domains in the fept thin film , but of different orientation of the c - axis ( i.e. the magnetization vector ) , compare fig . 1 . the reference sample of set one and two ( an as - prepared specimen ) was composed of 90(1)% of c - variant , 3.5(8)% a - variant and 4(1)% r - variant . a small quadrupole doublet in the center of the spectra with about 24% of the total spectral area results from surface oxidation during annealing , see fig . the line width of 0.15 mm s was measured for the as - prepared sample and confirms the high degree of order in the thin films . we chose samples from sample set one for thermal treatment at three annealing temperatures ( 773 k , 848 k and 898 k ) and one sample of sample set three at a different temperature of 808 k to verify the experimental results and our model . for the uncapped samples we find for the lowest temperature of 773 k an initial very strong decrease of c - variant content ( down to 62% ) , with corresponding increase of a - variant ( 15% ) and random distribution r ( 19% ) . the maximally reoriented state was reached after approximately 8 h and the final saturation state after about 20 h as shown in fig . longer than 8 h , the c - variant starts to recover with corresponding decrease of a - variant and r - variant . all fractions of the specimen annealed at 773 k are shown in fig . the samples annealed at 808 k and 898 k show a similar behavior as the one annealed at 773 k. the fractions of c , a and r saturate for times longer than 12 h at 808 k and 2 h at 898 k , respectively . a different behavior was found at 848 k. the c - variant never recovers at this temperature . nevertheless , the fractions saturate for times longer than 10 h. for the sample set capped with 2 nm pt the formation of a - variant is almost completely suppressed , as can clearly be seen in fig . according to the mc simulation this is due to the completely missing free fe - surface where the reorientation is initialized . the suppression - effect is smaller at higher temperatures the reason for this is the higher mobility of atoms : a random formation of a - variant domains in the topmost layers of the fept corresponds to the formation of fe - antisites on pt lattice sites . for all annealing temperatures 773 k , 808 k , 848 k and 898 k we find a fast decay of the c - variant 2 ( i.e. fast formation of a - variant and random distribution r ) and a slow recovery of the c - variant 1 ( i.e. decay of a - variant and r - variant ) . annealed at 848 k the dynamics contains an additional slowly decaying component 3 . the origin of this behavior is different from the results at other temperatures and in two sample sets and is an objective of further investigations . we find two different ae - values for short and long times , see fig . the results we achieved by means of cems measurements show to our best knowledge the reorientation effect described in our kinematical model . however , one has to carefully take into account other effects possibly causing similar changes in the cems spectra . first of all oxidation effects have to be investigated carefully . as the uncapped fept thin films were exposed to air between thermal - treatment steps , oxidation is not unrealistic . the cems technique is however very sensitive to oxidation effects due to characteristic changes of the hyperfine field values . we analyzed the spectra taking care on the arising fe o compounds , but only paramagnetic quadrupole doublet with a total spectral fraction of about 24% arising after the first annealing step could be identified . further , formation of anti - phase boundaries ( apb s ) could cause a change in the cems patterns . the formation of a - variant domains in an initially purely c - variant sample is only possible by formation of many defects . nevertheless , we can clearly attribute the arising sextets to a - variant , c - variant and disordered ( i.e. small , randomly oriented domains ) areas of the sample . apb s would certainly manifest mainly in the disordered region and only a minor fraction will contribute to the interface of a- and c - variant domains . we therefore conlude that the main contribution to our cems results originates from a structural reorientation ( i.e. order / order transformation ) of l10-ordered domains within the fept thin films . the ae - values are temperature independent within error bars as shown in fig . the ae can be interpreted as a measure of the growth dimensionality of domains . following this approach , n2 describes the dynamics at the start of the reorientation process where the initial formation of a and r takes place . the growth can be interpreted as 2d - like with a constant seed - rate ( e.g. the first monolayer is already completely a - variant ) or 1d with a growing number of seeds ( e.g. in small channels into the depth of the thin film while new a - variant domains are forming within the first monolayer ) . thinking of a pure topmost fe - layer in a real experimental setup with samples grown by mbe at elevated substrate temperatures is clearly unrealistic . nevertheless , we have an indirect proof for the prevailing of an fe - terminated surface . the preparation and heat treatment of the pt - capped sample set two was in the same way as for the uncapped sample sets one and three . the only possible difference explaining the complete missing of an ordering effect in sample set two , while at the same time one finds the well pronounced effect in sample set one and the reference sample set three , is at least enhanced concentration of surface terminating fe - atoms . while sample set two has definitely no surface fe - atoms due to 20 platinum capping , statistically in the sample sets one and three due to the preparation at high temperature and therefore enhanced diffusivities near the surface , see e.g. , a reasonable amount of surface atoms may be fe - atoms . n1 is close to the value of three for all annealing temperatures . in the jma - model thus the c - variant is either formed in the whole volume of a - variant ( and r - variant ) and with a random distribution and a constant number of seeds ( 3d - growth ) or is formed at interfaces between c - variant and a - variant ( and r - variant ) with a non - constant number of seeds ( 2d - growth ) . the values for n1 = 3.17(5 ) and n2 = 1.5(4 ) reproduce the mc result , that the growth starts from the surface and then proceed into the depth of the thin film . this result answers an open question arising from other mc simulations , which suggested ordering mechanisms in fept in terms of surface pt - precipitation , or in terms of surface induced disorder . the values describing the flow rates are shown in table 2 and in fig . we find for low temperatures f2 < 1 ( i.e. a r for the initial stage of the ordering dynamics or for annealing times short compared to the saturation time ) and f1 > 1 ( i.e. r a for long annealing times comparable to the saturation time ) . this corresponds to the fact that for short times the formation of the random distribution is dominating over the formation of a - variant ( disordering generated by the fast surface diffusion ) , while for long times the formation of a - variant dominates over the formation of the r - variant ( ordering generated by slow bulk diffusion ) . for the two highest temperatures we find fi < 1 ( i = 1 , 2 ) corresponding to an increase of disorder , since the thermal formation of defects is supported . the cause for the formation of r is that in the epitaxial film the a - variant has to form while having a different lattice constant ( a = 3.8504(8 ) , c = 3.7212(3 ) ) . this means that some kind of defects have to be introduced to allow the formation of a - variant in a region of the film which was c - variant before . further , the a - variant can only be formed by disordering the c - variant l10 structure first via the formation of fe - antisites on the pt sublattice . this makes it likely that a front of disordered l10 is located between a- and c - variant and contain many defects . the overall behavior is consistent with the results of epitaxial ordering of fept for different ordering stages and annealing temperatures , , . the time constants 1 and 2 , shown in fig . 7 , follow arrhenius behavior . from the slopes of the arrhenius plot we calculated activation energies for the dynamical processes ofq1=0.8(2)evandq2=1.5(1)ev . at the beginning of the annealing procedure ( short times , q2 ) the rearrangement takes place via the formation of fe - antisites on the pt sublattice . the atomic mechanism , therefore , is a net migration ( diffusion ) of fe - atoms along the c - axis of the thin film . we can compare our activation energy to similar results achieved by residual resistivity measurements for order order dynamics in bulk samples and thin films in the same temperature range which are 1.5(3 ) ev and 1.8(2 ) ev , respectively , . moreover , our former studies on iron diffusion along the c - axis in l10-ordered fept thin films by nuclear resonant scattering give an activation energy of 1.65(30 ) ev . the values again are the same within the error bars confirm that the underlying dynamical process is disordering . for long annealing times ( q1 ) the formation of the c - variant corresponds to ordering of the epitaxial l10 structure . we can directly compare our results with the activation energies found for ordering of disordered sputter - deposited fept thin films of 0.9(2 ) ev via xrd and 0.6(4 ) ev via mssbauer spectroscopy . the main result of the cems investigations , namely the reorientation behavior reproduces the mc simulation result . nevertheless , the unique and stunning result of the recovering of the c - variant i.e. a reversal of the reorientation in the epitaxial fept thin film has been not shown in the mc simulation . the discrepancy between the simulation and the experiment is apparently due to the fact that the real film is a complex system whose characteristics are determined by tetragonal distortion , epitaxy , magnetic stray fields , non - ideal surface termination and the interface to the substrate . however , the main result of the structural reorientation is in perfect agreement with mc simulations and with the cems experiment . simulations predict that a reorientation of the c - variant to a- and b - variants in l10-ordered fept occur . the reorientation effect eventually reverts ( recovering of the c - variant ) and then saturates for long annealing times . we have experimentally verified that the structural rearrangement in fept thin films also gives rise to the rearrangement of the magnetic field . we found two dynamical mechanisms ( ordering and disordering ) working simultaneously but with different time constants , dimensions of growth and activation energies . the activation energies are comparable to energies for ordering and disordering in l10 fept found in other measurements . we find that:(i)with respect to the degree of long - range order , the thin film evolves away from the configurational equilibrium because of the dynamical effect originating from the surface and epitaxial interface ( disordering);(ii)the reorientation overshoots the equilibrium lro state and the system partially restores its long - range order . both mechanisms work at the same time but with different time constants and activation energies . with respect to the degree of long - range order , the thin film evolves away from the configurational equilibrium because of the dynamical effect originating from the surface and epitaxial interface ( disordering ) ; the reorientation overshoots the equilibrium lro state and the system partially restores its long - range order . both mechanisms work at the same time but with different time constants and activation energies . the reason for the surprising recovery of the c - variant is that the ordering process has a lower activation energy than the disordering process and therefore dominates , an effect which is further enhanced due to epitaxy . the impact of the ordering component is only visible for long annealing times as its time constant is larger than the one of disordering component . the experimentally found behavior is consistent with the results of highly ordered epitaxial fept layer annealed at higher temperatures . the suppression - effect is smaller at higher temperatures due to the higher mobility of atoms .

we report on the development of structural and magnetic order in epitaxially grown l10 fept thin films . upon annealing , the easy axis of magnetization changes from the out - of - plain into the in - plain direction . we found that the overall fraction of reoriented domains first increases but after certain time decreases before achieving a saturated state . the results are based on conversion electron mssbauer spectroscopy studies and confirm monte carlo simulations in nano - layered fept . we present a modified version of the johnson mehl avrami ( jma ) model adequately describing the experimental findings . two dynamical processes , the first being a 2d - growth , dominate the initial state of sample annealing and the second being a 3d - growth , dominate the late stage close to saturation . from an arrhenius plots of jma coefficients for both processes we extracted the activation energies of the underlying dynamics which are 1.5(1 ) ev for disordering and 0.8(2 ) ev for ordering .

Introduction Theory Experimental results Discussion Conclusion

while fept prepared as a thin film is one of the main candidates for the next generation data storage devices , its physical properties regarding ordering dynamics were mainly investigated in the bulk . in the l10 phase the magnetocrystalline uniaxial anisotropy in the range between , 7 j cm and 80 j cm is the reason for its hard ferromagnetic constitution of about 0.5 mev anisotropy per 3d - atom . recent research is already dealing with practical designs for hard discs based on a new technique , . only then one can selectively design alloys that guarantee an acceptable thermal stability of the permanent magnetization in very small volumes enabling scale reduction of the memory bits in the recording media . in the last years various experiments with bulk l10 fept devoted to diffusion , , and ordering dynamics studies , , have been performed . on the contrary , the results we present here deal with the ordering mechanism of the epitaxial fept thin film system . our recent monte carlo ( mc ) simulations , , , predicted that the c - variant of a single - crystalline l10 structure with out - of - plane easy axis of thin film magnetization yielding technologically desired magnetic axis is unstable in its atomic stacking . the mono atomic planes spontaneously reorient creating a - variant domains with the easy axis of the magnetic field in the film plane . the underlying atomic - scale mechanism is as follows : some iron atoms from the surface iron layer replace platinum atoms in the layer beneath , see fig . here we present the experimental evidence of the reorientation within c - variant ordered l10 structures of fept thin films . we further report on the effect which was not observed in the mc simulations , i.e. partial recovery of the c - variant in the epitaxial thin film after long annealing times . one may consider this process as partly self - healing of the fept film driven by the ordering free energy overpowering the surface free energy . both processes originate from an initial disordering generated by ( fast ) surface diffusion followed by a partial recovery of the long - range order generated by ( slow ) bulk diffusion . the experimental data were evaluated by the johnson mehl avrami ( jma ) model . in our investigation we found that the ordering is driven by different kinds of dynamics for short and long time scales . the time dependence of the amount of c - variant is then determined by the sum of a decaying fraction dc(t ) and a growing fraction gc(t)(2)fc(t)=gc(t)+dc(t)=c0+c1e1(t)c2e2(t).c0 is the saturation value for t . the model allows that only a fraction c2 reorients , while the recovering of the c - variant dominates only in a fraction c1 of the film , i.e. according to equation ( 2 ) the fractions for the a - variant and the random r - variant were determined taking into account that a and r can be formed only by fractions a1 and r1 from the amount which was rearranged from the c - variant and vice versa , thus a1 + r1 = 1 . for the a - variant and r - variant the growing fractions ga(t ) and gr(t ) and the decaying fractions da(t ) and dr(t ) can be determined including also the initial fractions of a and r , a0 and r0 , respectively . to make the model flexible in describing rates of the formation or annihilation of a or r , a non - constant mass flow f between a and r was introduced ( again for the simplest case of two different time scales):1.for short times a part ( 1 f1 ) of the growing a - variant is forming the r - variant with corresponding fractions expressed by(4)ga(t)=f1ga(t)gr(t)=r0+a0+c2dc(t)ga(t).2.for long times a part ( 1 f2 ) of the decaying a - variant is not forming c but r(5)da(t)=f2da(t)dr(t)=gc(t)da(t).note that the model can only describe net - flows , i.e. the result of a r for short times is equivalent to the fact that the flow c r is larger than c a. for short times a part ( 1 f1 ) of the growing a - variant is forming the r - variant with corresponding fractions expressed by for long times a part ( 1 f2 ) of the decaying a - variant is not forming c but r the structure simulated in , , was a nano - layered l10 ab - binary stoichiometric thin film of 40 cubic fcc cells with periodic boundary conditions . the simulated system showed a discontinuous order disorder transition at a temperature tc close to the experimental one . thin layers of fept with different variants of the l10 superstructure were simulated by removing periodic boundary conditions in direction parallel ( c - variant ) or perpendicular ( a- and b - variant ) to the c - axis . the layer thickness was varied in such a way that the vacancy concentration was constant . for thin layers 0 k tequ relaxations were performed and the atoms were forced to remain in the initial box . generation of antisites within the volume of the layer ( as found for bulk - calculations ) ; ( ii ) formation of l10-ordered a- and b - variant domains due to the jumps of surface fe - atoms on pt antisites . the resulting ordering process nucleated heterogeneously and selectively on the fe - surface and advanced discontinuously inward the layer . for the simulation of very thin films of about 10 unit cells thickness , the reaction front reached the free pt - surface which resulted in an additional energy gain from the formation of a- and b - variant domains at the pt - surface ( the energy gain was due to the transformation / reorientation of the first layer of c - variant l10 unit cells at the fe - surface ) . the thin film froze in this configuration , which then is the final saturated state in thermodynamical equilibrium . only for thick films the reaction front does not reach the free pt - surface and the propagation depth saturates depending on the simulation temperature . monte carlo and ab - initio simulations of fe pt intermetallic alloys are not numerous but two recent papers should be commented . interesting is the derivation and application of four - body potentials on the basis of a combination of ab - initio and cluster - expansion calculations . in addition , the simulations start from a disordered fcc structure , so observed is a formation of a system of l10 domains . in general , the paper focuses on the correctness of the order disorder transition temperature resulting from the simulations . ab - initio energies of l10 fept - surfaces with diverse orientations are calculated and it is shown that the ( 100 ) surface ( i.e. the real structure can of course never provide the same results as the monte carlo simulation with two free surfaces . due to the presence of the substrate the films show also epitaxial strain enhancing the tetragonal distortion of the unit cell close to the substrate . to ensure that the reorientation effect originates genuinely from formation of a - variant domains at the surface , pt covered reference samples the pressure was better than 1 10 mbar and the substrate temperature was 623 k during preparation . for more details on the quality of the sample inclusive tem pictures , the thickness of 500 is about three times larger than the forty unit cells thick layer for mc simulations . nevertheless , the thicker sample was chosen to guarantee a relaxed , flat film of well ordered l10 structure , , . the samples were characterized regarding their chemical and magnetic order using conversion electron mssbauer spectroscopy ( cems ) , x - ray diffraction ( xrd ) , and vibrating sample magnetometer ( vsm ) . all methods give clear evidence of an almost perfect c - variant l10-ordered fept thin film . the long - range order ( lro ) of the samples was measured by xrd using cu - k radiation . the c - axis lattice constant of the as - prepared sample as calculated from the position of the bragg peaks is cexp = 3.723(2 ) . this corresponds to the ideal value of the c - axis in the l10 fept structure ( cid = 3.7212(3 ) ) , . from the intensities of the ( 001 ) and ( 002 ) reflections we estimate an lro parameter to be 0.77(5 ) for the as - prepared sample . magnetization curves ( not shown ) measured with the external field perpendicular to the film surface by means of vsm reproduced the values from the cems measurements quite well . it was , however , not possible to conclusively correlate the vsm data with the fraction of differently oriented variants . to double - check the stability of the specimen during annealing procedure , xrd and high resolution xrd ( hrxrd ) were aplied . the source was 50 mci co in rh - matrix and the calibration was done with a foil of high - purity natural iron . intensities of mssbauer lines , especially the intensity of the second and fifth line contains information about the orientation of the incident k vector of the 14.4 kev -quanta with respect to the b vector of the magnetic hyperfine field . detailed analysis of the cems spectra allows therefore to determine the fraction of different l10 variants in the structure of the fept thin film samples : c - variant ( kb ) , a - variant ( kb ) as well as the random distribution ( with a randomly oriented magnetic field vector ) . enhanced intensities of the second and fifth mssbauer lines are an evidence of the lower c - variant content and , consequently , higher a - variant ( or random domains ) level and vice versa . a larger fraction of very broad lines ( the lines are broad due to large hyperfine field distribution ) in fig . we want to point out that these variants are identical crystalline l10-ordered domains in the fept thin film , but of different orientation of the c - axis ( i.e. a small quadrupole doublet in the center of the spectra with about 24% of the total spectral area results from surface oxidation during annealing , see fig . the line width of 0.15 mm s was measured for the as - prepared sample and confirms the high degree of order in the thin films . we chose samples from sample set one for thermal treatment at three annealing temperatures ( 773 k , 848 k and 898 k ) and one sample of sample set three at a different temperature of 808 k to verify the experimental results and our model . the maximally reoriented state was reached after approximately 8 h and the final saturation state after about 20 h as shown in fig . longer than 8 h , the c - variant starts to recover with corresponding decrease of a - variant and r - variant . all fractions of the specimen annealed at 773 k are shown in fig . nevertheless , the fractions saturate for times longer than 10 h. for the sample set capped with 2 nm pt the formation of a - variant is almost completely suppressed , as can clearly be seen in fig . the suppression - effect is smaller at higher temperatures the reason for this is the higher mobility of atoms : a random formation of a - variant domains in the topmost layers of the fept corresponds to the formation of fe - antisites on pt lattice sites . for all annealing temperatures 773 k , 808 k , 848 k and 898 k we find a fast decay of the c - variant 2 ( i.e. fast formation of a - variant and random distribution r ) and a slow recovery of the c - variant 1 ( i.e. the origin of this behavior is different from the results at other temperatures and in two sample sets and is an objective of further investigations . the results we achieved by means of cems measurements show to our best knowledge the reorientation effect described in our kinematical model . as the uncapped fept thin films were exposed to air between thermal - treatment steps , oxidation is not unrealistic . the cems technique is however very sensitive to oxidation effects due to characteristic changes of the hyperfine field values . we analyzed the spectra taking care on the arising fe o compounds , but only paramagnetic quadrupole doublet with a total spectral fraction of about 24% arising after the first annealing step could be identified . small , randomly oriented domains ) areas of the sample . order / order transformation ) of l10-ordered domains within the fept thin films . following this approach , n2 describes the dynamics at the start of the reorientation process where the initial formation of a and r takes place . the growth can be interpreted as 2d - like with a constant seed - rate ( e.g. the first monolayer is already completely a - variant ) or 1d with a growing number of seeds ( e.g. in small channels into the depth of the thin film while new a - variant domains are forming within the first monolayer ) . the preparation and heat treatment of the pt - capped sample set two was in the same way as for the uncapped sample sets one and three . the only possible difference explaining the complete missing of an ordering effect in sample set two , while at the same time one finds the well pronounced effect in sample set one and the reference sample set three , is at least enhanced concentration of surface terminating fe - atoms . n1 is close to the value of three for all annealing temperatures . in the jma - model thus the c - variant is either formed in the whole volume of a - variant ( and r - variant ) and with a random distribution and a constant number of seeds ( 3d - growth ) or is formed at interfaces between c - variant and a - variant ( and r - variant ) with a non - constant number of seeds ( 2d - growth ) . the values for n1 = 3.17(5 ) and n2 = 1.5(4 ) reproduce the mc result , that the growth starts from the surface and then proceed into the depth of the thin film . the values describing the flow rates are shown in table 2 and in fig . a r for the initial stage of the ordering dynamics or for annealing times short compared to the saturation time ) and f1 > 1 ( i.e. this corresponds to the fact that for short times the formation of the random distribution is dominating over the formation of a - variant ( disordering generated by the fast surface diffusion ) , while for long times the formation of a - variant dominates over the formation of the r - variant ( ordering generated by slow bulk diffusion ) . this means that some kind of defects have to be introduced to allow the formation of a - variant in a region of the film which was c - variant before . further , the a - variant can only be formed by disordering the c - variant l10 structure first via the formation of fe - antisites on the pt sublattice . the overall behavior is consistent with the results of epitaxial ordering of fept for different ordering stages and annealing temperatures , , . from the slopes of the arrhenius plot we calculated activation energies for the dynamical processes ofq1=0.8(2)evandq2=1.5(1)ev . at the beginning of the annealing procedure ( short times , q2 ) the rearrangement takes place via the formation of fe - antisites on the pt sublattice . the atomic mechanism , therefore , is a net migration ( diffusion ) of fe - atoms along the c - axis of the thin film . we can compare our activation energy to similar results achieved by residual resistivity measurements for order order dynamics in bulk samples and thin films in the same temperature range which are 1.5(3 ) ev and 1.8(2 ) ev , respectively , . moreover , our former studies on iron diffusion along the c - axis in l10-ordered fept thin films by nuclear resonant scattering give an activation energy of 1.65(30 ) ev . the values again are the same within the error bars confirm that the underlying dynamical process is disordering . for long annealing times ( q1 ) the formation of the c - variant corresponds to ordering of the epitaxial l10 structure . we can directly compare our results with the activation energies found for ordering of disordered sputter - deposited fept thin films of 0.9(2 ) ev via xrd and 0.6(4 ) ev via mssbauer spectroscopy . the main result of the cems investigations , namely the reorientation behavior reproduces the mc simulation result . nevertheless , the unique and stunning result of the recovering of the c - variant i.e. a reversal of the reorientation in the epitaxial fept thin film has been not shown in the mc simulation . the discrepancy between the simulation and the experiment is apparently due to the fact that the real film is a complex system whose characteristics are determined by tetragonal distortion , epitaxy , magnetic stray fields , non - ideal surface termination and the interface to the substrate . however , the main result of the structural reorientation is in perfect agreement with mc simulations and with the cems experiment . simulations predict that a reorientation of the c - variant to a- and b - variants in l10-ordered fept occur . the reorientation effect eventually reverts ( recovering of the c - variant ) and then saturates for long annealing times . we have experimentally verified that the structural rearrangement in fept thin films also gives rise to the rearrangement of the magnetic field . we found two dynamical mechanisms ( ordering and disordering ) working simultaneously but with different time constants , dimensions of growth and activation energies . the activation energies are comparable to energies for ordering and disordering in l10 fept found in other measurements . we find that:(i)with respect to the degree of long - range order , the thin film evolves away from the configurational equilibrium because of the dynamical effect originating from the surface and epitaxial interface ( disordering);(ii)the reorientation overshoots the equilibrium lro state and the system partially restores its long - range order . with respect to the degree of long - range order , the thin film evolves away from the configurational equilibrium because of the dynamical effect originating from the surface and epitaxial interface ( disordering ) ; the reorientation overshoots the equilibrium lro state and the system partially restores its long - range order . both mechanisms work at the same time but with different time constants and activation energies . the reason for the surprising recovery of the c - variant is that the ordering process has a lower activation energy than the disordering process and therefore dominates , an effect which is further enhanced due to epitaxy . the impact of the ordering component is only visible for long annealing times as its time constant is larger than the one of disordering component . the experimentally found behavior is consistent with the results of highly ordered epitaxial fept layer annealed at higher temperatures .

creatine ( cr ) is endogenously produced from the biosynthesis of glycine , arginine , and methionine in kidneys , liver , pancreas , and possibly the brain . nowadays , cr dietary supplement is widely used by athletes to enhance their physical performance ( 2 ) . there are pieces of evidence on the protective effects of cr on muscles , heart ( 3 ) , nervous system , and brain function ( 4 ) . elevated cr level in human and rodents brain have been reported following oral supplementation of cr ( 5 - 7 ) . there are also studies on changes in serotonin and dopamine regulators of athletes brain ( 8) and in depression - like behavior of rodents after a period of cr supplementation ( 1 ) . some human studies have confirmed better cognitive performance following the supplementation of cr in adequate doses ( 4 , 9 - 11 ) . according to the literature , oral cr supplementation may impose neuroprotective effects on experimental animal models of huntington s disease ( 6 , 12 , 13 ) . alzheimer s disease ( ad ) is the main cause of dementia in elderly ( 14 ) . two core features of the ad are neurofibrillar tangles , which are intracellular aggregates of hyperphosphorylated tau protein , leading to neuronal malfunction ( 15 , 16 ) , and amyloid plaques , which are heterogenous aggregates found in extra - cellular space mainly containing -amyloid ( a ) peptide ( 17 ) . accumulation of amyloid inside neurons may contribute to the development or exacerbation of the ad ( 18 ) . cr is responsible for energy homeostasis in neurons and can protect the neurons exposed to neurotoxins by promoting their energy levels . it may be the probable mechanism behind neuroprotective role of cr against the toxic effects of -amyloid peptide in cell culture of hippocampal neurons ( 19 ) . furthermore , the discovery of cr deposits in the brain of transgenic ad mice and in the hippocampus of ad patients indicates an association between cellular energy levels , -amyloid , cr metabolism , and ad ( 20 ) . in this respect it may be speculated whether cr supplementation at an early time point of the disease can prevent or delay the course of ad - related neurodegeneration ( 21 ) . despite the numerous efforts and studies conducted in the field , there is no clinical or in vivo study to provide adequate proofs about the neuroprotective effects of cr supplementation on the ad . considering the neuropro - tective effect of cr against -amyloid toxicity in vitro and positive effects of cr supplementation on improving the cognitive performance in healthy individuals , the present study was performed to investigate the effects of oral supplementation of cr on -amyloid toxicity in vivo . thirty two male wistar rats ( 4 month of age and weighting of 200 - 250 g ) were obtained from pasteur institute of iran , research , production and education centre , ( tehran , iran ) . they were caged at a constant temperature of 211 c with controlled 12 hr/12 hr light - dark cycles , and ad libitum access to food and water . the rats were given at least one week to habituate to the facilities , and then experimental procedures began . all the guidelines of the committee of care in the use of experimental animals were followed , and the study procedures were approved by the ethics committee of tehran university of medical sciences . the animals were divided into four groups ( n=8 per group ) : control , sham , a ( -amyloid injection , no cr supplementation ) , and acr ( -amyloid injection , cr supplementation ) . acr group received cr monohydrate powder mixed in their chow ( 2% cr / diet ) and other groups received normal chow diet for four weeks . those in the a and acr groups were given a bilateral -amyloid peptide injection in the ca1 hippocampus ( 0.5 g/l ) , while the sham group received a normal saline injection in the same area of the brain . after surgery , the acr group continued receiving 2% cr diet while the other groups maintained their normal chow diet ( figure 1 ) . a : -amyloid ; cr : creatine human -amyoid peptide 1 - 42 ( sigma - aldrich , usa ) was solved in 0.1 m phosphate buffer saline ( pbs ; ph=7.4 ) and then aliquoted and stored at -70 c until use . the animals were anesthe - tized with intra peritoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and then injected bilaterally under stereotaxic conditions with -amyloid or normal saline into the ca1 hippocampus ( ap=3.9 mm , lr=2.2 mm , d=2.7 mm ) . injections were performed at a rate of 0.5 l / min using a hamilton syringe attached to the stereotax apparatus . four microliters of -amyloid ( 0.5 g/l ) solution was injected in every hippocampus , and the needle was kept in place for one min after injection before being slowly retracted ( figure 1 ) . two percent of cr ( sigma - aldrich , usa ) was mixed into the normal chow using an electric mill according the previous studies ( 6 , 12 , 13 ) . after adding some water and cutting the paste , the cr and chow mixture were being dried in a 30 - 45 min exposure to a continuous warm air stream . animals in the acr group had freely access to this mixed food ( figure 1 ) . the morris water maze procedure was performed 6 weeks after the -amyloid injection . water maze consisted of a pool ( 155 cm in diameter ) filled with water ( 211c ) to a level 10 cm from the edge of the tank . a transparent plexiglas platform ( 10 cm diameter ) was located 1.5 cm below the surface in the eastern quadrant of the tank ( target quadrant ) . the walls surrounding the pool were decorated with distinct extra maze spatial cues , which were kept in fixed positions during the entire experiment to allow the animals to find the hidden platform . the animals movements were recorded by a ccd camera ( panasonic inc , japan ) hanging from the ceiling above the mwm apparatus , and locomotion tracking was measured using ethovision software ( version xt7 , the netherlands ) , a video tracking system for automated analysis of animal behavior . twenty - four hr before starting the hidden platform training , rats were given 60 sec to swim in the tank without the platform in order to adapt to the environment . the reference spatial learning and memory tests were carried out based on the procedure previously conducted in our laboratory with some modifications in training ; the platform was submerged in the eastern quadrant of the pool . its place remained unchanged throughout training , but animals were released into the water ( while facing the tank wall ) from different locations chosen randomly from the south , north , west , northwest and southwest . the animals were allowed to swim for 60 sec to localize the platform s position in the tank . the rats which did not find the platform within 60 sec , were directed into it and were allowed to rest on it for 10 sec . twenty - four hr after the third session ; the spatial probe test was performed . during this test , the platform was removed , and rats were allowed to swim for 60 sec before getting removed . escape latencies , time spent in target quadrant , and swim speed were wholly recorded for subsequent analysis . the rats were anesthetized and their brains were fixed through a transcardial perfusion ( normal saline and 4% paraformaldehyde solution ) within 24 hr after the spatial probe test . after that , the brains were kept in a 4% paraformaldehyde solution . in preparation for the histological staining , the brains were embedded with paraffin , cut into consecutive 5 m transverse sections by a microtome , and placed on the poly - d - lysine - coated glass slides . alternate sections were used for thioflavin - t and tunel staining . in order to detect -amyloid plaques in brain sections thioflavin - t ( sigma - aldrich , usa ) brain sections were deparaffinized in xylene , rehydrated , covered with thioflavin - t solution ( 0.5% thioflavin - t in 0.1n hcl ) , and incubated in dark room at room temperature for 10 min . slides were immersed in distilled water twice before being observed through a fluorescent microscope . in order to detect apoptotic neurons in brain sections , the tunel staining protocol was performed using tunel apoptosis detection kit , ( millipore , uk ) . brain sections were deparaffinized in xylene , rehydrated , immersed in pbs , and then incubated at 37 c for 30 min . out of pbs , sections were covered with diluted protein kinase and incubated at 37 c for 20 min . to halt protein kinase activity each brain section was covered with 50 l tdt ( terminal deoxynucleotidyl transferase ) buffer for 10 min . the tdt buffer was removed using a sampler , and then each section was covered with 50 l tdt mixture solution ( 90% tdt buffer , 5% dutp - biotin , 5% tdt ) and incubated for 60 min at 37 c . slides were washed for 5 min by immersing in tb buffer at room temperature and then dipping in pbs ( 42 min ) . after the washing process , sections were covered with 50 l of blocking solution and incubated for 20 min at room temperature . after removing the blocking buffer with a sampler , 50 l avidin - ftc mixture ( 1 part avidin ftc , 9 parts blocking solution ) was used for each section , and slides were incubated at 37 c for 30 min in a dark room . to obtain the mean percentage of apoptotic neurons in the hippocampus area , 3 brain sections were chosen randomly from each subject and the number of tunel positive and negative cells was counted in three adjacent 400x microscopic fields . the data were presented as meanse and processed by the statistical package for social sciences ( version 16.0 ; spss inc . , mean escape latency , distance traveled to the platform , time spent in target quadrant in mwm test and also mean percentage of apoptotic cells were analyzed . all the groups were compared using one - way anova test . comparing sham and a and also a and acr was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison . thirty two male wistar rats ( 4 month of age and weighting of 200 - 250 g ) were obtained from pasteur institute of iran , research , production and education centre , ( tehran , iran ) . they were caged at a constant temperature of 211 c with controlled 12 hr/12 hr light - dark cycles , and ad libitum access to food and water . the rats were given at least one week to habituate to the facilities , and then experimental procedures began . all the guidelines of the committee of care in the use of experimental animals were followed , and the study procedures were approved by the ethics committee of tehran university of medical sciences . the animals were divided into four groups ( n=8 per group ) : control , sham , a ( -amyloid injection , no cr supplementation ) , and acr ( -amyloid injection , cr supplementation ) . acr group received cr monohydrate powder mixed in their chow ( 2% cr / diet ) and other groups received normal chow diet for four weeks . those in the a and acr groups were given a bilateral -amyloid peptide injection in the ca1 hippocampus ( 0.5 g/l ) , while the sham group received a normal saline injection in the same area of the brain . after surgery , the acr group continued receiving 2% cr diet while the other groups maintained their normal chow diet ( figure 1 ) . human -amyoid peptide 1 - 42 ( sigma - aldrich , usa ) was solved in 0.1 m phosphate buffer saline ( pbs ; ph=7.4 ) and then aliquoted and stored at -70 c until use . the animals were anesthe - tized with intra peritoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and then injected bilaterally under stereotaxic conditions with -amyloid or normal saline into the ca1 hippocampus ( ap=3.9 mm , lr=2.2 mm , d=2.7 mm ) . injections were performed at a rate of 0.5 l / min using a hamilton syringe attached to the stereotax apparatus . four microliters of -amyloid ( 0.5 g/l ) solution was injected in every hippocampus , and the needle was kept in place for one min after injection before being slowly retracted ( figure 1 ) . two percent of cr ( sigma - aldrich , usa ) was mixed into the normal chow using an electric mill according the previous studies ( 6 , 12 , 13 ) . after adding some water and cutting the paste , the cr and chow mixture were being dried in a 30 - 45 min exposure to a continuous warm air stream . animals in the acr group had freely access to this mixed food ( figure 1 ) . water maze consisted of a pool ( 155 cm in diameter ) filled with water ( 211c ) to a level 10 cm from the edge of the tank . a transparent plexiglas platform ( 10 cm diameter ) was located 1.5 cm below the surface in the eastern quadrant of the tank ( target quadrant ) . the walls surrounding the pool were decorated with distinct extra maze spatial cues , which were kept in fixed positions during the entire experiment to allow the animals to find the hidden platform . the animals movements were recorded by a ccd camera ( panasonic inc , japan ) hanging from the ceiling above the mwm apparatus , and locomotion tracking was measured using ethovision software ( version xt7 , the netherlands ) , a video tracking system for automated analysis of animal behavior . twenty - four hr before starting the hidden platform training , rats were given 60 sec to swim in the tank without the platform in order to adapt to the environment . the reference spatial learning and memory tests were carried out based on the procedure previously conducted in our laboratory with some modifications in training ; the platform was submerged in the eastern quadrant of the pool . its place remained unchanged throughout training , but animals were released into the water ( while facing the tank wall ) from different locations chosen randomly from the south , north , west , northwest and southwest . the animals were allowed to swim for 60 sec to localize the platform s position in the tank . the rats which did not find the platform within 60 sec , were directed into it and were allowed to rest on it for 10 sec . twenty - four hr after the third session ; the spatial probe test was performed . during this test , the platform was removed , and rats were allowed to swim for 60 sec before getting removed . escape latencies , time spent in target quadrant , and swim speed were wholly recorded for subsequent analysis . the rats were anesthetized and their brains were fixed through a transcardial perfusion ( normal saline and 4% paraformaldehyde solution ) within 24 hr after the spatial probe test . after that , the brains were kept in a 4% paraformaldehyde solution . in preparation for the histological staining , the brains were embedded with paraffin , cut into consecutive 5 m transverse sections by a microtome , and placed on the poly - d - lysine - coated glass slides . alternate sections were used for thioflavin - t and tunel staining . in order to detect -amyloid plaques in brain sections thioflavin - t ( sigma - aldrich , usa ) brain sections were deparaffinized in xylene , rehydrated , covered with thioflavin - t solution ( 0.5% thioflavin - t in 0.1n hcl ) , and incubated in dark room at room temperature for 10 min . slides were immersed in distilled water twice before being observed through a fluorescent microscope . in order to detect apoptotic neurons in brain sections , the tunel staining protocol was performed using tunel apoptosis detection kit , ( millipore , uk ) . brain sections were deparaffinized in xylene , rehydrated , immersed in pbs , and then incubated at 37 c for 30 min . out of pbs , sections were covered with diluted protein kinase and incubated at 37 c for 20 min . to halt protein kinase activity each brain section was covered with 50 l tdt ( terminal deoxynucleotidyl transferase ) buffer for 10 min . the tdt buffer was removed using a sampler , and then each section was covered with 50 l tdt mixture solution ( 90% tdt buffer , 5% dutp - biotin , 5% tdt ) and incubated for 60 min at 37 c . slides were washed for 5 min by immersing in tb buffer at room temperature and then dipping in pbs ( 42 min ) . after the washing process , sections were covered with 50 l of blocking solution and incubated for 20 min at room temperature . after removing the blocking buffer with a sampler , 50 l avidin - ftc mixture ( 1 part avidin ftc , 9 parts blocking solution ) was used for each section , and slides were incubated at 37 c for 30 min in a dark room . to obtain the mean percentage of apoptotic neurons in the hippocampus area , 3 brain sections were chosen randomly from each subject and the number of tunel positive and negative cells was counted in three adjacent 400x microscopic fields . the data were presented as meanse and processed by the statistical package for social sciences ( version 16.0 ; spss inc . , mean escape latency , distance traveled to the platform , time spent in target quadrant in mwm test and also mean percentage of apoptotic cells were analyzed . all the groups were compared using one - way anova test . comparing sham and a and also a and acr was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison . according to the obtained results , -amyloid plaques were found in the brain sections of the animals in either acr or a groups , except for the sham - operated group and the control group ( figure 2 ) . arrows show the amyloid plaques in the brain section derived from -amyloid injected ( without creatine supplementation ) rats . no amyloid plaque is visible in the brain section of the rat from sham group . obiective 10 although the rats in a group had higher mean escape latency than the sham ( 27.90.53 vs 20.90.99 , p=0.003 ) and control ( 27.90.53 vs 17.251.19 , p=0.0001 ) groups , however , they showed better performance compared to the acr group ( 27.90.53 vs 33.221.56 , p= 0.009 ) . overall , the results indicated learning impairment in both acr and a groups , but , cr supplementation imposed a deterioration effect on learning function of rats after -amyloid injection ( figure 3 ) . this was confirmed by comparing the average distance traveled by the rats to find the platform in training trials ( figure 4 ) . mean escape latency during three days of training in study groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse mean distance traveled to the platform during three days of training in study groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse comparing sham & control groups ( p=0.444 ) , sham & a groups ( p=0.003 ) and a & acr groups ( p=0.019 ) was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison during the probe test , animals in a group spent less time in the target quadrant ( 31.990.78 ) than those in the sham ( 35.90.91 ) and control ( 33.572.58 ) groups but these differences were not statistically significant ( p=0.16 & p=0.56 respectively ) . however , acr group members spent less time in the target quadrant ( 27.671.55 ) compared to sham group ( p=0.003 ) and the a group , but the difference between a and acr groups did not reach statistical significance ( p=0.083 ) . this reflects the negative effect of oral cr supplementation on memory retrieval ability of rats after -amyloid injection ( figure 5 ) . as mentioned earlier , the tunel staining protocol was performed to detect apoptotic neurons in the brain sections ( figure 6 ) . as the figure suggests , the brain sections of the rats in the acr and a groups contained more tunel - positive neurons than those in the sham group . the percentage of tunel - positive neurons counted in the brain sections of the rats in the a ( 24.004.45 percent ) and acr ( 25.335.04 percent ) groups was significantly higher than those in the sham ( 4.831.33 percent ) group ( p=0.001 & p=0.001 respectively ) . comparing to the a group , the mean percentage of tunel - positive neurons was not significantly different in the acr group ( p=0.799 ) . based on the foregoing , cr supplementation had no influence on apoptotic effects of -amyloid in neurons . mean time spent in target quadrant in probe test in different groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse tunel staining . arrows show the tunel positive neurons in the picture of the brain section -amyloid injected rats ( a & acr groups ) have more tunel positive neurons compared to the rats in the sham group . the results of this study revealed a deficit in rats ability to orientate in the morris water maze following a single injection of -amyloid 1 - 42 in the hippocampal ca1 area . many studies have examined the behavioral consequences of injecting or infusing different -amyloid fragments in various brain areas of rodents . despite few studies that have found no behavioral changes after a low dose single injection of -amyloid ( 22 ) , the results of the present study revealed that a single 2 g / side hipocampal ca1 area injection of prefibrillar -amyloid 1 - 42 ( incubated for 48 hr in room temperature ) ( 23 ) , could induce spatial short - term memory impairment , when tested six weeks after the injection . due to limited permeability of the blood - brain barrier to cr , the increase of brain cr followed by oral supplementation would be a slow process ( 21 ) . for better metabolic support at the time of injection , supplementation of cr contrary to previous studies on healthy individuals , the results of the present study revealed negative effects of cr supplementation on learning and memory retrieval ( 4 , 9 - 11 ) . furthermore , the tunel - positive neurons counted in the brain sections showed no protective effect of cr . this contrasts with the results of those studies that emphasize on protective effects of cr in cell culture ( 19 ) . given the same rate of tunel - positive neurons in either group , more severe memory impairment in the group received cr can be for any reason ; a temporary inflammation or non - apoptotic death of neurons . although the final judgment in this case can not be achieved based solely on the results of this research , however , by putting together the results of this study and previous literature , a mechanism could be expected for this phenomenon . gallant et al detected soluble cr deposits in autopsy of transgenic mice and ad brain sections ( 20 ) . in an earlier study on these tgcrnd8 mice , the inflammation and microglial activation of their brain was reported by wyss and kaddurah - daouk ( 21 ) . gallant et al attributed this inflammation to the detected cr deposits that might be formed due to the low energy levels of neurons and declined creatine kinase ( ck ) activity ( 20 ) . in the present study , ck activity was not measured , which may be one of the constraints of this research . however , there is some evidence to suggest that , -amyloid promotes protein oxidation and causes irreversible inhibition of brain ck ( bb - ck ) at concentrations toxic to cultured neurons . this evidence is in line with the findings of yatin and his colleagues in 2000 on modification of bb - ck in ad ( 24 - 26 ) . so , it would not be irrational to conclude that the activity of bb - ck may be declined in a rats and in consequent ; more cr supply ( acr ) caused more cr deposition in the neurons , resulting in more inflammation and/or cell death . similar studies are needed to determine the relationship between declined bb - ck activity , cr supply of the neurons , and cr deposition in neurons based on the measurement results of bb - ck activity and cr deposits after -amyloid injection . oral cr supplementation in rats , before and after -amyloid injection into the ca1 area of hippocampus , can deteriorate learning and memory impairment and does not protect neuronal apoptosis induced by -amyloid .

objective(s):neuroprotective effect of creatine ( cr ) against -amyloid ( a ) is reported in an in vitro study . this study investigated the effect of cr supplementation on -amyloid toxicity in vivo.materials and methods : thirty two , male wistar rats were divided into 4 groups . during ten weeks of study , control group went through no surgical or dietary intervention . at the 4th week of study sham group had a hippocampal normal saline injection , while a and acr groups had an -amyloid injection in the hippocampus . acr group were fed by cr diet during the study . after 10 weeks , morris water maze ( mwm ) test was administered to measure learning ability and memory retrieval . animals were sacrificed for tunel anti apoptotic assay and staining of amyloid plaques by thioflavin-t.results:there was a significant retention deficit among acr and a group while the escape latency and the distance traveled to the platform were significantly higher in acr group compared to a group . acr group had same percent of tunel positive neurons compared to a group.conclusion:cr supplementation before and after -amyloid injection into the ca1 area of hippocampus deteriorates the learning and memory impairment of rats and it does not protect neuronal apoptosis caused by -amyloid .

Introduction Materials and Methods Study design Surgery procedure Supplementation Morris Water Maze (MWM) Apparatus, Habituation and Procedure Histological tests Statistical analysis Results Discussion Conclusion

creatine ( cr ) is endogenously produced from the biosynthesis of glycine , arginine , and methionine in kidneys , liver , pancreas , and possibly the brain . there are pieces of evidence on the protective effects of cr on muscles , heart ( 3 ) , nervous system , and brain function ( 4 ) . elevated cr level in human and rodents brain have been reported following oral supplementation of cr ( 5 - 7 ) . there are also studies on changes in serotonin and dopamine regulators of athletes brain ( 8) and in depression - like behavior of rodents after a period of cr supplementation ( 1 ) . some human studies have confirmed better cognitive performance following the supplementation of cr in adequate doses ( 4 , 9 - 11 ) . according to the literature , oral cr supplementation may impose neuroprotective effects on experimental animal models of huntington s disease ( 6 , 12 , 13 ) . alzheimer s disease ( ad ) is the main cause of dementia in elderly ( 14 ) . two core features of the ad are neurofibrillar tangles , which are intracellular aggregates of hyperphosphorylated tau protein , leading to neuronal malfunction ( 15 , 16 ) , and amyloid plaques , which are heterogenous aggregates found in extra - cellular space mainly containing -amyloid ( a ) peptide ( 17 ) . accumulation of amyloid inside neurons may contribute to the development or exacerbation of the ad ( 18 ) . it may be the probable mechanism behind neuroprotective role of cr against the toxic effects of -amyloid peptide in cell culture of hippocampal neurons ( 19 ) . furthermore , the discovery of cr deposits in the brain of transgenic ad mice and in the hippocampus of ad patients indicates an association between cellular energy levels , -amyloid , cr metabolism , and ad ( 20 ) . in this respect it may be speculated whether cr supplementation at an early time point of the disease can prevent or delay the course of ad - related neurodegeneration ( 21 ) . despite the numerous efforts and studies conducted in the field , there is no clinical or in vivo study to provide adequate proofs about the neuroprotective effects of cr supplementation on the ad . considering the neuropro - tective effect of cr against -amyloid toxicity in vitro and positive effects of cr supplementation on improving the cognitive performance in healthy individuals , the present study was performed to investigate the effects of oral supplementation of cr on -amyloid toxicity in vivo . thirty two male wistar rats ( 4 month of age and weighting of 200 - 250 g ) were obtained from pasteur institute of iran , research , production and education centre , ( tehran , iran ) . the rats were given at least one week to habituate to the facilities , and then experimental procedures began . all the guidelines of the committee of care in the use of experimental animals were followed , and the study procedures were approved by the ethics committee of tehran university of medical sciences . the animals were divided into four groups ( n=8 per group ) : control , sham , a ( -amyloid injection , no cr supplementation ) , and acr ( -amyloid injection , cr supplementation ) . acr group received cr monohydrate powder mixed in their chow ( 2% cr / diet ) and other groups received normal chow diet for four weeks . those in the a and acr groups were given a bilateral -amyloid peptide injection in the ca1 hippocampus ( 0.5 g/l ) , while the sham group received a normal saline injection in the same area of the brain . after surgery , the acr group continued receiving 2% cr diet while the other groups maintained their normal chow diet ( figure 1 ) . the animals were anesthe - tized with intra peritoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and then injected bilaterally under stereotaxic conditions with -amyloid or normal saline into the ca1 hippocampus ( ap=3.9 mm , lr=2.2 mm , d=2.7 mm ) . injections were performed at a rate of 0.5 l / min using a hamilton syringe attached to the stereotax apparatus . four microliters of -amyloid ( 0.5 g/l ) solution was injected in every hippocampus , and the needle was kept in place for one min after injection before being slowly retracted ( figure 1 ) . two percent of cr ( sigma - aldrich , usa ) was mixed into the normal chow using an electric mill according the previous studies ( 6 , 12 , 13 ) . after adding some water and cutting the paste , the cr and chow mixture were being dried in a 30 - 45 min exposure to a continuous warm air stream . animals in the acr group had freely access to this mixed food ( figure 1 ) . the morris water maze procedure was performed 6 weeks after the -amyloid injection . water maze consisted of a pool ( 155 cm in diameter ) filled with water ( 211c ) to a level 10 cm from the edge of the tank . a transparent plexiglas platform ( 10 cm diameter ) was located 1.5 cm below the surface in the eastern quadrant of the tank ( target quadrant ) . the walls surrounding the pool were decorated with distinct extra maze spatial cues , which were kept in fixed positions during the entire experiment to allow the animals to find the hidden platform . twenty - four hr before starting the hidden platform training , rats were given 60 sec to swim in the tank without the platform in order to adapt to the environment . the reference spatial learning and memory tests were carried out based on the procedure previously conducted in our laboratory with some modifications in training ; the platform was submerged in the eastern quadrant of the pool . its place remained unchanged throughout training , but animals were released into the water ( while facing the tank wall ) from different locations chosen randomly from the south , north , west , northwest and southwest . the animals were allowed to swim for 60 sec to localize the platform s position in the tank . the rats which did not find the platform within 60 sec , were directed into it and were allowed to rest on it for 10 sec . twenty - four hr after the third session ; the spatial probe test was performed . during this test , the platform was removed , and rats were allowed to swim for 60 sec before getting removed . the rats were anesthetized and their brains were fixed through a transcardial perfusion ( normal saline and 4% paraformaldehyde solution ) within 24 hr after the spatial probe test . in preparation for the histological staining , the brains were embedded with paraffin , cut into consecutive 5 m transverse sections by a microtome , and placed on the poly - d - lysine - coated glass slides . alternate sections were used for thioflavin - t and tunel staining . to obtain the mean percentage of apoptotic neurons in the hippocampus area , 3 brain sections were chosen randomly from each subject and the number of tunel positive and negative cells was counted in three adjacent 400x microscopic fields . , mean escape latency , distance traveled to the platform , time spent in target quadrant in mwm test and also mean percentage of apoptotic cells were analyzed . comparing sham and a and also a and acr was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison . thirty two male wistar rats ( 4 month of age and weighting of 200 - 250 g ) were obtained from pasteur institute of iran , research , production and education centre , ( tehran , iran ) . the rats were given at least one week to habituate to the facilities , and then experimental procedures began . all the guidelines of the committee of care in the use of experimental animals were followed , and the study procedures were approved by the ethics committee of tehran university of medical sciences . the animals were divided into four groups ( n=8 per group ) : control , sham , a ( -amyloid injection , no cr supplementation ) , and acr ( -amyloid injection , cr supplementation ) . acr group received cr monohydrate powder mixed in their chow ( 2% cr / diet ) and other groups received normal chow diet for four weeks . those in the a and acr groups were given a bilateral -amyloid peptide injection in the ca1 hippocampus ( 0.5 g/l ) , while the sham group received a normal saline injection in the same area of the brain . after surgery , the acr group continued receiving 2% cr diet while the other groups maintained their normal chow diet ( figure 1 ) . the animals were anesthe - tized with intra peritoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and then injected bilaterally under stereotaxic conditions with -amyloid or normal saline into the ca1 hippocampus ( ap=3.9 mm , lr=2.2 mm , d=2.7 mm ) . injections were performed at a rate of 0.5 l / min using a hamilton syringe attached to the stereotax apparatus . four microliters of -amyloid ( 0.5 g/l ) solution was injected in every hippocampus , and the needle was kept in place for one min after injection before being slowly retracted ( figure 1 ) . two percent of cr ( sigma - aldrich , usa ) was mixed into the normal chow using an electric mill according the previous studies ( 6 , 12 , 13 ) . after adding some water and cutting the paste , the cr and chow mixture were being dried in a 30 - 45 min exposure to a continuous warm air stream . animals in the acr group had freely access to this mixed food ( figure 1 ) . water maze consisted of a pool ( 155 cm in diameter ) filled with water ( 211c ) to a level 10 cm from the edge of the tank . a transparent plexiglas platform ( 10 cm diameter ) was located 1.5 cm below the surface in the eastern quadrant of the tank ( target quadrant ) . the walls surrounding the pool were decorated with distinct extra maze spatial cues , which were kept in fixed positions during the entire experiment to allow the animals to find the hidden platform . the animals movements were recorded by a ccd camera ( panasonic inc , japan ) hanging from the ceiling above the mwm apparatus , and locomotion tracking was measured using ethovision software ( version xt7 , the netherlands ) , a video tracking system for automated analysis of animal behavior . twenty - four hr before starting the hidden platform training , rats were given 60 sec to swim in the tank without the platform in order to adapt to the environment . the reference spatial learning and memory tests were carried out based on the procedure previously conducted in our laboratory with some modifications in training ; the platform was submerged in the eastern quadrant of the pool . its place remained unchanged throughout training , but animals were released into the water ( while facing the tank wall ) from different locations chosen randomly from the south , north , west , northwest and southwest . the animals were allowed to swim for 60 sec to localize the platform s position in the tank . the rats which did not find the platform within 60 sec , were directed into it and were allowed to rest on it for 10 sec . twenty - four hr after the third session ; the spatial probe test was performed . during this test , the platform was removed , and rats were allowed to swim for 60 sec before getting removed . the rats were anesthetized and their brains were fixed through a transcardial perfusion ( normal saline and 4% paraformaldehyde solution ) within 24 hr after the spatial probe test . in order to detect -amyloid plaques in brain sections thioflavin - t ( sigma - aldrich , usa ) brain sections were deparaffinized in xylene , rehydrated , covered with thioflavin - t solution ( 0.5% thioflavin - t in 0.1n hcl ) , and incubated in dark room at room temperature for 10 min . to halt protein kinase activity each brain section was covered with 50 l tdt ( terminal deoxynucleotidyl transferase ) buffer for 10 min . to obtain the mean percentage of apoptotic neurons in the hippocampus area , 3 brain sections were chosen randomly from each subject and the number of tunel positive and negative cells was counted in three adjacent 400x microscopic fields . , mean escape latency , distance traveled to the platform , time spent in target quadrant in mwm test and also mean percentage of apoptotic cells were analyzed . all the groups were compared using one - way anova test . comparing sham and a and also a and acr was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison . according to the obtained results , -amyloid plaques were found in the brain sections of the animals in either acr or a groups , except for the sham - operated group and the control group ( figure 2 ) . arrows show the amyloid plaques in the brain section derived from -amyloid injected ( without creatine supplementation ) rats . no amyloid plaque is visible in the brain section of the rat from sham group . obiective 10 although the rats in a group had higher mean escape latency than the sham ( 27.90.53 vs 20.90.99 , p=0.003 ) and control ( 27.90.53 vs 17.251.19 , p=0.0001 ) groups , however , they showed better performance compared to the acr group ( 27.90.53 vs 33.221.56 , p= 0.009 ) . overall , the results indicated learning impairment in both acr and a groups , but , cr supplementation imposed a deterioration effect on learning function of rats after -amyloid injection ( figure 3 ) . this was confirmed by comparing the average distance traveled by the rats to find the platform in training trials ( figure 4 ) . mean escape latency during three days of training in study groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse mean distance traveled to the platform during three days of training in study groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse comparing sham & control groups ( p=0.444 ) , sham & a groups ( p=0.003 ) and a & acr groups ( p=0.019 ) was carried out using one - way anova , least significant difference ( lsd ) post hoc comparison during the probe test , animals in a group spent less time in the target quadrant ( 31.990.78 ) than those in the sham ( 35.90.91 ) and control ( 33.572.58 ) groups but these differences were not statistically significant ( p=0.16 & p=0.56 respectively ) . however , acr group members spent less time in the target quadrant ( 27.671.55 ) compared to sham group ( p=0.003 ) and the a group , but the difference between a and acr groups did not reach statistical significance ( p=0.083 ) . this reflects the negative effect of oral cr supplementation on memory retrieval ability of rats after -amyloid injection ( figure 5 ) . as mentioned earlier , the tunel staining protocol was performed to detect apoptotic neurons in the brain sections ( figure 6 ) . as the figure suggests , the brain sections of the rats in the acr and a groups contained more tunel - positive neurons than those in the sham group . the percentage of tunel - positive neurons counted in the brain sections of the rats in the a ( 24.004.45 percent ) and acr ( 25.335.04 percent ) groups was significantly higher than those in the sham ( 4.831.33 percent ) group ( p=0.001 & p=0.001 respectively ) . comparing to the a group , the mean percentage of tunel - positive neurons was not significantly different in the acr group ( p=0.799 ) . based on the foregoing , cr supplementation had no influence on apoptotic effects of -amyloid in neurons . mean time spent in target quadrant in probe test in different groups a : a injection , no creatine supplementation . control : control group with no surgical and nutritional intervention ; results are presented as meanse tunel staining . arrows show the tunel positive neurons in the picture of the brain section -amyloid injected rats ( a & acr groups ) have more tunel positive neurons compared to the rats in the sham group . the results of this study revealed a deficit in rats ability to orientate in the morris water maze following a single injection of -amyloid 1 - 42 in the hippocampal ca1 area . despite few studies that have found no behavioral changes after a low dose single injection of -amyloid ( 22 ) , the results of the present study revealed that a single 2 g / side hipocampal ca1 area injection of prefibrillar -amyloid 1 - 42 ( incubated for 48 hr in room temperature ) ( 23 ) , could induce spatial short - term memory impairment , when tested six weeks after the injection . due to limited permeability of the blood - brain barrier to cr , the increase of brain cr followed by oral supplementation would be a slow process ( 21 ) . for better metabolic support at the time of injection , supplementation of cr contrary to previous studies on healthy individuals , the results of the present study revealed negative effects of cr supplementation on learning and memory retrieval ( 4 , 9 - 11 ) . furthermore , the tunel - positive neurons counted in the brain sections showed no protective effect of cr . this contrasts with the results of those studies that emphasize on protective effects of cr in cell culture ( 19 ) . given the same rate of tunel - positive neurons in either group , more severe memory impairment in the group received cr can be for any reason ; a temporary inflammation or non - apoptotic death of neurons . although the final judgment in this case can not be achieved based solely on the results of this research , however , by putting together the results of this study and previous literature , a mechanism could be expected for this phenomenon . in an earlier study on these tgcrnd8 mice , the inflammation and microglial activation of their brain was reported by wyss and kaddurah - daouk ( 21 ) . gallant et al attributed this inflammation to the detected cr deposits that might be formed due to the low energy levels of neurons and declined creatine kinase ( ck ) activity ( 20 ) . in the present study , ck activity was not measured , which may be one of the constraints of this research . however , there is some evidence to suggest that , -amyloid promotes protein oxidation and causes irreversible inhibition of brain ck ( bb - ck ) at concentrations toxic to cultured neurons . so , it would not be irrational to conclude that the activity of bb - ck may be declined in a rats and in consequent ; more cr supply ( acr ) caused more cr deposition in the neurons , resulting in more inflammation and/or cell death . similar studies are needed to determine the relationship between declined bb - ck activity , cr supply of the neurons , and cr deposition in neurons based on the measurement results of bb - ck activity and cr deposits after -amyloid injection . oral cr supplementation in rats , before and after -amyloid injection into the ca1 area of hippocampus , can deteriorate learning and memory impairment and does not protect neuronal apoptosis induced by -amyloid .

protein protein complexes are fundamental to life where they are key to processes ranging from central metabolism to cell signaling . protein interactions generally underpin the electron - transfer ( et ) pathways of respiration . one of the many well - characterized examples of a transient et complex is that between cytochrome c and cytochrome c oxidase . this ensures the frequent exchange of partner proteins as required to support electron flux in cases where the sole function of one of the proteins is to shuttle electrons between redox partners . protein interactions are specific , for paracoccus denitrificans it has recently been shown that seven proteins in a respiratory network interact in a seemingly ill - defined manner . this results in an intricate electron - transfer network that may be better suited to successful colonization of habitats with changing resources . typical examples are the quinol nitrate oxidoreductase , narghi , and the quinol nitrite oxidoreductase , nrfha . quinol oxidation occurs in a trans - membrane subunit that is permanently bound to one or more periplasmic subunits that contain the site for catalytic reduction of the water - soluble substrate . a chain of redox centers extends between the catalytic sites to support electron exchange between them . however , during anaerobic respiration in shewanella , a single enzyme , cyma , oxidizes the membrane - bound menaquinol-7 pool and donates the electrons to a variety of terminal reductases , which can , for instance , reduce periplasmic nitrate , nitrite , fumarate , or extracellular ferric oxides and dimethyl sulfoxide ( dmso ) . the promiscuity with which cyma passes electrons from mq-7 to a wide variety of proteins poses an interesting question : does cyma form long - lived et complexes , analogous to the nrfha complex and quinol dehydrogenases in other species , or transient complexes of the type commonly observed when a redox protein functions as a shuttle to transfer electrons between two enzymes ? this question is particularly pertinent as cyma donates electrons to proteins that function solely in et , such as scya , and to enzymes , including tetraheme flavocytochrome c3 ( fcc3 ) fumarate reductase ( figure 1a ) , iron - reducing small tetraheme cytochrome ( stc ) , and a nitrite reductase ( nrfa ) . ( a ) schematic of the inner membrane respiratory chain of shewanella grown anaerobically with fumarate as the terminal electron acceptor . electrons generated during catabolism are donated to the mq-7 pool , which is reoxidized by cyma . based on primary sequence analysis , homology to nrfh for which a crystal structure is available , and biochemical analysis of both cyma and other napc / nirt superfamily members , cyma is known to contain a single n - terminal transmembrane -helix with a single globular head cyma transfers the electrons to the periplasmic enzyme flavocytochrome c3 ( fcc3 ) , which reduces fumarate to succinate . the site of action of the competitive inhibitor , 2-n - heptyl-4-hydroxyquinoline n - oxide ( hqno ) , is indicated in magenta . ( b ) schematic of cyma containing inner - membrane architecture on an electrode surface in the presence of fcc3 and a chemical reducing agent dithionite ( s2o4 ) . in both panels , chemical reactions and et steps are shown with black and green arrows , respectively . previously , we have studied the interaction between cyma and its native quinol substrate by adsorbing cyma on gold electrodes and exposing the protein film to liposomes containing mq-7 . cyclic voltammetry revealed that cyma in isolation catalyzed the reduction of mq-7 but failed to perform the reaction that underpins anaerobic respiration , the oxidation of mq-7 . here , we report the extension of these studies in which the quinone oxidoreductase activity of cyma is determined in the absence and presence of its et partner , fcc3 fumarate reductase . our approach was to construct a cyma containing inner - membrane architecture on planar surfaces ( figure 1b ) . the interaction between cyma and fcc3 was characterized with a quartz - crystal microbalance with dissipation ( qcm - d ) and the quinol dehydrogenase activity of cyma monitored by electrochemistry . formation of a long - lived complex between cyma and fcc3 was found to retune the catalytic bias of cyma toward oxidation of mq-7 ( schematically shown in figure 1b ) . this ability of protein protein interactions to modulate the catalytic bias of redox enzymes reveals a new mechanism by which the magnitude and direction of electron flux through respiratory et networks can be regulated . menaquinone-7 ( mq-7 , wako ) stock solutions were prepared at concentrations of 1 mg / ml in chloroform . 2-n - heptyl-4-hydroxyquinoline n - oxide ( hqno ) was purchased from alexis biochemicals , and a stock solution of 50 mm was prepared in dmso . mixtures of 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ; avanti polar lipids ) and cardiolipin ( cl ; avanti polar lipids ) at a 90/10 ratio were dissolved in chloroform , aliquoted to 5 mg total lipid , and dried under an n2 stream before storage under an n2 atmosphere at 20 c . mr-1 cultures were grown microaerobically , and fcc3 and membranous cyma were isolated as described elsewhere . a cyma truncation mutant ( cymasol ) , in which the n - terminal transmembrane helix was removed and replaced with a his - tagged maltose - binding protein ( mbp ) and a tev cleavage motif between the cymasol and the mbp , was expressed and purified as described in the supporting information of ref ( 15 ) . the plasmid used to express the mbp - cyma construct is described by londer et al . the mpb was cleaved from cyma by incubating with tev protease , purified as described previously , at 1:2 ( w / w ) ratio for 16 h at 4 c . cymasol was then purified by passing the protein mixture though a ni - sepharose ( fast flow , ge healthcare ) column , to remove his6-tagged mpb , his6-tagged tev protease , and any remaining uncleaved cymasol - mbp from the cleaved cymasol fraction . full - length recombinant cyma , cymasol - mbp fusion protein , and fcc3 were confirmed by mass spectrometry , n - terminal sequencing , and/or immunoblotting . sds - page analysis identified approximately 90% pure hemoprotein with a band at 24 kda ( cyma ) , 64 kda ( cymasol - mbp fusion ) , or 62 kda ( fcc3 ) . electronic absorption spectroscopy characterized cyma and cymasol as typical c - type cytochromes ( a407 nm / a275 nm = 4 ) consistent with four inserted hemes per protein monomer . protein concentrations were determined using a bicinchoninic acid ( bca ) protein assay kit ( sigma ) with bovine serum albumin as the standard . mq-7 was introduced into aliquots of a mixture of 90%/10% ( w / w ) popc and cl using a 50:50 chloroform / methanol mixture before being dried under a stream of nitrogen for at least 1 h. the resulting film was resuspended by vortexing in buffer ( 20 mm 3-(n - morpholino)propanesulfonic acid ( mops ) , 30 mm na2so4 , ph 7.4 ) to a concentration of 5 mg / ml . a homogeneous vesicle solution was prepared by extrusion through a 200 nm nucleopore track - etched polycarbonate membrane ( whatman ) using a mini - extruder ( avanti ) at 20 c . cyma was reconstituted by the method based on that of carter et al . on the day of reconstitution , vesicles were prepared as described above except a lipid concentration of 20 mg / ml was used . lipid vesicles , octylglucoside ( og ) , and cyma were mixed by inversion to final concentrations of 16.4 mg / ml lipid , 45 mm og , and 0.16 or 0.32 mg / ml cyma ( i.e. , 1 or 2% ( w / w ) cyma to lipid ) . qcm - d experiments were performed with 2% ( w / w ) cyma , while the electrochemistry was done with 1% ( w / w ) . after 15 min incubation on ice , the lipid / protein mixture was rapidly diluted 200-fold with buffer , precooled to 4 c , and centrifuged at 142 000 g for 1 h to pellet the proteoliposomes . the proteoliposomes were resuspended in the same volume of fresh cold buffer and centrifuged again at 142 000 g for 1 h. proteoliposomes were resuspended in 1 ml of buffer and extruded through a 200 nm track - etched polycarbonate membrane using a mini - extruder ( avanti ) . qcm - d experiments were conducted using a q - sense e4 ( q - sense ab ) . experiments were performed using silicon - oxide sensor crystals at 21 c , with the flow rate held at 70 l / min . experiments were conducted outside and inside an n2-filled glovebox ( mbraun labmaster ; < 1 ppm o2 ) , and no difference in behavior was observed . all solutions were purged with n2 and stored in the glovebox at least 24 h before use . silicon - oxide qcm - d crystals were cleaned by bath sonicating them with milli - q water ( 30 min ) , 0.4% sds detergent ( 20 min ) , and again milli - q water ( 20 min ) . after that , crystals were treated for 20 min with uv / ozone ( uv / ozone cleaning system , low pressure quartz - mercury vapor lamp emitting 254 and 185 nm uv ; uvocs ) followed by a 30 min bath sonication with milli - q water . all bilayers were formed using 0.5 mg / ml lipid vesicles in water containing 10 mm cacl2 . after being rinsed with water , the formed ssms were rinsed with 20 mm mops , 30 mm na2so4 , ph 7.4 ( buffer ) containing 1 mm edta and then buffer alone to remove excess vesicles and calcium ions . all protein - binding experiments were performed in buffer . on graphs , changes in the dissipation ( d ) and frequency ( f ) of the seventh overtone are presented , while third , fifth , ninth , eleventh , and thirteenth overtones were also recorded . d and f values are given as the shift as compared to values obtained in buffer . f was used to calculate adsorbed weight under the assumptions of the sauerbrey equation ( i.e. , 17.7 ng cm hz for the equipment and crystals used ) and then into protein coverages by taking into account that approximately 25% of the adsorbed weight is due to water entrapped within the protein matrix . routinely , electrochemical experiments were carried out with ultraflat template stripped gold ( tsg ) working electrodes , prepared as described previously . 150 nm of 99.99% gold ( goodfellows ) was evaporated on silicon wafers using an edwards auto 306 evaporator at < 2 10 mbar . 1.2 cm glass slides were glued to the gold layer with epo - tek 377 and cured for 2 h at 120 c . before use , the glass slides were detached from the silicon wafers to expose the tsg surface . the formation of the self - assembled monolayers ( sams ) containing the cholesterol tether and the formation of the ssm onto the electrode were performed as described previously . sams were formed by incubating a freshly exposed tsg slide in 0.11 mm eo3-cholesteryl and 0.89 mm 6-mercaptohexanol ( 6mh ) in propanol for 16 h. after incubation , the excess thiol was gently washed away with isopropanol and methanol , and the electrodes were then dried in a stream of n2 . this procedure results in a 60%/40% eo3-cholesteryl/6-mercaptohexanol area ratio on the surface as confirmed by impedance spectroscopy before each experiment . to form solid - supported lipid membranes ( ssms ) , vesicles or proteoliposomes were added to the sam surface at a final concentration of 0.5 mg / ml in the presence of 10 mm cacl2 and incubated for 12 h until a capacitance drop to less than 1.2 f / cm was observed . the surface was then rinsed three times with water , then buffer containing 1 mm edta to remove any traces of calcium ions in the cell . finally , the ssm - modified electrode was rinsed three times with buffer and used in the electrochemistry experiments . care was taken to keep the electrodes immersed in an aqueous environment at all times during rinsing . a bespoke glass spectro - electrochemical cell was assembled using a standard three - electrode setup : the ultraflat template - stripped gold ( tsg ) working electrode was embedded in a ptfe holder with a rubber o - ring seal , ( a = 0.2 cm ) ; a platinum wire counter electrode and a saturated silver / silver chloride electrode ( ag / agcl ) completed the circuit in the buffer volume . all potentials are quoted versus standard hydrogen electrode ( she ) using 0.199 v vs she for the ag / agcl reference electrode . routinely , experiments were conducted in 20 mm mops , 30 mm na2so4 , ph 7.4 ( buffer ) . the cell was used in a steel mesh faraday cage to minimize electrical noise , and all experiments were conducted inside an n2-filled glovebox ( mbraun labmaster ) where the o2 levels were < 1 ppm . all solutions were purged with n2 and stored in the glovebox at least 24 h before use . electrochemical measurements were recorded at 21 c using an autolab electrochemical analyzer with a pgstat30 potentiostat , scangen and adc750 modules , and fra2 frequency analyzer ( ecochemie ) . analogue cyclic voltammetry experiments were routinely carried out by holding the potential at 0.5 or 0.3 v for 5 s before cycling at a scan rate of 1 or 10 mv / s in the potential window from 0.5 or 0.3 to 0.4 v. after addition of cymasol , fcc3 , or hqno to the buffer , an incubation time of 30 min was allowed . after incubating the ssm with cymasol or fcc3 , protein that was not bound to the ssm was washed out by rinsing the electrochemical cell five times with buffer . where hqno was introduced in dmso , qcm - d was used to monitor the association of fcc3 with cyma - containing inner membrane architectures assembled on silicon - oxide surfaces ( figure 2a ) . the qcm - d technique gives simultaneous and real - time information about changes in adsorbed mass , through the resonant frequency ( f ) , and the rigidity or viscoelastic properties of the adsorbed layer through the dissipation ( d ) . solid - supported bilayer membranes ( ssms ) were formed on the silicon - oxide surfaces from vesicles comprised of 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) and cardiolipin ( cl ) that contained 2% ( w / w ) cyma to lipid . ssm formation was confirmed by a drop in frequency of 2627 hz and a small change in dissipation ( figure 2a ; for clarity , the formation of the bilayer itself is not shown and the trace starts after the bilayer is formed ) . the frequency shift is very similar to values reported for the formation of phosphatidylcholine - only ssms under similar conditions . this is expected because the low cyma content of the ssm - forming vesicles corresponds to 0.3 pmol of cyma / cm , and this would only cause a frequency shift of approximately 0.5 hz . the assembly and catalytic activities of cyma(sol ) containing ssms . ( a ) qcm - d results of a sio2 surface in buffer , plotting ( black line , left axis ) frequency and ( red line , right axis ) dissipation against time . changes in the solution composition flowing over the ssm are indicated : ( fcc3 ) 5 m fcc3/1 mm fumarate ; ( wash 1 ) 1 mm fumarate ; ( wash 2 ) buffer only . ssm was formed with cyma proteoliposomes ( 90:10 popc : cardiolipin ; 2% ( w / w ) cyma ; 1% ( b ) cvs ( 10 mv / s ) of a ssm on a gold electrode modified with cholesterol tethers . ( w / w ) cyma ; 1% ( w / w ) mq-7 ) . cvs are shown ( a ) before and after addition of ( b ) 5 m fcc3 , ( c ) 1 mm fumarate ( after rinsing unbound fcc3 from solution ) , and ( d ) 1 mm fumarate/10 m hqno . ( c ) relative current measured from cvs as in ( b , trace c ) at 0.25 v vs she of the reductive scan as a function of mq-7 concentration in the ssm . mq-7 content is given in weight percentages relative to the lipid weight in the proteoliposomes . ( d ) qcm - d results of a sio2 surface in buffer , plotting ( black line , left axis ) frequency and ( red line , right axis ) dissipation against time . for clarity reasons , only the traces after the formation of the ssm are shown . changes in the solution composition flowing over the ssm are indicated : ( cymasol ) 0.1 m cymasol ; ( wash 1 ) , 1 mm fumarate ; ( fcc3 ) 5 m fcc3/1 mm fumarate ; ( wash 2 ) 1 mm fumarate . ssm was formed with liposomes ( 90:10 popc : cardiolipin ; 1% ( w / w ) mq-7 ) . ( e ) cvs ( 1 mv / s ) of a ssm on a gold electrode modified with cholesterol tethers . the ssm was formed with liposomes ( 90:10 popc : cardiolipin ; 2% ( w / w ) mq-7 ) . cvs are shown ( a ) before and after addition of ( b ) 0.1 m cymasol , ( c ) 5 m fcc3 , ( d ) 1 mm fumarate ( after rinsing unbound fcc3 from solution ) , and ( e ) 1 mm fumarate/10 m hqno . ( f ) relative current measured from cvs as in ( e , trace d ) at 0.25 v vs she of the reductive scan as a function of mq-7 concentration in the ssm . mq-7 content is given in weight percentages relative to the lipid weight in the liposomes . exposure of cyma - containing ssms to fcc3 results in a further decrease in frequency to 55 hz , and the dissipation rises by approximately 2 units ( figure 2a ) . this indicates that fcc3 binds to ssms as a layer exhibiting some viscosity , such that it is not fully elastic , which suggests that some of the fcc3 is loosely bound . this was confirmed when subsequently the ssm assemblies were washed with buffer solutions lacking fcc3 ( figure 2a ) . the dissipation returned to its previous value , and the frequency settled at a value of 35 hz indicating that some , but not all , fcc3 had detached from the membrane . the shift in frequency ( 9 hz ) due to remaining tightly bound fcc3 is equivalent to the adsorption of approximately 0.16 g fcc3/cm and so approximately 2 pmol fcc3/cm . control experiments with ssms that did not contain cyma revealed that no fcc3 remained bound to the membrane after washing ( figure s1 in the supporting information ) . trans - membrane proteins are known to introduce defects into ssms , and although the qcm - d data indicate that the cyma - containing ssms are essentially homogeneous , it can not be excluded that a small number of defects cause some a - specific adsorption of fcc3 . thus , in terms of the molecular architecture of the ssm and its associated proteins , the qcm - d data do not allow for unambiguous interpretation of the ratio of bound fcc3 to cyma . furthermore , fcc3 is reported to be a monomer in solution , and it is unlikely that cyma forms complexes with fcc3 oligomers of the high order that is implied by the frequency shifts ( approximately six fcc3 per cyma ) . a more reasonable interpretation is that cyma forms complexes with fcc3 with a stoichiometry close to 1:1 ( or 1:2 as observed for nrfha ) , while additional fcc3 molecules are bound to defects in the ssm induced by the presence of cyma . defects in the ssm that are capable of binding fcc3 may arise because the location of cyma in the ssms used for these experiments can not be controlled . previous electrophoresis of cyma - containing ssms equivalent to those studied here has shown that approximately one - half the cyma is immobile . one - half the population of cyma may present its head domain toward the silicon oxide surface leading to interactions that immobilize the protein , while the mobile population of cyma presents its head domain to the solution . to eliminate the possibility that this issue influences the qcm - d results , a series of experiments were performed with a truncated form of cyma that lacks the n - terminal transmembrane helix . , cymasol could be introduced to ssms after their formation such that the protein can only associate with the solvent - exposed face of the ssm . tight binding of cymasol to ssms was observed within minutes from solutions containing approximately 0.1 m cymasol ( figure 2d ) . after washing the cymasol - containing ssm assembly to remove loosely bound protein , the frequency was 21 hz lower than that displayed by the ssm alone . this shift in frequency corresponds to the adsorption of 12 pmol cymasol / cm . on exposing the cymasol - containing ssms to fcc3 , followed by washing to remove loosely bound material , the frequency dropped by a further 18 hz this behavior is consistent with fcc3 binding tightly to cymasol - containing ssms and confirms the data obtained with the full - length cyma . assuming that all cymasol remains bound to the ssm upon fcc3 adsorption , the drop in frequency on fcc3 binding corresponds to 4 pmol fcc3/cm . from this , the ratio of fcc3 bound per cymasol is 1:3 . even allowing for the possibility that fcc3 partly replaces cymasol on the ssm surface , the fcc3 to cymasol ratio is still significantly lower than that for fcc3 bound to full - length cyma . these observations most likely reflect steric constraints on the binding of fcc3 to cymasol that are not present for cyma - containing ssms . in fact , assuming that cymasol has dimensions comparable to those of the nrfh head domain , the qcm - d data correspond to the formation of a close - packed monolayer of cymasol covered by another close - packed monolayer of fcc3 . in summary , the qcm - d is consistent with fcc3 binding strongly to ssms containing either cyma or cymasol , but not to ssms without cyma(sol ) , such that an inner membrane architecture may be present on the solid surfaces as shown schematically in figure 1b . if physiologically relevant cyma(sol)-fcc3 complexes are formed , both enzymes should be catalytically active and should support intermolecular et to couple mq-7 oxidation to fumarate reduction . to assess whether this was the case , ssms containing cyma(sol)-fcc3 complexes and mq-7 were assembled on gold electrode surfaces as described below and studied by electrochemistry . ssms were formed from cyma proteoliposomes by self - assembly on ultrasmooth gold electrodes that had been modified with submonolayers of cholesterol tethers . formation of planar ssms was confirmed by electrochemical impedance spectroscopy , which showed a drop in capacitance to below 1.2 f / cm after addition of the proteoliposomes ( figure s2 in the supporting information ) . cyclic voltammograms ( cvs ) of the ssms containing cyma and mq-7 showed little change after exposure to fcc3 ( figure 2b ) . however , addition of fumarate had a large effect on the appearance of the cv that was converted to a waveshape typical for catalytic reduction reactions . this confirmed that the catalytic response had its origin in fcc3 bound irreversibly to the cyma - containing ssm . to exclude the possibility that fcc3 received electrons directly from the electrode , or mq-7 , an inhibitor of cyma s quinol - dehydrogenase activity the addition of hqno inhibited catalytic fumarate reduction ( figure 2b ) , demonstrating that the electrons for fumarate reduction are indeed supplied to fcc3 from mq-7 oxidation by cyma as illustrated in figure 1 . in agreement with this conclusion , when the mq-7 concentration within the lipid membrane was altered , the catalytic current increased in a manner consistent with michaelis menten behavior ( figure 2c ) . the maximum catalytic current ( 0.25 a or 1.25 a / cm at 250 mv ) is achieved at mq-7 contents between 0.75% and 2% ( w / w ) . the turnover frequency for mq-7 oxidation can be calculated if the current can be related to the number of catalytically active cyma - fcc3 complexes on the surface . as described above , the precise number of such complexes in these experiments is not defined . an upper limit for the concentration of complex is 0.15 pmol / cm based on the ssms being formed from vesicles containing 1% ( w / w ) cyma . from this , we calculate a minimum turnover number for mq-7 oxidation by the cyma - fcc3 complex of 40 s. the experiments described above reveal that the cyma - fcc3 complex is able to oxidize mq-7 in ssms . this is in stark contrast to our previous studies of cyma in contact with mq-7 containing liposomes where only mq-7 reduction was observed . to establish whether fcc3 is responsible for the change in the catalytic bias of cyma , the activity of cyma in the membrane - modified electrodes was measured in the absence of fcc3 using chemical reductants and oxidants ( figure 3 ) . addition of ferricyanide had almost no effect on the voltammogram ( figure 3a ) . electronic absorbance spectroscopy demonstrates that solutions of reduced cyma are rapidly oxidized by ferricyanide ( not shown ) . as a consequence , the absence of a reductive signal in the cv confirms that cyma without fcc3 is unable to oxidize mq-7 . the reduction potential of ferricyanide ( + 420 mv ) is higher than that of fumarate ( 30 mv ) , indicating that mq-7 oxidation by the cyma - fcc3 complex is not driven solely by thermodynamics . instead , formation of the long - lived cyma - fcc3 complex shifts the catalytic bias of cyma toward mq-7 oxidation . when , instead of ferricyanide , dithionite is added to the ssm , a catalytic oxidation signal appears that is inhibited by addition of hqno . this confirms our previous findings , that cyma is only able to reduce mq-7 in the absence of fcc3 . cvs ( 10 mv / s ) of a ssm on a gold electrode modified with cholesterol tethers . the ssm was formed with cyma proteoliposomes ( 90:10 popc : cardiolipin ; 1% ( w / w ) cyma ; 1% ( w / w ) mq-7 ) . ( a ) cvs ( a ) before and ( b ) after addition of 1 mm potassium ferricyanide . ( b ) cvs ( a ) before and ( b , c ) after addition of ( b ) 1 mm sodium dithionite and ( c ) 1 mm sodium dithionite/10 m hqno . very similar results were obtained when experiments were repeated with cymasol ( in these experiments , cymasol is adsorbed after ssm formation ; figure 2e and f and figure s3 in the supporting information ) . this demonstrates that structural details of the interaction between the lipid membrane and cyma are not important determinants of the enzyme s catalytic bias . it is notable that the catalytic currents displayed by cymasol are 45-fold lower than those for the ssms containing full - length cyma . this is despite the fact that the surface coverages of cymasol and fcc3 are higher than those for the cyma assemblies . this lower activity could reflect very slow quinol oxidation by cymasol and/or adsorption of cymasol in a range of orientations , many of which preclude efficient interaction with mq-7 and/or et to fcc3 . the latter possibility is consistent with the absence of a significant change in the cv from the ssms after immobilization of cymasol . a turnover frequency of 0.1 s for mq-7 oxidation can be calculated on the basis of a cymasol coverage of 12 pmol / cm . however , on the basis of arguments discussed above , this activity represents a lower limit . s. oneidensis mr-1 has become an important model organism for bioreactor , bioengineering , and bioremediation studies . its ability to transfer electrons from the oxidation of organic compounds or hydrogen to extracellular minerals and electrodes has inspired many to investigate applications in microbial - fuel cell technology . underpinning these capabilities is a conduit for electron transfer that extends from the quinol pool and cyma at the inner membrane to extracellular minerals and electrodes . however , cyma also distributes electrons from quinol oxidation to the terminal reductases for fumarate , nitrate , nitrite , and extracellular dmso . cyma thus plays a significant role in the highly branched network that supports anaerobic respiration in s. oneidensis mr-1 . our finding that complex formation between cyma and one of its partner proteins is required for cyma to act as a quinol dehydrogenase significantly advances our understanding of the factors that regulate the distribution of electrons across the respiratory network of s. oneidensis mr-1 . that cyma does not oxidize mq-7 in the absence of a partner protein will occlude et to nonphysiological redox partners and prevent side - reactions such as the production of radical oxygen species . because complex formation between cyma and each of its partner proteins is required to trigger mq-7 oxidation , respiratory electron flux should be dependent to some extent on the lifetime of the complexes formed by cyma . the cyma - stc et complex is believed to be transient because it has not been possible to cross - link these proteins . however , for cyma - fcc3 , evidence has been presented to support transient as well as long - lived et complexes . a recent nmr study by fonseca et al . shows that detergent - solubilized cyma forms a transient low - affinity complex with fcc3 with a kd of 0.4 mm . describe a solution assay in which cymasol is continuously oxidized by a much smaller number of fcc3 enzymes in the presence of fumarate . in contrast , ross and colleagues provided evidence for static cyma - fcc3 and cyma - mtrcab complexes in whole cells of s. oneidensis mr-1 . given the critical role of protein protein complex formation in activating the mq-7-dehydrogenase activity of cyma , it is now important to fully understand those factors that determine the lifetime of the complexes formed by cyma under cellular conditions . the electrochemical data indicate that the mq-7 oxidation activity of the cyma - fcc3 complex is at least 40 s. if we assume that cyma oxidizes mq-7 at similar rates in complex with its other redox partners , we anticipate that the electron flux through cyma will be sufficient to support anaerobic respiration of s. oneidensis mr-1 , including respiration on inorganic minerals and electrodes where a flux of 1 electron s per mtrc is reported . previously , the rate of fumarate reduction by fcc3 and cyma was measured as 0.01 s in a spectrophotometric assay that employed the water - soluble quinol analogue menadiol . the turnover frequencies resolved here for cyma with the natural substrate , mq-7 , are orders of magnitude greater . similar behavior has been observed in studies with nrfha where rates of menaquinol oxidation coupled to nitrite reduction are of the order of 390 s but drop to 17 s when assayed with the water - soluble analogue 2,3-dimethyl-1,4-naphthoquinone . together these studies illustrate the care that should be taken when interpreting results obtained with water - soluble analogues . that the formation of a protein protein complex can influence the catalytic activity or bias of redox enzymes , or their subunits , adds an intriguing dimension to the mechanisms by which organisms regulate their et networks . the group of lger has recently suggested that in a multicentered redox enzyme , nife - hydrogenase , the forward ( h2-formation ) and reverse ( h2-oxidation ) reactions are limited by different rate - limiting steps and that this phenomena results in a tuning of catalytic bias , which can be altered by a series of mutations that do not alter the reduction potential of the active site . for nife - hydrogenase , it is proposed that the rate of h2 oxidation is defined by the rate of et along a redox chain consisting of three fe s clusters . the reduction potentials of the hemes in cyma lie between 110 and 265 mv , which is lower than that of mq-7 ( 70 mv ) . this is also the case for other members of the napc / nirt superfamily . we have previously shown that menadione , which has a reduction potential equal to mq-7 , is unable to reduce the hemes in cyma . the results presented here extend this observation to the native substrate mq-7 and demonstrate that mq-7 oxidation in cyma is limited by et to the heme groups . fcc3 binding to cyma is unlikely to change the properties of the buried mq-7 active site , but will influence the properties of the surface - exposed hemes that are presented to the periplasm . consequently , we propose that mq-7 oxidation by cyma , and possibly all members of the napc / nirt superfamily , is regulated by an increase in the reduction potential of these hemes upon complex formation . we note that not all heme reduction potentials need to be higher than that of mq-7 as rollercoaster profiles , in which redox sites of alternating high and low potentials are commonly observed to make up a redox chain . an extreme example of the latter is the iron sulfur redox chain in complex i. in conclusion , we find that inner membrane architectures can be constructed on planar surfaces and that this provides a powerful approach to resolve protein complex formation and quinone oxidoreductase activity of membrane enzymes . our finding that protein protein interactions modulate the catalytic bias of cyma reveals a new mechanism by which the magnitude and direction of electron flux through respiratory et networks can be regulated . mq-7 oxidation is regulated by controlling the rate of et along the heme redox chain , which is rate limiting for cyma in isolation .

protein protein interactions are well - known to regulate enzyme activity in cell signaling and metabolism . here , we show that protein protein interactions regulate the activity of a respiratory - chain enzyme , cyma , by changing the direction or bias of catalysis . cyma , a member of the widespread napc / nirt superfamily , is a menaquinol-7 ( mq-7 ) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of shewanella oneidensis . we report the incorporation of cyma within solid - supported membranes that mimic the inner membrane architecture of s. oneidensis . quartz - crystal microbalance with dissipation ( qcm - d ) resolved the formation of a stable complex between cyma and one of its native redox partners , flavocytochrome c3 ( fcc3 ) fumarate reductase . cyclic voltammetry revealed that cyma alone could only reduce mq-7 , while the cyma - fcc3 complex catalyzed the reaction required to support anaerobic respiration , the oxidation of mq-7 . we propose that mq-7 oxidation in cyma is limited by electron transfer to the hemes and that complex formation with fcc3 facilitates the electron - transfer rate along the heme redox chain . these results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein protein interactions .

Introduction Methodology Results Discussion

protein protein complexes are fundamental to life where they are key to processes ranging from central metabolism to cell signaling . protein interactions generally underpin the electron - transfer ( et ) pathways of respiration . one of the many well - characterized examples of a transient et complex is that between cytochrome c and cytochrome c oxidase . this ensures the frequent exchange of partner proteins as required to support electron flux in cases where the sole function of one of the proteins is to shuttle electrons between redox partners . protein interactions are specific , for paracoccus denitrificans it has recently been shown that seven proteins in a respiratory network interact in a seemingly ill - defined manner . however , during anaerobic respiration in shewanella , a single enzyme , cyma , oxidizes the membrane - bound menaquinol-7 pool and donates the electrons to a variety of terminal reductases , which can , for instance , reduce periplasmic nitrate , nitrite , fumarate , or extracellular ferric oxides and dimethyl sulfoxide ( dmso ) . this question is particularly pertinent as cyma donates electrons to proteins that function solely in et , such as scya , and to enzymes , including tetraheme flavocytochrome c3 ( fcc3 ) fumarate reductase ( figure 1a ) , iron - reducing small tetraheme cytochrome ( stc ) , and a nitrite reductase ( nrfa ) . ( a ) schematic of the inner membrane respiratory chain of shewanella grown anaerobically with fumarate as the terminal electron acceptor . based on primary sequence analysis , homology to nrfh for which a crystal structure is available , and biochemical analysis of both cyma and other napc / nirt superfamily members , cyma is known to contain a single n - terminal transmembrane -helix with a single globular head cyma transfers the electrons to the periplasmic enzyme flavocytochrome c3 ( fcc3 ) , which reduces fumarate to succinate . the site of action of the competitive inhibitor , 2-n - heptyl-4-hydroxyquinoline n - oxide ( hqno ) , is indicated in magenta . ( b ) schematic of cyma containing inner - membrane architecture on an electrode surface in the presence of fcc3 and a chemical reducing agent dithionite ( s2o4 ) . previously , we have studied the interaction between cyma and its native quinol substrate by adsorbing cyma on gold electrodes and exposing the protein film to liposomes containing mq-7 . cyclic voltammetry revealed that cyma in isolation catalyzed the reduction of mq-7 but failed to perform the reaction that underpins anaerobic respiration , the oxidation of mq-7 . here , we report the extension of these studies in which the quinone oxidoreductase activity of cyma is determined in the absence and presence of its et partner , fcc3 fumarate reductase . the interaction between cyma and fcc3 was characterized with a quartz - crystal microbalance with dissipation ( qcm - d ) and the quinol dehydrogenase activity of cyma monitored by electrochemistry . formation of a long - lived complex between cyma and fcc3 was found to retune the catalytic bias of cyma toward oxidation of mq-7 ( schematically shown in figure 1b ) . this ability of protein protein interactions to modulate the catalytic bias of redox enzymes reveals a new mechanism by which the magnitude and direction of electron flux through respiratory et networks can be regulated . menaquinone-7 ( mq-7 , wako ) stock solutions were prepared at concentrations of 1 mg / ml in chloroform . qcm - d experiments were performed with 2% ( w / w ) cyma , while the electrochemistry was done with 1% ( w / w ) . qcm - d experiments were conducted using a q - sense e4 ( q - sense ab ) . on graphs , changes in the dissipation ( d ) and frequency ( f ) of the seventh overtone are presented , while third , fifth , ninth , eleventh , and thirteenth overtones were also recorded . the formation of the self - assembled monolayers ( sams ) containing the cholesterol tether and the formation of the ssm onto the electrode were performed as described previously . to form solid - supported lipid membranes ( ssms ) , vesicles or proteoliposomes were added to the sam surface at a final concentration of 0.5 mg / ml in the presence of 10 mm cacl2 and incubated for 12 h until a capacitance drop to less than 1.2 f / cm was observed . finally , the ssm - modified electrode was rinsed three times with buffer and used in the electrochemistry experiments . analogue cyclic voltammetry experiments were routinely carried out by holding the potential at 0.5 or 0.3 v for 5 s before cycling at a scan rate of 1 or 10 mv / s in the potential window from 0.5 or 0.3 to 0.4 v. after addition of cymasol , fcc3 , or hqno to the buffer , an incubation time of 30 min was allowed . where hqno was introduced in dmso , qcm - d was used to monitor the association of fcc3 with cyma - containing inner membrane architectures assembled on silicon - oxide surfaces ( figure 2a ) . the qcm - d technique gives simultaneous and real - time information about changes in adsorbed mass , through the resonant frequency ( f ) , and the rigidity or viscoelastic properties of the adsorbed layer through the dissipation ( d ) . ssm formation was confirmed by a drop in frequency of 2627 hz and a small change in dissipation ( figure 2a ; for clarity , the formation of the bilayer itself is not shown and the trace starts after the bilayer is formed ) . the frequency shift is very similar to values reported for the formation of phosphatidylcholine - only ssms under similar conditions . ( a ) qcm - d results of a sio2 surface in buffer , plotting ( black line , left axis ) frequency and ( red line , right axis ) dissipation against time . changes in the solution composition flowing over the ssm are indicated : ( fcc3 ) 5 m fcc3/1 mm fumarate ; ( wash 1 ) 1 mm fumarate ; ( wash 2 ) buffer only . ( c ) relative current measured from cvs as in ( b , trace c ) at 0.25 v vs she of the reductive scan as a function of mq-7 concentration in the ssm . mq-7 content is given in weight percentages relative to the lipid weight in the proteoliposomes . ( d ) qcm - d results of a sio2 surface in buffer , plotting ( black line , left axis ) frequency and ( red line , right axis ) dissipation against time . for clarity reasons , only the traces after the formation of the ssm are shown . changes in the solution composition flowing over the ssm are indicated : ( cymasol ) 0.1 m cymasol ; ( wash 1 ) , 1 mm fumarate ; ( fcc3 ) 5 m fcc3/1 mm fumarate ; ( wash 2 ) 1 mm fumarate . ( f ) relative current measured from cvs as in ( e , trace d ) at 0.25 v vs she of the reductive scan as a function of mq-7 concentration in the ssm . mq-7 content is given in weight percentages relative to the lipid weight in the liposomes . control experiments with ssms that did not contain cyma revealed that no fcc3 remained bound to the membrane after washing ( figure s1 in the supporting information ) . trans - membrane proteins are known to introduce defects into ssms , and although the qcm - d data indicate that the cyma - containing ssms are essentially homogeneous , it can not be excluded that a small number of defects cause some a - specific adsorption of fcc3 . thus , in terms of the molecular architecture of the ssm and its associated proteins , the qcm - d data do not allow for unambiguous interpretation of the ratio of bound fcc3 to cyma . furthermore , fcc3 is reported to be a monomer in solution , and it is unlikely that cyma forms complexes with fcc3 oligomers of the high order that is implied by the frequency shifts ( approximately six fcc3 per cyma ) . a more reasonable interpretation is that cyma forms complexes with fcc3 with a stoichiometry close to 1:1 ( or 1:2 as observed for nrfha ) , while additional fcc3 molecules are bound to defects in the ssm induced by the presence of cyma . previous electrophoresis of cyma - containing ssms equivalent to those studied here has shown that approximately one - half the cyma is immobile . one - half the population of cyma may present its head domain toward the silicon oxide surface leading to interactions that immobilize the protein , while the mobile population of cyma presents its head domain to the solution . to eliminate the possibility that this issue influences the qcm - d results , a series of experiments were performed with a truncated form of cyma that lacks the n - terminal transmembrane helix . on exposing the cymasol - containing ssms to fcc3 , followed by washing to remove loosely bound material , the frequency dropped by a further 18 hz this behavior is consistent with fcc3 binding tightly to cymasol - containing ssms and confirms the data obtained with the full - length cyma . in fact , assuming that cymasol has dimensions comparable to those of the nrfh head domain , the qcm - d data correspond to the formation of a close - packed monolayer of cymasol covered by another close - packed monolayer of fcc3 . in summary , the qcm - d is consistent with fcc3 binding strongly to ssms containing either cyma or cymasol , but not to ssms without cyma(sol ) , such that an inner membrane architecture may be present on the solid surfaces as shown schematically in figure 1b . formation of planar ssms was confirmed by electrochemical impedance spectroscopy , which showed a drop in capacitance to below 1.2 f / cm after addition of the proteoliposomes ( figure s2 in the supporting information ) . cyclic voltammograms ( cvs ) of the ssms containing cyma and mq-7 showed little change after exposure to fcc3 ( figure 2b ) . this confirmed that the catalytic response had its origin in fcc3 bound irreversibly to the cyma - containing ssm . to exclude the possibility that fcc3 received electrons directly from the electrode , or mq-7 , an inhibitor of cyma s quinol - dehydrogenase activity the addition of hqno inhibited catalytic fumarate reduction ( figure 2b ) , demonstrating that the electrons for fumarate reduction are indeed supplied to fcc3 from mq-7 oxidation by cyma as illustrated in figure 1 . the turnover frequency for mq-7 oxidation can be calculated if the current can be related to the number of catalytically active cyma - fcc3 complexes on the surface . from this , we calculate a minimum turnover number for mq-7 oxidation by the cyma - fcc3 complex of 40 s. the experiments described above reveal that the cyma - fcc3 complex is able to oxidize mq-7 in ssms . to establish whether fcc3 is responsible for the change in the catalytic bias of cyma , the activity of cyma in the membrane - modified electrodes was measured in the absence of fcc3 using chemical reductants and oxidants ( figure 3 ) . as a consequence , the absence of a reductive signal in the cv confirms that cyma without fcc3 is unable to oxidize mq-7 . the reduction potential of ferricyanide ( + 420 mv ) is higher than that of fumarate ( 30 mv ) , indicating that mq-7 oxidation by the cyma - fcc3 complex is not driven solely by thermodynamics . instead , formation of the long - lived cyma - fcc3 complex shifts the catalytic bias of cyma toward mq-7 oxidation . when , instead of ferricyanide , dithionite is added to the ssm , a catalytic oxidation signal appears that is inhibited by addition of hqno . this confirms our previous findings , that cyma is only able to reduce mq-7 in the absence of fcc3 . underpinning these capabilities is a conduit for electron transfer that extends from the quinol pool and cyma at the inner membrane to extracellular minerals and electrodes . however , cyma also distributes electrons from quinol oxidation to the terminal reductases for fumarate , nitrate , nitrite , and extracellular dmso . cyma thus plays a significant role in the highly branched network that supports anaerobic respiration in s. oneidensis mr-1 . our finding that complex formation between cyma and one of its partner proteins is required for cyma to act as a quinol dehydrogenase significantly advances our understanding of the factors that regulate the distribution of electrons across the respiratory network of s. oneidensis mr-1 . that cyma does not oxidize mq-7 in the absence of a partner protein will occlude et to nonphysiological redox partners and prevent side - reactions such as the production of radical oxygen species . because complex formation between cyma and each of its partner proteins is required to trigger mq-7 oxidation , respiratory electron flux should be dependent to some extent on the lifetime of the complexes formed by cyma . the cyma - stc et complex is believed to be transient because it has not been possible to cross - link these proteins . however , for cyma - fcc3 , evidence has been presented to support transient as well as long - lived et complexes . in contrast , ross and colleagues provided evidence for static cyma - fcc3 and cyma - mtrcab complexes in whole cells of s. oneidensis mr-1 . given the critical role of protein protein complex formation in activating the mq-7-dehydrogenase activity of cyma , it is now important to fully understand those factors that determine the lifetime of the complexes formed by cyma under cellular conditions . the electrochemical data indicate that the mq-7 oxidation activity of the cyma - fcc3 complex is at least 40 s. if we assume that cyma oxidizes mq-7 at similar rates in complex with its other redox partners , we anticipate that the electron flux through cyma will be sufficient to support anaerobic respiration of s. oneidensis mr-1 , including respiration on inorganic minerals and electrodes where a flux of 1 electron s per mtrc is reported . that the formation of a protein protein complex can influence the catalytic activity or bias of redox enzymes , or their subunits , adds an intriguing dimension to the mechanisms by which organisms regulate their et networks . the group of lger has recently suggested that in a multicentered redox enzyme , nife - hydrogenase , the forward ( h2-formation ) and reverse ( h2-oxidation ) reactions are limited by different rate - limiting steps and that this phenomena results in a tuning of catalytic bias , which can be altered by a series of mutations that do not alter the reduction potential of the active site . the reduction potentials of the hemes in cyma lie between 110 and 265 mv , which is lower than that of mq-7 ( 70 mv ) . this is also the case for other members of the napc / nirt superfamily . we have previously shown that menadione , which has a reduction potential equal to mq-7 , is unable to reduce the hemes in cyma . the results presented here extend this observation to the native substrate mq-7 and demonstrate that mq-7 oxidation in cyma is limited by et to the heme groups . fcc3 binding to cyma is unlikely to change the properties of the buried mq-7 active site , but will influence the properties of the surface - exposed hemes that are presented to the periplasm . consequently , we propose that mq-7 oxidation by cyma , and possibly all members of the napc / nirt superfamily , is regulated by an increase in the reduction potential of these hemes upon complex formation . an extreme example of the latter is the iron sulfur redox chain in complex i. in conclusion , we find that inner membrane architectures can be constructed on planar surfaces and that this provides a powerful approach to resolve protein complex formation and quinone oxidoreductase activity of membrane enzymes . our finding that protein protein interactions modulate the catalytic bias of cyma reveals a new mechanism by which the magnitude and direction of electron flux through respiratory et networks can be regulated . mq-7 oxidation is regulated by controlling the rate of et along the heme redox chain , which is rate limiting for cyma in isolation .

in the coming decades , the percentage of elderly people will rapidly increase , while the percentage of population of working age will decrease . although prolongation of life is a positive effect , the aging population is a challenge for health care systems . due to aging , the importance of cardiovascular disease ( cvd ) as the leading cause of death in adults becomes very important . according to the seventh report of the joint national committee on prevention , detection , evaluation and treatment of high blood pressure , hypertension occurs in more than two - thirds of individuals older than 50 years.1 polsenior study2 reveals that in population older than 80 years , hypertension affects about a million people . if the observed increase continues , the number of elderly hypertensive patients will increase by 50% by 2035.3 increase in prevalence of hypertension along with age is mainly caused by demographic changes . application of antihypertensive therapy in individuals older than 50 years reduces the risk of strokes , limits exacerbations of heart insufficiency , and lowers mortality due to cvds.3 in elderly people ( according to the world health organization [ who ] over the age of 65 years , while according to un over the age of 60 years ) , proper diagnosis and treatment of hypertension are difficult.4 older patients require careful pharmacotherapy with low doses of drugs and monotherapy , and in case of ineffectiveness , increase the dose or introduce another antihypertensive drug . patient adherence and regular drug intake are prerequisites for effective therapy , especially in older age.5 a degree of adherence depends on patients involvement in the therapeutic process , understanding of the therapeutic goals , and well - being during treatment . among the most frequent reasons for skipping doses of medication and non - compliance with the recommendations include : side effects , financial reasons , old age , comorbidities , reduced physical , and cognitive abilities , strength , and proper cognitive functioning ( frailty syndrome [ fs]).6 about 55% of elderly patients do not adhere to therapeutic plan.7 medication non - adherence is cited as the primary cause for lack of hypertension control.8 due to physiological changes progressing with age and comorbidities , treatment of hypertension in elderly patients is a challenge for the entire therapeutic team . what is characteristic in elderly patients is the occurrence of geriatric syndrome ( gs ) , which gradually leads to functional disability and lowers quality of life . gss , which commonly include frailty , dementia , delirium , incontinence , falls , and dizziness , are highly prevalent , multifactorial , and associated with substantial morbidity , hospitalization , and poor outcomes . understanding basic mechanisms involved in gss will be critical to advancing research and developing targeted therapeutic options , although given the complexity of these multifactorial conditions , attempts to define relevant mechanisms will need to incorporate more complex models , including a focus on synergistic interactions between different risk factors.9 it may be assumed that gs could modify the medication status of elderly hypertensive patients . in the recent years , it develops in 15%20% of patients older than 60 years and in 30% of patients older than 80 years.9 fs has been implicated as a causative and prognostic factor in patients with cvd . the american heart association and the society of geriatric cardiology have called for a better understanding of frailty as it pertains to cardiac care in the elderly . patients diagnosed with fs are at a higher risk of falling , decreased mobility , decreased ability to perform the basic activities of daily living , frequent hospitalizations , and death.10 in all , 25%50% of cardiac patients suffer from fs.9 some studies have also shown that the assessment of frailty can delay the development of disability and reduce the need for institutionalization and nursing care among elderly people living at home.11,12 chao et al demonstrated that the absence of frailty / pre - frailty and presence of polypharmacy were significantly associated with poorer medication adherence.13 research studies available indicate that despite the availability of effective medical therapy , over half of all hypertensives do not take any treatment and more than half of those on treatment have blood pressures over the 140/90 mmhg threshold.14 who describes poor adherence as the most important cause of uncontrolled blood pressure and estimates that 50%70% of people do not take their antihypertensive medication as prescribed.15 there is research available that emphasizes the negative influence of sociodemographic and clinical variables on adherence to treatment recommendations . medication adherence rates have been shown to be related to age , sex , race , geographical region of residence , and illness perceptions.16 the psychosocial factors that strongly correlate with non - adherence include depression , lack of social support , and low quality of life.17 only a few studies are available on the relation between the gs and medication adherence . they pertain to chronic diseases other than hypertension.13,18 there is a discussion in the research available on the relation between age and medication adherence . some studies have shown that younger patients display higher adherence,16,19,20 while others indicate that young age is a determinant of poor adherence to hypotensive medication.21,22 several authors report higher adherence in elderly patients,17,2325 which can be explained by the presence of comorbidities , making the patients perceive themselves as very ill and take the prescribed treatment seriously.22 considering the increasing age of patients with hypertension and contradictory study results regarding the effect of age on adherence , a special approach to their treatment is required with more attention paid to gs . the purposes of this study were to evaluate the influence of frailty as a component of gs on medication adherence among elderly patients with hypertension and to assess whether other factors ( especially polypharmacy as a component of gs ) influence adherence to treatment in this group of patients . data were collected between january 2015 and november 2015 . the study included consecutive 296 patients ( 131/45.3% men , 165/55.7% women ) with a mean age of 68.88.0 years diagnosed with hypertension . inclusion criteria were as follows : 1 ) clinically confirmed hypertension , 2 ) written informed consent , and 3 ) age 60 years . exclusion criteria were as follows : 1 ) moderate to severe dementia ( defined as mini - mental score 15),26 2 ) previous stroke , and 3 ) lack of consent to participation in the study . patients were selected by a panel consisting of a physician and a nurse specialist in the field of cardiac nursing . all patients received information about the purposes and nature of the study and provided written informed consent to participate in the study . sociodemographic data ( age , sex , education , and marital status ) and clinical data ( hypertension level according to the european society of cardiology , comorbidities , duration of hypertension , blood pressure monitoring , number and methods of taking medications , and method of non - pharmacological treatment ) were obtained from patients records and during personal interviews performed by a nurse . the spectrum of gs is expanding , and the definitions of each component are constantly evolving . in the present study , we included the following gs for analysis : frailty and polypharmacy , whose influence on the prognosis had already been documented.13,27 frailty was measured using the polish version of the tilburg frailty indicator ( tfi ) , which consists of two parts.28 one addresses sociodemographic characteristics of a participant ( sex , age , marital status , country of origin , educational level , and monthly income ) and other potential determinants of frailty ( lifestyle , multimorbidity , life events , and home living environment ) . the second part addresses components of frailty and comprises 15 self - reported questions , divided into three domains . the physical domain ( 08 points ) consists of eight questions related to physical health , unexplained weight loss , difficulty in walking , balance , hearing problems , vision problems , strength in hands , and physical tiredness . the psychological domain ( 04 points ) comprises four items related to cognition , depressive symptoms , anxiety , and coping . the social domain ( 03 points ) comprises three questions related to living alone , social relations , and social support . eleven items of part two of the tfi have two response categories ( yes and no ) , while the remaining items have three ( yes , no , and sometimes ) . yes or sometimes responses are scored 1 point each , while no responses are scored 0 . the instrument s total score may range from 0 to 15 ; the higher the score , the higher the patient s frailty . previous studies have suggested that the tfi is a valid and reliable instrument for measuring frailty.28,29 adherence to treatment was assessed using the polish version of 8-item morisky medication adherence scale ( mmas-8 ) . the self - reported mmas-8 tool is an 8-item tool , simple to administer , reliable , and economical for use in clinical practice . the mmas-8 was designed to facilitate the identification of barriers and behaviors associated with adherence to chronic medication . the tool has been determined to be reliable and significantly associated with blood pressure control in individuals with hypertension , as well as with antihypertensive medication pharmacy fill rates . scores on the mmas-8 range from 0 to 8 , with scores of less than 6 reflecting low adherence , 6 to < 8 reflecting medium , and 8 reflecting high adherence.3032 for the quantitative variables , verification of empirical normality distribution was tested by shapiro - wilk test . mean and standard deviations ( sds ) were calculated . for comparisons of means between the two groups of patients , one - way analysis of variance was used . in case of rejection of the null hypothesis of no differences in the groups , qualitative and ordinal variables were grouped in cross - tabulation table the number of cases was summed for each category and the percentage was calculated . independence of qualitative variables was verified by pearson chi - square test . for evaluation of the strength and significance of associations , spearman s rank correlation coefficient ( rho ) was used . to test the influence of independent variables on the level of adherence , linear regression was used ( general stepwise regression ) . prior to regression analysis , assumption of applicability of least squares method was checked as well as outliers were detected . for predictor variables ( frailty index and age ) , standardized coefficient and regression coefficient b were calculated . statistical significance of given variables in the model was tested by student s t - test . statistical analysis was carried out with computer package statistica software v.10 ( statsoft inc . , the study was approved by the bioethics committee of wroclaw medical university ( approval no kb 521/2014 ) and it conforms to the principles outlined in the declaration of helsinki . the analysis of fs with tilburg questionnaire for the entire group of 296 patients showed that the mean level of physical domain score was 3.31 ( sd = 2.21 ) , mean level of psychological domain was 1.79 ( sd = 1.07 ) , and mean level of social domain was 1.02 ( sd = 0.83 ) . the mean total tfi score was 6.11 ( sd = 3.12 ) with the range from 0 to 14 scores . on this basis , patients were classified into the following two groups : 1 ) frail ( tfi 5 ) , n=198 and 2 ) non - frail ( tfi < 5 ) , n=98 . tilburg questionnaire domain scores for patients from the frail group were statistically significantly higher compared to those from the non - frail group . patients from the frail group were older ( 70.08.2 vs 66.37.1 years ; p<0.001 ) and more often professionally inactive ( retired : 58.1% vs 48% ; disabled 17.7% vs 12.2% ; p=0.025 ) . majority of them were single / divorced / alone ( 43.9% vs 32.7% ; p=0.122 ) ; they were more often living alone ( 26.3% vs 9.2% ; p=0.004 ) . frail patients had lower household income per capita and almost 60% of them received less than 1,500 pln , while similar group of non - frail subjects ( 66% ) received more than 1,500 pln . frail patients are considerably more likely to experience a traumatic or stressful event such as serious illness ( 40.9% ) , and partner s severe disease ( 32.8% ) or partner s death ( 25.3% ) than non - frail subjects . patients with hypertension less often reported normal blood pressure values ( 5.6% vs 10.2% ) and majority of them were diagnosed with stage i ( 55% vs 39.8% ) or stage ii ( 27.3% vs 20.4% ; p=0.04 ) hypertension . frail patients prevailed in a group of 169 patients who were treated with monotherapy ( 61.2% vs 55.1% ; not significant ) . frail patients also prevailed in a group of 96 patients who were treated with several medicines at a time ( 37.9% vs 21.4% ; p=0.047 ) and considerably gave way to non - frail patients in a group of 31 patients who received polytherapy in one tablet ( 7.1% vs 17.4% ; p=0.021 ) . frail patients more often took beta - blockers than non - frail ones ( 30.8% vs 21.4% ) . in terms of other variables , the analysis of the level of adherence with mmas-8 questionnaire showed that frail subjects received lower score of adherence in comparison to non - frail subjects ( 6.601.89 vs 7.111.42 ; p=0.028 ) . in the frail group , 21% of patients had low level of adherence , while in the non - frail group , only 12.3% of studied subjects had low level of adherence ( table 3 ) . next , spearman s rank correlation coefficient was calculated in order to assess the influence of selected variables on the level of adherence ( mmas-8 ) ; the results are presented in table 4 . the analysis of correlations showed that some variables such as marital status ( rho = 0.132 ) , satisfaction with the home living environment ( rho = 0.169 ) , knowledge about complications ( rho = 0.116 ) , and treatment with angiotensin receptor antagonist ( rho = 0.120 ) had significant positive association with adherence , while living alone ( rho = 0.117 ) , traumatic / stressful event in the last year : death of a spouse ( rho = 0.138 ) , serious illness ( rho = 0.117 ) , serious illness in a spouse / partner ( rho = 0.178 ) , divorce ( rho = 0.140 ) , and traffic accident ( rho = 0.120 ) had negative association with adherence ( table 5 ) . spearman s rank correlation coefficients calculated for components of tilburg questionnaire showed that significant determinants with negative influence on the level of adherence ( mmas-8 ) were physical ( rho = 0.117 ) , psychological ( rho = 0.183 ) , and social domain ( rho = 0.163 ) of tilburg questionnaire as well as the total score of the questionnaire ( rho = 0.183 ) ( table 4 ) . as a result of the analysis of multiple regression , independent stimulants to the level of adherence such as knowledge about complications ( =0.395 ) and satisfaction with the home environment ( =0.897 ) were found , while it is surprising that none of the components of tilburg questionnaire was a statistically significant independent determinant lowering the level of adherence . the model appeared to be significant : f(4 ; 291 ) = 8.17 ; p<0.0001 . adherence = 6.2 + 0.39 knowledge about complications + 0.90 satisfaction with the home environment . poor adherence to prescribed medications reduces treatment benefits and can lower the clinician s assessment of therapeutic effectiveness . non - adherence is thought to account for 30%50% of treatment failures.33 non - adherence leads to worse medical treatment outcomes such as higher hospitalization rates , institutionalization for the frail elderly patients , and increased health care costs.3336 an improved understanding of the determinants associated with medication adherence and health behaviors has become an important outcome in management strategies for hypertension patients . effective identification of patients at risk of pharmacological non - adherence might be particularly helpful in planning interventions to enhance illness control , prevent complications , improve long - term treatment outcomes , and limit adverse outcomes in these patients.13,16,20 attention to adherence is especially important in frail elderly patients . there is an ongoing discussion in the literature on the influence of gs and/or its components on the level of adherence . in the present study , koizumi et al evaluated the association between hypertension and prevalence of fs and confirmed that impaired mobility ( inability to walk for more than 15 min without rest ) , weight loss of more than 23 kg in the past 6 months , difficulty in eating solid food , and limitations in performing complex activities of daily living were correlated with prevalence , treatment , and control of hypertension.37 in other studies on frailty , women were more frequently affected than man and age was found as determinant of frailty.38,39 this has not been confirmed in our study because the number of women was similar in both the groups ( frail vs non - frail ) . the lack of adherence may negatively affect the course of disease , especially in elderly patients . many factors can interfere with adherence in this group of patients , eg , cognitive impairment , lack of knowledge about illness and medication , and coexistence of frailty . talegawkar et al assessed the association between frailty and adherence to mediterranean diet and concluded that subjects without fs had higher level of adherence to diet recommendations than frailty patients.40 on the other hand , chao et al demonstrated that , contrary to our research results , in a group of chronically dialyzed patients , the absence of frailty / pre - frailty was significantly associated with poorer medication adherence.13 results obtained by chao et al are surprising as they defy the assumption that frailty negatively affects the level of adherence.13 however , the authors explain this fact with the old age of patients , which also in other studies appears as a well - established factor contributing to better adherence . additionally , elderly patients might be more concerned about their health and complexity of comorbidities , which make them more focused on adhering to complex therapy regimens.13 wu et al , in turn , believe that elderly patients with fs and cognitive disorders may not report the level of adherence as accurately as younger patients , which may be the cause of artificially high results.41 but the authors rightly stress that the connection between poor adherence to therapy and the level of cognitive function is not well documented in clinical practice , and contradictory results may stem from an insufficient representation of elderly patients with cognitive disorders in the majority of adherence studies42,43 and difficulty in differentiating between simple forgetfulness and cognitive disorders.44 other studies on patients with af demonstrated that cognitive disorders were a significant determinant of poorer adherence to pharmacological recommendations,18 and in the research by salas et al , cognitive function was an independent predictor of compliance with antihypertensive drugs in elderly patients who are living alone.45 in the present study , we proved that frailty negatively affects adherence in elderly hypertensive patients but only in the univariate analysis . to our best knowledge , studies on association between fs and adherence in patients with hypertension have not been conducted to date . medication adherence is considered essential for management of hypertension as is patient cooperation . in our study , we found that patients adherence was lower in the frail group . among factors that negatively affected adherence were loneliness and some frailty determinants according to tilburg scale such as being alone , death of the beloved one , serious illness , serious illness in a partner , and divorce or ending of a relationship during the past year . additionally in the study by talegawkar et al , the lack of adherence was related to decrease in physical activity and difficulty in walking , which is similar to our findings where impaired mobility and physical component of tilburg questionnaire negatively affected adherence.40 in the present study , coexistence of fs as measured by tilburg scale was the determinant of worse adherence . it is worth noting that in our study , the social , physical , and physical frailty components were also the determinants of lower adherence to antihypertensive treatment . regression analysis of answers from tilburg questionnaire showed that knowledge about complications of hypertension and satisfaction with the home environment are predictors of good adherence , while lack of support from others and loneliness are predictors of bad adherence . the role of social support in care of frail patients is therefore a crucial element in health care planning.46,47 in the studies from the literature , patients living with families had higher adherence than those living alone.34 also , elderly patients using the possibility of transportation as well as care and support from caregivers more often adhered to treatment recommendation than patient with limited support.48,49 other authors have reported that low level of social support is associated with increased risk for fs.50,51 also , low socioeconomic status contributes to development of frailty.50,52 moreira et al reported significant association between marital status and development of frailty ; in their research , single men and widowers had increased risk for fs.53 in the present study , satisfaction with the home environment and living in a relationship stimulated better adherence . reports from the literature show outcomes on positive effect of social support on the course of the disease and recovery in cardiac patients.54 support experienced by patients accelerates the recovery process and therefore becomes an additional help , increasing natural forces in the fight against diseases and enables them to recover to full health or live with a disease . in the meta - analysis performed by scheurer et al , similar to our finding , positive role of social support in adherence was documented.55 in other studies , the level of adherence was considerably higher among patients with social support and living in supportive families , while it was considerably lower in patients from conflicted families . outcomes of research by dimatteo et al correspond with our results and reveal that marriage or living in a partnership is a factor improving adherence.34 one of the components of gs is polypharmacy . in our study , we evaluated the relation between polypharmacy and adherence . similarly to our previous research,16 we did not observe any correlation in this respect . however , there are authors who have demonstrated that polypharmacy is an important barrier to adherence . in the present study , an independent predictor of good adherence was knowledge about complications of untreated hypertension . systematic identification of adherence determinants , including frailty , is important in terms of risk stratification and necessity of introducing multidisciplinary interventions to prevent symptoms of frailty . higher level of frailty among elderly patients may be considered as a determinant of lower adherence . however , social support and knowledge about complications of untreated hypertension are the most important independent determinants of adherence to pharmacological treatment . potential barriers to adherence should be assessed and addressed , with a particular reference to engagement of social support systems . understanding the concept of frailty may help to optimize medication prescribed for older people and target health care resources more effectively and appropriately , improving the care of all older people with hypertension . when frailty can be measured with precision , we can start to explore which interventions are the most beneficial for patients according to their different levels of resilience . the analysis of treatment of other chronic diseases apart from hypertension as well as possible polypharmacy , drug interactions , and side effects was not included ; however , these factors may also contribute to the level of adherence and severity of frailty among elderly patients with hypertension .

backgroundhypertension affects about 80% of people older than 80 years ; however , diagnosis and treatment are difficult because about 55% of them do not adhere to treatment recommendations due to low socioeconomic status , comorbidities , age , physical limitations , and frailty syndrome.aimsthe purposes of this study were to evaluate the influence of frailty on medication adherence among elderly hypertensive patients and to assess whether other factors influence adherence in this group of patients.methods and resultsthe study included 296 patients ( mean age 68.88.0 ) divided into frail ( n=198 ) and non - frail ( n=98 ) groups . the polish versions of the tilburg frailty indicator ( tfi ) for frailty assessment and 8-item morisky medication adherence scale for adherence assessment were used . the frail patients had lower medication adherence in comparison to the non - frail subjects ( 6.601.89 vs 7.111.42 ; p=0.028 ) . spearman s rank correlation coefficients showed that significant determinants with negative influence on the level of adherence were physical ( rho = 0.117 ) , psychological ( rho = 0.183 ) , and social domain ( rho = 0.163 ) of tfi as well as the total score of the questionnaire ( rho = 0.183 ) . however , multiple regression analysis revealed that only knowledge about complications of untreated hypertension ( =0.395 ) and satisfaction with the home environment ( =0.897 ) were found to be independent stimulants of adherence level.conclusionfrailty is highly prevalent among elderly hypertensive patients . higher level of frailty among elderly patients can be considered as a determinant of lower adherence . however , social support and knowledge about complications of untreated hypertension are the most important independent determinants of adherence to pharmacological treatment .

Introduction Methods Instruments Statistical analysis Results Sociodemographic characteristics of patients with hypertension with respect to FS Clinical characteristic of patients with hypertension with respect to FS Analysis of the level of adherence in relation to FS Univariate analysis the impact of the studied factors on the level of adherence (MMAS-8 score) Spearmans rank correlation coefficients of analyzed component of Tilburg questionnaire with the level of adherence Discussion Conclusion Implications for practice Study limitations

due to aging , the importance of cardiovascular disease ( cvd ) as the leading cause of death in adults becomes very important . according to the seventh report of the joint national committee on prevention , detection , evaluation and treatment of high blood pressure , hypertension occurs in more than two - thirds of individuals older than 50 years.1 polsenior study2 reveals that in population older than 80 years , hypertension affects about a million people . if the observed increase continues , the number of elderly hypertensive patients will increase by 50% by 2035.3 increase in prevalence of hypertension along with age is mainly caused by demographic changes . application of antihypertensive therapy in individuals older than 50 years reduces the risk of strokes , limits exacerbations of heart insufficiency , and lowers mortality due to cvds.3 in elderly people ( according to the world health organization [ who ] over the age of 65 years , while according to un over the age of 60 years ) , proper diagnosis and treatment of hypertension are difficult.4 older patients require careful pharmacotherapy with low doses of drugs and monotherapy , and in case of ineffectiveness , increase the dose or introduce another antihypertensive drug . patient adherence and regular drug intake are prerequisites for effective therapy , especially in older age.5 a degree of adherence depends on patients involvement in the therapeutic process , understanding of the therapeutic goals , and well - being during treatment . among the most frequent reasons for skipping doses of medication and non - compliance with the recommendations include : side effects , financial reasons , old age , comorbidities , reduced physical , and cognitive abilities , strength , and proper cognitive functioning ( frailty syndrome [ fs]).6 about 55% of elderly patients do not adhere to therapeutic plan.7 medication non - adherence is cited as the primary cause for lack of hypertension control.8 due to physiological changes progressing with age and comorbidities , treatment of hypertension in elderly patients is a challenge for the entire therapeutic team . what is characteristic in elderly patients is the occurrence of geriatric syndrome ( gs ) , which gradually leads to functional disability and lowers quality of life . gss , which commonly include frailty , dementia , delirium , incontinence , falls , and dizziness , are highly prevalent , multifactorial , and associated with substantial morbidity , hospitalization , and poor outcomes . understanding basic mechanisms involved in gss will be critical to advancing research and developing targeted therapeutic options , although given the complexity of these multifactorial conditions , attempts to define relevant mechanisms will need to incorporate more complex models , including a focus on synergistic interactions between different risk factors.9 it may be assumed that gs could modify the medication status of elderly hypertensive patients . in the recent years , it develops in 15%20% of patients older than 60 years and in 30% of patients older than 80 years.9 fs has been implicated as a causative and prognostic factor in patients with cvd . patients diagnosed with fs are at a higher risk of falling , decreased mobility , decreased ability to perform the basic activities of daily living , frequent hospitalizations , and death.10 in all , 25%50% of cardiac patients suffer from fs.9 some studies have also shown that the assessment of frailty can delay the development of disability and reduce the need for institutionalization and nursing care among elderly people living at home.11,12 chao et al demonstrated that the absence of frailty / pre - frailty and presence of polypharmacy were significantly associated with poorer medication adherence.13 research studies available indicate that despite the availability of effective medical therapy , over half of all hypertensives do not take any treatment and more than half of those on treatment have blood pressures over the 140/90 mmhg threshold.14 who describes poor adherence as the most important cause of uncontrolled blood pressure and estimates that 50%70% of people do not take their antihypertensive medication as prescribed.15 there is research available that emphasizes the negative influence of sociodemographic and clinical variables on adherence to treatment recommendations . medication adherence rates have been shown to be related to age , sex , race , geographical region of residence , and illness perceptions.16 the psychosocial factors that strongly correlate with non - adherence include depression , lack of social support , and low quality of life.17 only a few studies are available on the relation between the gs and medication adherence . they pertain to chronic diseases other than hypertension.13,18 there is a discussion in the research available on the relation between age and medication adherence . some studies have shown that younger patients display higher adherence,16,19,20 while others indicate that young age is a determinant of poor adherence to hypotensive medication.21,22 several authors report higher adherence in elderly patients,17,2325 which can be explained by the presence of comorbidities , making the patients perceive themselves as very ill and take the prescribed treatment seriously.22 considering the increasing age of patients with hypertension and contradictory study results regarding the effect of age on adherence , a special approach to their treatment is required with more attention paid to gs . the purposes of this study were to evaluate the influence of frailty as a component of gs on medication adherence among elderly patients with hypertension and to assess whether other factors ( especially polypharmacy as a component of gs ) influence adherence to treatment in this group of patients . the study included consecutive 296 patients ( 131/45.3% men , 165/55.7% women ) with a mean age of 68.88.0 years diagnosed with hypertension . sociodemographic data ( age , sex , education , and marital status ) and clinical data ( hypertension level according to the european society of cardiology , comorbidities , duration of hypertension , blood pressure monitoring , number and methods of taking medications , and method of non - pharmacological treatment ) were obtained from patients records and during personal interviews performed by a nurse . in the present study , we included the following gs for analysis : frailty and polypharmacy , whose influence on the prognosis had already been documented.13,27 frailty was measured using the polish version of the tilburg frailty indicator ( tfi ) , which consists of two parts.28 one addresses sociodemographic characteristics of a participant ( sex , age , marital status , country of origin , educational level , and monthly income ) and other potential determinants of frailty ( lifestyle , multimorbidity , life events , and home living environment ) . the second part addresses components of frailty and comprises 15 self - reported questions , divided into three domains . the physical domain ( 08 points ) consists of eight questions related to physical health , unexplained weight loss , difficulty in walking , balance , hearing problems , vision problems , strength in hands , and physical tiredness . the social domain ( 03 points ) comprises three questions related to living alone , social relations , and social support . eleven items of part two of the tfi have two response categories ( yes and no ) , while the remaining items have three ( yes , no , and sometimes ) . previous studies have suggested that the tfi is a valid and reliable instrument for measuring frailty.28,29 adherence to treatment was assessed using the polish version of 8-item morisky medication adherence scale ( mmas-8 ) . the tool has been determined to be reliable and significantly associated with blood pressure control in individuals with hypertension , as well as with antihypertensive medication pharmacy fill rates . scores on the mmas-8 range from 0 to 8 , with scores of less than 6 reflecting low adherence , 6 to < 8 reflecting medium , and 8 reflecting high adherence.3032 for the quantitative variables , verification of empirical normality distribution was tested by shapiro - wilk test . for evaluation of the strength and significance of associations , spearman s rank correlation coefficient ( rho ) was used . to test the influence of independent variables on the level of adherence , linear regression was used ( general stepwise regression ) . prior to regression analysis , assumption of applicability of least squares method was checked as well as outliers were detected . , the study was approved by the bioethics committee of wroclaw medical university ( approval no kb 521/2014 ) and it conforms to the principles outlined in the declaration of helsinki . the analysis of fs with tilburg questionnaire for the entire group of 296 patients showed that the mean level of physical domain score was 3.31 ( sd = 2.21 ) , mean level of psychological domain was 1.79 ( sd = 1.07 ) , and mean level of social domain was 1.02 ( sd = 0.83 ) . on this basis , patients were classified into the following two groups : 1 ) frail ( tfi 5 ) , n=198 and 2 ) non - frail ( tfi < 5 ) , n=98 . tilburg questionnaire domain scores for patients from the frail group were statistically significantly higher compared to those from the non - frail group . patients from the frail group were older ( 70.08.2 vs 66.37.1 years ; p<0.001 ) and more often professionally inactive ( retired : 58.1% vs 48% ; disabled 17.7% vs 12.2% ; p=0.025 ) . majority of them were single / divorced / alone ( 43.9% vs 32.7% ; p=0.122 ) ; they were more often living alone ( 26.3% vs 9.2% ; p=0.004 ) . frail patients had lower household income per capita and almost 60% of them received less than 1,500 pln , while similar group of non - frail subjects ( 66% ) received more than 1,500 pln . frail patients are considerably more likely to experience a traumatic or stressful event such as serious illness ( 40.9% ) , and partner s severe disease ( 32.8% ) or partner s death ( 25.3% ) than non - frail subjects . patients with hypertension less often reported normal blood pressure values ( 5.6% vs 10.2% ) and majority of them were diagnosed with stage i ( 55% vs 39.8% ) or stage ii ( 27.3% vs 20.4% ; p=0.04 ) hypertension . frail patients prevailed in a group of 169 patients who were treated with monotherapy ( 61.2% vs 55.1% ; not significant ) . frail patients also prevailed in a group of 96 patients who were treated with several medicines at a time ( 37.9% vs 21.4% ; p=0.047 ) and considerably gave way to non - frail patients in a group of 31 patients who received polytherapy in one tablet ( 7.1% vs 17.4% ; p=0.021 ) . frail patients more often took beta - blockers than non - frail ones ( 30.8% vs 21.4% ) . in terms of other variables , the analysis of the level of adherence with mmas-8 questionnaire showed that frail subjects received lower score of adherence in comparison to non - frail subjects ( 6.601.89 vs 7.111.42 ; p=0.028 ) . in the frail group , 21% of patients had low level of adherence , while in the non - frail group , only 12.3% of studied subjects had low level of adherence ( table 3 ) . next , spearman s rank correlation coefficient was calculated in order to assess the influence of selected variables on the level of adherence ( mmas-8 ) ; the results are presented in table 4 . the analysis of correlations showed that some variables such as marital status ( rho = 0.132 ) , satisfaction with the home living environment ( rho = 0.169 ) , knowledge about complications ( rho = 0.116 ) , and treatment with angiotensin receptor antagonist ( rho = 0.120 ) had significant positive association with adherence , while living alone ( rho = 0.117 ) , traumatic / stressful event in the last year : death of a spouse ( rho = 0.138 ) , serious illness ( rho = 0.117 ) , serious illness in a spouse / partner ( rho = 0.178 ) , divorce ( rho = 0.140 ) , and traffic accident ( rho = 0.120 ) had negative association with adherence ( table 5 ) . spearman s rank correlation coefficients calculated for components of tilburg questionnaire showed that significant determinants with negative influence on the level of adherence ( mmas-8 ) were physical ( rho = 0.117 ) , psychological ( rho = 0.183 ) , and social domain ( rho = 0.163 ) of tilburg questionnaire as well as the total score of the questionnaire ( rho = 0.183 ) ( table 4 ) . as a result of the analysis of multiple regression , independent stimulants to the level of adherence such as knowledge about complications ( =0.395 ) and satisfaction with the home environment ( =0.897 ) were found , while it is surprising that none of the components of tilburg questionnaire was a statistically significant independent determinant lowering the level of adherence . adherence = 6.2 + 0.39 knowledge about complications + 0.90 satisfaction with the home environment . non - adherence is thought to account for 30%50% of treatment failures.33 non - adherence leads to worse medical treatment outcomes such as higher hospitalization rates , institutionalization for the frail elderly patients , and increased health care costs.3336 an improved understanding of the determinants associated with medication adherence and health behaviors has become an important outcome in management strategies for hypertension patients . effective identification of patients at risk of pharmacological non - adherence might be particularly helpful in planning interventions to enhance illness control , prevent complications , improve long - term treatment outcomes , and limit adverse outcomes in these patients.13,16,20 attention to adherence is especially important in frail elderly patients . there is an ongoing discussion in the literature on the influence of gs and/or its components on the level of adherence . in the present study , koizumi et al evaluated the association between hypertension and prevalence of fs and confirmed that impaired mobility ( inability to walk for more than 15 min without rest ) , weight loss of more than 23 kg in the past 6 months , difficulty in eating solid food , and limitations in performing complex activities of daily living were correlated with prevalence , treatment , and control of hypertension.37 in other studies on frailty , women were more frequently affected than man and age was found as determinant of frailty.38,39 this has not been confirmed in our study because the number of women was similar in both the groups ( frail vs non - frail ) . the lack of adherence may negatively affect the course of disease , especially in elderly patients . many factors can interfere with adherence in this group of patients , eg , cognitive impairment , lack of knowledge about illness and medication , and coexistence of frailty . talegawkar et al assessed the association between frailty and adherence to mediterranean diet and concluded that subjects without fs had higher level of adherence to diet recommendations than frailty patients.40 on the other hand , chao et al demonstrated that , contrary to our research results , in a group of chronically dialyzed patients , the absence of frailty / pre - frailty was significantly associated with poorer medication adherence.13 results obtained by chao et al are surprising as they defy the assumption that frailty negatively affects the level of adherence.13 however , the authors explain this fact with the old age of patients , which also in other studies appears as a well - established factor contributing to better adherence . additionally , elderly patients might be more concerned about their health and complexity of comorbidities , which make them more focused on adhering to complex therapy regimens.13 wu et al , in turn , believe that elderly patients with fs and cognitive disorders may not report the level of adherence as accurately as younger patients , which may be the cause of artificially high results.41 but the authors rightly stress that the connection between poor adherence to therapy and the level of cognitive function is not well documented in clinical practice , and contradictory results may stem from an insufficient representation of elderly patients with cognitive disorders in the majority of adherence studies42,43 and difficulty in differentiating between simple forgetfulness and cognitive disorders.44 other studies on patients with af demonstrated that cognitive disorders were a significant determinant of poorer adherence to pharmacological recommendations,18 and in the research by salas et al , cognitive function was an independent predictor of compliance with antihypertensive drugs in elderly patients who are living alone.45 in the present study , we proved that frailty negatively affects adherence in elderly hypertensive patients but only in the univariate analysis . among factors that negatively affected adherence were loneliness and some frailty determinants according to tilburg scale such as being alone , death of the beloved one , serious illness , serious illness in a partner , and divorce or ending of a relationship during the past year . additionally in the study by talegawkar et al , the lack of adherence was related to decrease in physical activity and difficulty in walking , which is similar to our findings where impaired mobility and physical component of tilburg questionnaire negatively affected adherence.40 in the present study , coexistence of fs as measured by tilburg scale was the determinant of worse adherence . it is worth noting that in our study , the social , physical , and physical frailty components were also the determinants of lower adherence to antihypertensive treatment . regression analysis of answers from tilburg questionnaire showed that knowledge about complications of hypertension and satisfaction with the home environment are predictors of good adherence , while lack of support from others and loneliness are predictors of bad adherence . the role of social support in care of frail patients is therefore a crucial element in health care planning.46,47 in the studies from the literature , patients living with families had higher adherence than those living alone.34 also , elderly patients using the possibility of transportation as well as care and support from caregivers more often adhered to treatment recommendation than patient with limited support.48,49 other authors have reported that low level of social support is associated with increased risk for fs.50,51 also , low socioeconomic status contributes to development of frailty.50,52 moreira et al reported significant association between marital status and development of frailty ; in their research , single men and widowers had increased risk for fs.53 in the present study , satisfaction with the home environment and living in a relationship stimulated better adherence . reports from the literature show outcomes on positive effect of social support on the course of the disease and recovery in cardiac patients.54 support experienced by patients accelerates the recovery process and therefore becomes an additional help , increasing natural forces in the fight against diseases and enables them to recover to full health or live with a disease . in the meta - analysis performed by scheurer et al , similar to our finding , positive role of social support in adherence was documented.55 in other studies , the level of adherence was considerably higher among patients with social support and living in supportive families , while it was considerably lower in patients from conflicted families . in the present study , an independent predictor of good adherence was knowledge about complications of untreated hypertension . systematic identification of adherence determinants , including frailty , is important in terms of risk stratification and necessity of introducing multidisciplinary interventions to prevent symptoms of frailty . higher level of frailty among elderly patients may be considered as a determinant of lower adherence . however , social support and knowledge about complications of untreated hypertension are the most important independent determinants of adherence to pharmacological treatment . when frailty can be measured with precision , we can start to explore which interventions are the most beneficial for patients according to their different levels of resilience . the analysis of treatment of other chronic diseases apart from hypertension as well as possible polypharmacy , drug interactions , and side effects was not included ; however , these factors may also contribute to the level of adherence and severity of frailty among elderly patients with hypertension .

the online version of this article ( doi:10.1007/s10875 - 012 - 9775-z ) contains supplementary material , which is available to authorized users . primary immunodeficiencies ( pids ) are a heterogeneous group of inherited disorders of the immune system leading to enhanced susceptibility to infections . the complement system is a crucial component of innate immunity and one of the main effector mechanisms of antibody - mediated immunity ( reviewed in ) . inherited complement deficiencies represent immunodeficiencies characterized by susceptibility to invasive infections by encapsulated bacteria such as streptococcus pneumoniae ( reviewed in [ 35 ] ) . the third component of the complement system ( c3 ) is indispensable to all the known pathways of complement activation . c3 deficiency ( omim : 120700 ) is a rare pid , leading to predisposition to recurrent pyogenic infections [ 1 , 4 ] . a few biallelic defects in the c3 gene have been described in patients suffering not only from s. pneumoniae infections [ 612 ] but also from autoimmune and immune - complex - related disorders , in particular affecting the kidney [ 1315 ] . a similar phenotype can also be observed in patients with deficiency of complement factor h or i , respectively . here , we describe a patient with selective immunoglobulin a ( iga ) deficiency presenting with recurrent airway infections caused by s. pneumoniae and bronchiectasis with no autoimmune or immune complex manifestations . our molecular analyses revealed that the patient suffers from c3 deficiency caused by a novel , homozygous mutation in the c3 gene . this study has been approved by the ethics committee at the medical university of vienna , austria . the patient and the other family members gave informed consent to the genetic analysis described here . clinical data from the patients were provided in anonymized form by the responsible physician(s ) . measurement of capsular polysaccharide serotype - specific immunoglobulin g ( igg ) antibodies against s. pneumoniae was performed by enzyme - linked immunosorbent assay ( elisa ) in serum samples before and 6 weeks after vaccination with pneumo 23 ( unconjugated polysaccharide vaccine against s. pneumoniae ) as described previously with minor modifications . all sera were analyzed in duplicates in the same elisa run . in order to eliminate antibodies to cell wall polysaccharides , microtiter plates were coated with capsular polysaccharide antigens ( from the american type culture collection ( atcc ) , rockville , md ) and the samples were pre - incubated overnight with species - specific common cell wall polysaccharide from s. pneumoniae ( cwps ; c - polysaccharide purified ; statens serum institute , denmark ) . antibody concentrations are indicated as the percentage of reference serum , the hyperimmune plasma pool ( u.s . pneumococcal reference serum fda7 cber , bethesda , md ) in units per milliliter ( u / ml ) , where the reference plasma pool represents 100 u / ml for each serotype . since patients with high pre - immunization titers may not generate a drastic increase after immunization , the final concentration of antibodies after immunization ( regardless of increase from pre - immunization concentration ) was taken into account . a minimal concentration of 20 u / ml in at least 50 % of the serotypes tested was considered as a positive response to the vaccination . this criterion was selected according to results obtained in 40 healthy turkish children ( age range : 5 to 15 years ; median : 10 years and mean , 9.7 years ) ( o. sanal , unpublished data ) . genomic dna was isolated from whole blood obtained from the patient and parents using a commercially available kit ( wizard genomic dna purification kit , promega corporation ) according to the manufacturer s instructions . the primers used for sequencing of the c3 gene were previously described by goldberg et al . with one additional pair covering part of exon 41 and the 3utr . this additional pair has the following sequences : forward 5-ctcagctacatcatcgggaag-3 and reverse 5-ccttggctaaagaagtcagca-3. all primers were purchased from sigma aldrich , austria . capillary sequencing was performed with the big dye terminator v3.1 cycle sequencing kit ( applied biosystems , germany ) and analyzed on a 3130 l genetic analyzer ( applied biosystems ) . for sequence analysis , the nucleotide variations found were further sequenced on both parents in order to evaluate the segregation . polyphen2 ( polymorphism phenotyping v2 , http://genetics.bwh.harvard.edu/pph2/ ) and sift ( j. craig venter institute , http://sift.jcvi.org/ ) were used to predict the effect of the mutation on protein function . phylogenetic conservation was assessed using protein sequences from ensembl ( http://www.ensembl.org ) and uniprot ( http://www.uniprot.org/ ) and aligned using uniprot multiple sequence alignment tool . for the protein modeling , we used molsoft icm browser pro software and a crystal structure model of the c3 convertase ( 2win ) from the protein database website ( http://www.rcsb.org/pdb/ ) . this study has been approved by the ethics committee at the medical university of vienna , austria . the patient and the other family members gave informed consent to the genetic analysis described here . clinical data from the patients were provided in anonymized form by the responsible physician(s ) . measurement of capsular polysaccharide serotype - specific immunoglobulin g ( igg ) antibodies against s. pneumoniae was performed by enzyme - linked immunosorbent assay ( elisa ) in serum samples before and 6 weeks after vaccination with pneumo 23 ( unconjugated polysaccharide vaccine against s. pneumoniae ) as described previously with minor modifications . all sera were analyzed in duplicates in the same elisa run . in order to eliminate antibodies to cell wall polysaccharides , microtiter plates were coated with capsular polysaccharide antigens ( from the american type culture collection ( atcc ) , rockville , md ) and the samples were pre - incubated overnight with species - specific common cell wall polysaccharide from s. pneumoniae ( cwps ; c - polysaccharide purified ; statens serum institute , denmark ) . antibody concentrations are indicated as the percentage of reference serum , the hyperimmune plasma pool ( u.s . pneumococcal reference serum fda7 cber , bethesda , md ) in units per milliliter ( u / ml ) , where the reference plasma pool represents 100 u / ml for each serotype . since patients with high pre - immunization titers may not generate a drastic increase after immunization , the final concentration of antibodies after immunization ( regardless of increase from pre - immunization concentration ) was taken into account . a minimal concentration of 20 u / ml in at least 50 % of the serotypes tested was considered as a positive response to the vaccination . this criterion was selected according to results obtained in 40 healthy turkish children ( age range : 5 to 15 years ; median : 10 years and mean , 9.7 years ) ( o. sanal , unpublished data ) . genomic dna was isolated from whole blood obtained from the patient and parents using a commercially available kit ( wizard genomic dna purification kit , promega corporation ) according to the manufacturer s instructions . the primers used for sequencing of the c3 gene were previously described by goldberg et al . with one additional pair covering part of exon 41 and the 3utr . this additional pair has the following sequences : forward 5-ctcagctacatcatcgggaag-3 and reverse 5-ccttggctaaagaagtcagca-3. all primers were purchased from sigma aldrich , austria . capillary sequencing was performed with the big dye terminator v3.1 cycle sequencing kit ( applied biosystems , germany ) and analyzed on a 3130 l genetic analyzer ( applied biosystems ) . for sequence analysis , the nucleotide variations found were further sequenced on both parents in order to evaluate the segregation . polyphen2 ( polymorphism phenotyping v2 , http://genetics.bwh.harvard.edu/pph2/ ) and sift ( j. craig venter institute , http://sift.jcvi.org/ ) were used to predict the effect of the mutation on protein function . phylogenetic conservation was assessed using protein sequences from ensembl ( http://www.ensembl.org ) and uniprot ( http://www.uniprot.org/ ) and aligned using uniprot multiple sequence alignment tool . for the protein modeling , we used molsoft icm browser pro software and a crystal structure model of the c3 convertase ( 2win ) from the protein database website ( http://www.rcsb.org/pdb/ ) . at the age of 16 years , a male turkish patient born to consanguineous parents ( first - degree cousins ) as the third of eight children , was admitted to hospital with a history of fever , cough and respiratory distress for 48 h. physical examination revealed weight and height within normal range , a wound scar resulting from a thoracotomy performed at the age of 6 months , and crackles and bronchial respiratory sounds . a chest x - ray revealed pneumonia ( fig 1lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung with regards to the past medical history , the patient had needed a tube thoracostomy due to pleural effusion at the age of 6 months and had suffered from recurrent lower respiratory infections with a frequency of 4 to 5 times per year . at the age of 8 years , subsequently , the frequency of infections was reduced however , in a period of 6 years ( from 14 to 20 years ) he still presented with two episodes of otitis media and 8 episodes of lower respiratory tract infections for which hospitalization was necessary . at the age of 20 years , the patient was vaccinated for s. pneumoniae and has not suffered from severe infections or needed hospitalization since ( 7 years of follow up so far ) . the patient has never presented with hematuria , hypertension or other clinical feature indicative of renal involvement or autoimmune disorder . notably , two of his brothers had died during the neonatal period for unknown reasons . his parents , three brothers and two sisters are healthy ( see fig . 2a and supplementary figure 1 for pedigrees).fig . 2pedigree and genetic analysis of the core family ( 2a ) and phylogenetic conservation of the cysteine 1518 in c3 ( 2b ) . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . females are represented as circles and males as squares . filled and half - filled symbols represent homozygous and heterozygous individuals , respectively . cysteine 1518 is indicated with a red box pedigree and genetic analysis of the core family ( 2a ) and phylogenetic conservation of the cysteine 1518 in c3 ( 2b ) . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . cysteine 1518 is indicated with a red box the patient had normal red cell counts but elevated erythrocyte sedimentation rate ( 52 mm / h ; reference values : 015 mm / h ) and c - reactive protein levels ( 66 mg / dl ; reference values : 8.510.6 mg / dl ) , respectively . total leukocyte ( 7500/mm ) and lymphocyte counts ( 3500/mm ) were within the normal range . routine urine analysis and renal function were unremarkable ( see table 1 for blood urea nitrogen and creatinine levels ; urine analysis data are not shown ) . immunoglobulin m , g and e levels were within the normal range as well as the igg subclasses , while serum iga levels were reduced ( table 1 ) . as there were no signs or symptoms of autoimmunity , autoantibody levels were not evaluated.table 1description of the laboratory findings of the patient and parentspatientfathermotherreference valuesblood urea nitrogen ( mg / dl)262040creatinine ( mg / dl)0,80,61,2ch50 ( u / ml)07171>15c3 levels ( mg / dl)819637190180c4 levels ( mg / dl)201040iga ( mg / dl)<5.823034144244igg ( mg / dl)18116402010igg11220315855igg266864495igg312523196igg42511157igm ( mg / dl)8652297ige ( iu / ml)460100anti - a titer1/641/10anti - b titer1/1281/10tuberculin test ( mm)10510 in vitro pha ( % ) 6165.8 9.2unstimulated nbt ( % ) 50038stimulated nbt ( % ) 706090total lymphocyte counts ( cell / mm)350017005700 t lymphocytes ( cd3 + ) ( % ) 685579(cells / mm)204011004100 t helper cells ( cd4 + ) ( % ) 382851(cells / mm)11406002400cytotoxic t cells ( cd8 + ) ( % ) 261642(cells / mm)7804001500natural killer cells ( cd16 + 56 + ) ( % ) 7528(cells / mm)2102001000b lymphocytes ( cd19 + ) ( % ) 201028(cells / mm)6002001400total hemolytic complement activity test . in vitro lymphocyte stimulation with 20 g / ml of phytohemagglutinin for 72 h. the values refer to the percentages of the blastic transformation of lymphocytes ( enlarged nucleus , condensed chromatin and/or with pores in the cytoplasm ) evaluated using light microscopy . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activitypatient examination at the age of 16 yearsnormal values in age - matched turkish subjects description of the laboratory findings of the patient and parents total hemolytic complement activity test . in vitro lymphocyte stimulation with 20 g / ml of phytohemagglutinin for 72 h. the values refer to the percentages of the blastic transformation of lymphocytes ( enlarged nucleus , condensed chromatin and/or with pores in the cytoplasm ) evaluated using light microscopy . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activity patient examination at the age of 16 years normal values in age - matched turkish subjects six weeks after vaccination for s. pneumoniae ( 20 years of age ) , the patient demonstrated a positive antibody response ( table 2).table 2specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / mlserotypes36b1419f23f7fbefore vaccination2216321916186 weeks after vaccination29>10046656820 specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / ml c3 levels were severely decreased in the patient , varying from 8 to 19 mg / dl at 6 different measurements ( table 1 ; reference values : 90180 mg / dl ) , with absent complement hemolytic activity measured using the ch50 test . the other complement levels ( c1 , c2 , c4 , c5 , c6 , c7 , c8 , c9 , factor h and factor i ) were found to be normal ( see table 1 for c4 ; other data not shown ) . the proliferative response of peripheral blood lymphocytes to phytohemagglutinin and the spontaneous and stimulated nitroblue tetrazolium test were normal ( table 1 ) . the parents showed normal levels of iga and normal ch50 , but reduced c3 levels ( 63 mg / dl and 71 mg / dl ) , respectively . details of the laboratory findings of the patient and his parents are shown in table 1 . sanger sequencing identified a homozygous missense mutation in exon 38 ( c. c4554 g , p. cys1518trp ) . molecular segregation analysis showed perfect segregation , with both parents and three siblings being carriers of one affected allele ( fig . 2a and supplementary figure 1 ) . to date , this variant has neither been annotated as a polymorphism in ncbi ( http://www.ncbi.nlm.nih.gov/ ) , nor in ucsc genomic bioinformatics site ( http://genome.ucsc.edu/ ) or ensembl ( http://www.ensembl.org ) , respectively . polyphen2 and sorting intolerant from tolerant ( sift ) analysis predict that the substitution of cysteine to tryptophan at position 1518 of c3 is very likely to affect protein function , with the maximum score in both analyses ( 1.000 in polyphen2 and 0.000 in sift , respectively ) . as indicated in fig . 2b , the cysteine residue at position 1518 is conserved among vertebrates and it has been shown to form a disulfide bridge with cysteine 1590 [ 17 , 18 ] . in fig . 3 , a detail of the c345c domain in complex with factor b ( model based on the pdb 2win ) is depicted and the localization of the disulphide bridge is shown.fig . 3details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the figure shows the c345c domain on the right hand side ( in red / orange ) and factor b on the left hand side ( in blue / green ) . note that , in the pdb model , the residues cys1518 and cys1590 correspond to cys1496 and cys 1568 , respectively . the arrow points the disulphide bridge formed by both cysteine residues details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the figure shows the c345c domain on the right hand side ( in red / orange ) and factor b on the left hand side ( in blue / green ) . note that , in the pdb model , the residues cys1518 and cys1590 correspond to cys1496 and cys 1568 , respectively . at the age of 16 years , a male turkish patient born to consanguineous parents ( first - degree cousins ) as the third of eight children , was admitted to hospital with a history of fever , cough and respiratory distress for 48 h. physical examination revealed weight and height within normal range , a wound scar resulting from a thoracotomy performed at the age of 6 months , and crackles and bronchial respiratory sounds . a chest x - ray revealed pneumonia ( fig 1lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung with regards to the past medical history , the patient had needed a tube thoracostomy due to pleural effusion at the age of 6 months and had suffered from recurrent lower respiratory infections with a frequency of 4 to 5 times per year . at the age of 8 years , subsequently , the frequency of infections was reduced however , in a period of 6 years ( from 14 to 20 years ) he still presented with two episodes of otitis media and 8 episodes of lower respiratory tract infections for which hospitalization was necessary . at the age of 20 years , the patient was vaccinated for s. pneumoniae and has not suffered from severe infections or needed hospitalization since ( 7 years of follow up so far ) . the patient has never presented with hematuria , hypertension or other clinical feature indicative of renal involvement or autoimmune disorder . notably , two of his brothers had died during the neonatal period for unknown reasons . his parents , three brothers and two sisters are healthy ( see fig . 2a and supplementary figure 1 for pedigrees).fig . 2pedigree and genetic analysis of the core family ( 2a ) and phylogenetic conservation of the cysteine 1518 in c3 ( 2b ) . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . females are represented as circles and males as squares . filled and half - filled symbols represent homozygous and heterozygous individuals , respectively . cysteine 1518 is indicated with a red box pedigree and genetic analysis of the core family ( 2a ) and phylogenetic conservation of the cysteine 1518 in c3 ( 2b ) . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . the patient had normal red cell counts but elevated erythrocyte sedimentation rate ( 52 mm / h ; reference values : 015 mm / h ) and c - reactive protein levels ( 66 mg / dl ; reference values : 8.510.6 mg / dl ) , respectively . total leukocyte ( 7500/mm ) and lymphocyte counts ( 3500/mm ) were within the normal range . routine urine analysis and renal function were unremarkable ( see table 1 for blood urea nitrogen and creatinine levels ; urine analysis data are not shown ) . immunoglobulin m , g and e levels were within the normal range as well as the igg subclasses , while serum iga levels were reduced ( table 1 ) . as there were no signs or symptoms of autoimmunity , autoantibody levels were not evaluated.table 1description of the laboratory findings of the patient and parentspatientfathermotherreference valuesblood urea nitrogen ( mg / dl)262040creatinine ( mg / dl)0,80,61,2ch50 ( u / ml)07171>15c3 levels ( mg / dl)819637190180c4 levels ( mg / dl)201040iga ( mg / dl)<5.823034144244igg ( mg / dl)18116402010igg11220315855igg266864495igg312523196igg42511157igm ( mg / dl)8652297ige ( iu / ml)460100anti - a titer1/641/10anti - b titer1/1281/10tuberculin test ( mm)10510 in vitro pha ( % ) 6165.8 9.2unstimulated nbt ( % ) 50038stimulated nbt ( % ) 706090total lymphocyte counts ( cell / mm)350017005700 t lymphocytes ( cd3 + ) ( % ) 685579(cells / mm)204011004100 t helper cells ( cd4 + ) ( % ) 382851(cells / mm)11406002400cytotoxic t cells ( cd8 + ) ( % ) 261642(cells / mm)7804001500natural killer cells ( cd16 + 56 + ) ( % ) 7528(cells / mm)2102001000b lymphocytes ( cd19 + ) ( % ) 201028(cells / mm)6002001400total hemolytic complement activity test . in vitro lymphocyte stimulation with 20 g / ml of phytohemagglutinin for 72 h. the values refer to the percentages of the blastic transformation of lymphocytes ( enlarged nucleus , condensed chromatin and/or with pores in the cytoplasm ) evaluated using light microscopy . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activitypatient examination at the age of 16 yearsnormal values in age - matched turkish subjects description of the laboratory findings of the patient and parents total hemolytic complement activity test . in vitro lymphocyte stimulation with 20 g / ml of phytohemagglutinin for 72 h. the values refer to the percentages of the blastic transformation of lymphocytes ( enlarged nucleus , condensed chromatin and/or with pores in the cytoplasm ) evaluated using light microscopy . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activity patient examination at the age of 16 years normal values in age - matched turkish subjects six weeks after vaccination for s. pneumoniae ( 20 years of age ) , the patient demonstrated a positive antibody response ( table 2).table 2specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / mlserotypes36b1419f23f7fbefore vaccination2216321916186 weeks after vaccination29>10046656820 specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / ml c3 levels were severely decreased in the patient , varying from 8 to 19 mg / dl at 6 different measurements ( table 1 ; reference values : 90180 mg / dl ) , with absent complement hemolytic activity measured using the ch50 test . the other complement levels ( c1 , c2 , c4 , c5 , c6 , c7 , c8 , c9 , factor h and factor i ) were found to be normal ( see table 1 for c4 ; other data not shown ) . the proliferative response of peripheral blood lymphocytes to phytohemagglutinin and the spontaneous and stimulated nitroblue tetrazolium test were normal ( table 1 ) . the parents showed normal levels of iga and normal ch50 , but reduced c3 levels ( 63 mg / dl and 71 mg / dl ) , respectively . details of the laboratory findings of the patient and his parents are shown in table 1 . sanger sequencing identified a homozygous missense mutation in exon 38 ( c. c4554 g , p. cys1518trp ) . molecular segregation analysis showed perfect segregation , with both parents and three siblings being carriers of one affected allele ( fig . 2a and supplementary figure 1 ) . to date , this variant has neither been annotated as a polymorphism in ncbi ( http://www.ncbi.nlm.nih.gov/ ) , nor in ucsc genomic bioinformatics site ( http://genome.ucsc.edu/ ) or ensembl ( http://www.ensembl.org ) , respectively . polyphen2 and sorting intolerant from tolerant ( sift ) analysis predict that the substitution of cysteine to tryptophan at position 1518 of c3 is very likely to affect protein function , with the maximum score in both analyses ( 1.000 in polyphen2 and 0.000 in sift , respectively ) . as indicated in fig . 2b , the cysteine residue at position 1518 is conserved among vertebrates and it has been shown to form a disulfide bridge with cysteine 1590 [ 17 , 18 ] . in fig . 3 , a detail of the c345c domain in complex with factor b ( model based on the pdb 2win ) is depicted and the localization of the disulphide bridge is shown.fig . 3details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the figure shows the c345c domain on the right hand side ( in red / orange ) and factor b on the left hand side ( in blue / green ) . note that , in the pdb model , the residues cys1518 and cys1590 correspond to cys1496 and cys 1568 , respectively . the arrow points the disulphide bridge formed by both cysteine residues details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the figure shows the c345c domain on the right hand side ( in red / orange ) and factor b on the left hand side ( in blue / green ) . note that , in the pdb model , the residues cys1518 and cys1590 correspond to cys1496 and cys 1568 , respectively . the complement system is a protein network crucial for both innate and adaptive immune responses . c3 is the convergence point for all the known complement activation cascades , resulting in cleavage of c3 into c3a ( anaphylatoxin ) and c3b ( reviewed in ) . the latter is an important product for opsonization of bacteria including encapsulated bacteria such as s. pneumoniae [ 18 , 19 ] , for amplification of complement activation through the alternative pathway , where the association of c3b with complement factor b is essential , and for cell lysis through the formation of c5 convertase ( reviewed in ) . patients with c3 deficiency frequently develop severe episodes of recurrent pneumonia , meningitis or sepsis . clinically , these patients present at an early age with overwhelming infections caused by s. pneumoniae [ 4 , 5 ] . autoimmunity and other immune manifestations , frequently affecting the kidney , are also observed in c3-deficient patients [ 1315 ] . since the parents of the patient described here were first - degree cousins and the index patient s severe clinical manifestations were unlikely to be explained by the diagnosis of isolated iga deficiency , an autosomal recessive disorder was suspected . we discovered and here describe a novel , homozygous missense mutation in c3 , altering a highly conserved amino acid found in the first position of the c345c domain of the c3 protein which is hypothesized to function as a binding site for factor b , as required for c3 convertase formation [ 2022 ] . this domain is known to undergo large rearrangements upon activation and is present in the c3b molecule [ 23 , 24 ] . furthermore , this cysteine residue forms one of the disulfide bonds in the c3 protein [ 20 , 25 ] , thus its loss will likely affect protein folding and/or stability . after vaccination for s. pneumoniae , our patient showed a marked clinical improvement . in line with this observation , it has been shown that mice depleted for c3 by intraperitoneal injection of cobra venom factor which are immunized against s. pneumoniae have reduced sepsis when colonized with this bacterium compared to control or neutrophil - depleted mice . previous studies have illustrated that c3-deficient patients are able to mount adaptive immune responses to conjugated vaccines against s. pneumoniae [ 8 , 9 ] . as mentioned , in addition to c3 deficiency the patient showed selective iga deficiency ( sigad ) . sigad is the most common form of primary immunodeficiency defined by decreased levels of iga in the presence of normal levels of other immunoglobulin isotypes ( reviewed in ) . patients are predisposed to recurrent sinopulmonary infections , gastrointestinal disorders , autoimmune diseases , atopy and malignancies [ 27 , 28 ] . amongst the gastrointestinal disorders , giardiasis , malabsorption , lactose intolerance , celiac disease , ulcerative respiratory tract infections are the most frequent morbidities in sigad patients , however , bronchiectasis is a rare complication . although sigad is often asymptomatic , patients with concomitant igg2 deficiency may present with impaired antibody responses against polysaccharide antigens and show predisposition to more severe bacterial infections . besides c3 deficiency , other deficiencies of adaptive or innate immunity can also lead to increased susceptibility to infections caused by s. pneumoniae , albeit with differences in the clinical and laboratory findings ( reviewed in [ 4 , 12 ] ) . two interesting examples of such innate immune deficiencies are irak4 and myd88 deficiency , respectively . irak4- or myd88-deficient patients are predisposed to recurrent invasive infections with s. pneumoniae , especially meningitis ( reviewed in ) . these patients also frequently present with impaired ability to increase plasma c - reactive protein and to mount fever in response to infection , with spontaneous improvement in adolescence ( reviewed in ) . by contrast , our patient presented mainly with pneumonias , and similar to other c3-deficient patients [ 4 , 5 ] , he showed high levels of crp and had episodes of fever , with infectious episodes persisting throughout adolescence . taken together , the following observations support our hypothesis that the clinical phenotype of our patient was - at least predominantly - caused by the underlying deficiency in c3 rather than associated with sigad : 1 ) sinusitis or gastrointestinal disorders are absent in the patient ; 2 ) bronchiectasis is observed although our patient presented with normal igg2 levels and normal antibody responses to polysaccharide antigens ; 3 ) the patient displays a marked and relatively specific susceptibility to infections with encapsulated bacteria such as s. pneumoniae ; and 4 ) there was a marked clinical amelioration upon vaccination against s. pneumoniae . we here report a novel homozygous mutation in c3 in a patient with recurrent and severe infections caused by streptococcus pneumoniae and associated iga deficiency . the case presented here highlights the importance of a more thorough evaluation of sigad patients when the clinical presentation is unusual or more severe than expected . in case of severe infections caused by encapsulated agents such as s. pneumoniae , our data lend further support to the concept that vaccination against this microorganism is critical for immunodeficient patients , in particular for inherited complement deficiencies . the c3 mutation ( c. c4554 g , p. cys1518trp ) shows perfect segregation with the disease . the filled symbol represents the homozygous affected individual ( iv-6 ) and half - filled symbols represent heterozygous individuals . note that three siblings , one nephew and one niece of the affected subject are also heterozygous for the c3 mutation . the c3 mutation ( c. c4554 g , p. cys1518trp ) shows perfect segregation with the disease . the filled symbol represents the homozygous affected individual ( iv-6 ) and half - filled symbols represent heterozygous individuals . note that three siblings , one nephew and one niece of the affected subject are also heterozygous for the c3 mutation . ( gif 289 kb ) high resolution ( eps 289 kb ) high resolution ( eps 289 kb ) this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposeimmunological and molecular evaluation of a patient presenting with recurrent infections caused by streptococcus pneumoniae and low complement component 3 ( c3 ) levels.methodsimmunological evaluation included complement components and immunoglobulin level quantification as well as number and function of t cells , b cells and neutrophils . serotype - specific immunoglobulin g antibodies against s. pneumoniae capsular polysaccharides were quantified by elisa in serum samples before and after vaccination with unconjugated polysaccharide vaccine . for the molecular analysis , genomic dna from the patient and parents were isolated and all exons as well as exon - intron boundaries of the c3 gene were sequenced by sanger sequencing.resultsa 16-year - old male , born to consanguineous parents , presented with recurrent episodes of pneumonia caused by s. pneumoniae and bronchiectasis . the patient showed severely reduced c3 and immunoglobulin a levels , while the parents showed moderately reduced levels of c3 . mutational analysis revealed a novel , homozygous missense mutation in the c3 gene ( c. c4554 g , p. cys1518trp ) , substituting a highly conserved amino acid in the c345c domain of c3 and interrupting one of its disulfide bonds . both parents were found to be carriers of the affected allele . vaccination against s. pneumoniae resulted in considerable clinical improvement.conclusionswe report a novel homozygous mutation in the c3 gene in a patient with concomitant selective iga deficiency who presented with a marked clinical improvement after vaccination against s. pneumoniae . this observation underlines the notion that vaccination against this microorganism is an important strategy for treatment of pid patients , particularly those presenting with increased susceptibility to infections caused by this agent.electronic supplementary materialthe online version of this article ( doi:10.1007/s10875 - 012 - 9775-z ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Methods Ethics Committee Determination of Antibody Titers Against Molecular Analysis Results Clinical Characterization of Patient and Family Laboratory Evaluation Mutation Identification in the Discussion Conclusions Electronic supplementary material Conflict of interest Open Access

the online version of this article ( doi:10.1007/s10875 - 012 - 9775-z ) contains supplementary material , which is available to authorized users . the third component of the complement system ( c3 ) is indispensable to all the known pathways of complement activation . a few biallelic defects in the c3 gene have been described in patients suffering not only from s. pneumoniae infections [ 612 ] but also from autoimmune and immune - complex - related disorders , in particular affecting the kidney [ 1315 ] . here , we describe a patient with selective immunoglobulin a ( iga ) deficiency presenting with recurrent airway infections caused by s. pneumoniae and bronchiectasis with no autoimmune or immune complex manifestations . our molecular analyses revealed that the patient suffers from c3 deficiency caused by a novel , homozygous mutation in the c3 gene . measurement of capsular polysaccharide serotype - specific immunoglobulin g ( igg ) antibodies against s. pneumoniae was performed by enzyme - linked immunosorbent assay ( elisa ) in serum samples before and 6 weeks after vaccination with pneumo 23 ( unconjugated polysaccharide vaccine against s. pneumoniae ) as described previously with minor modifications . in order to eliminate antibodies to cell wall polysaccharides , microtiter plates were coated with capsular polysaccharide antigens ( from the american type culture collection ( atcc ) , rockville , md ) and the samples were pre - incubated overnight with species - specific common cell wall polysaccharide from s. pneumoniae ( cwps ; c - polysaccharide purified ; statens serum institute , denmark ) . genomic dna was isolated from whole blood obtained from the patient and parents using a commercially available kit ( wizard genomic dna purification kit , promega corporation ) according to the manufacturer s instructions . the primers used for sequencing of the c3 gene were previously described by goldberg et al . for the protein modeling , we used molsoft icm browser pro software and a crystal structure model of the c3 convertase ( 2win ) from the protein database website ( http://www.rcsb.org/pdb/ ) . measurement of capsular polysaccharide serotype - specific immunoglobulin g ( igg ) antibodies against s. pneumoniae was performed by enzyme - linked immunosorbent assay ( elisa ) in serum samples before and 6 weeks after vaccination with pneumo 23 ( unconjugated polysaccharide vaccine against s. pneumoniae ) as described previously with minor modifications . in order to eliminate antibodies to cell wall polysaccharides , microtiter plates were coated with capsular polysaccharide antigens ( from the american type culture collection ( atcc ) , rockville , md ) and the samples were pre - incubated overnight with species - specific common cell wall polysaccharide from s. pneumoniae ( cwps ; c - polysaccharide purified ; statens serum institute , denmark ) . genomic dna was isolated from whole blood obtained from the patient and parents using a commercially available kit ( wizard genomic dna purification kit , promega corporation ) according to the manufacturer s instructions . the primers used for sequencing of the c3 gene were previously described by goldberg et al . for the protein modeling , we used molsoft icm browser pro software and a crystal structure model of the c3 convertase ( 2win ) from the protein database website ( http://www.rcsb.org/pdb/ ) . at the age of 16 years , a male turkish patient born to consanguineous parents ( first - degree cousins ) as the third of eight children , was admitted to hospital with a history of fever , cough and respiratory distress for 48 h. physical examination revealed weight and height within normal range , a wound scar resulting from a thoracotomy performed at the age of 6 months , and crackles and bronchial respiratory sounds . a chest x - ray revealed pneumonia ( fig 1lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung with regards to the past medical history , the patient had needed a tube thoracostomy due to pleural effusion at the age of 6 months and had suffered from recurrent lower respiratory infections with a frequency of 4 to 5 times per year . at the age of 20 years , the patient was vaccinated for s. pneumoniae and has not suffered from severe infections or needed hospitalization since ( 7 years of follow up so far ) . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . cysteine 1518 is indicated with a red box the patient had normal red cell counts but elevated erythrocyte sedimentation rate ( 52 mm / h ; reference values : 015 mm / h ) and c - reactive protein levels ( 66 mg / dl ; reference values : 8.510.6 mg / dl ) , respectively . immunoglobulin m , g and e levels were within the normal range as well as the igg subclasses , while serum iga levels were reduced ( table 1 ) . as there were no signs or symptoms of autoimmunity , autoantibody levels were not evaluated.table 1description of the laboratory findings of the patient and parentspatientfathermotherreference valuesblood urea nitrogen ( mg / dl)262040creatinine ( mg / dl)0,80,61,2ch50 ( u / ml)07171>15c3 levels ( mg / dl)819637190180c4 levels ( mg / dl)201040iga ( mg / dl)<5.823034144244igg ( mg / dl)18116402010igg11220315855igg266864495igg312523196igg42511157igm ( mg / dl)8652297ige ( iu / ml)460100anti - a titer1/641/10anti - b titer1/1281/10tuberculin test ( mm)10510 in vitro pha ( % ) 6165.8 9.2unstimulated nbt ( % ) 50038stimulated nbt ( % ) 706090total lymphocyte counts ( cell / mm)350017005700 t lymphocytes ( cd3 + ) ( % ) 685579(cells / mm)204011004100 t helper cells ( cd4 + ) ( % ) 382851(cells / mm)11406002400cytotoxic t cells ( cd8 + ) ( % ) 261642(cells / mm)7804001500natural killer cells ( cd16 + 56 + ) ( % ) 7528(cells / mm)2102001000b lymphocytes ( cd19 + ) ( % ) 201028(cells / mm)6002001400total hemolytic complement activity test . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activitypatient examination at the age of 16 yearsnormal values in age - matched turkish subjects description of the laboratory findings of the patient and parents total hemolytic complement activity test . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activity patient examination at the age of 16 years normal values in age - matched turkish subjects six weeks after vaccination for s. pneumoniae ( 20 years of age ) , the patient demonstrated a positive antibody response ( table 2).table 2specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / mlserotypes36b1419f23f7fbefore vaccination2216321916186 weeks after vaccination29>10046656820 specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / ml c3 levels were severely decreased in the patient , varying from 8 to 19 mg / dl at 6 different measurements ( table 1 ; reference values : 90180 mg / dl ) , with absent complement hemolytic activity measured using the ch50 test . the other complement levels ( c1 , c2 , c4 , c5 , c6 , c7 , c8 , c9 , factor h and factor i ) were found to be normal ( see table 1 for c4 ; other data not shown ) . the parents showed normal levels of iga and normal ch50 , but reduced c3 levels ( 63 mg / dl and 71 mg / dl ) , respectively . details of the laboratory findings of the patient and his parents are shown in table 1 . sanger sequencing identified a homozygous missense mutation in exon 38 ( c. c4554 g , p. cys1518trp ) . molecular segregation analysis showed perfect segregation , with both parents and three siblings being carriers of one affected allele ( fig . 3 , a detail of the c345c domain in complex with factor b ( model based on the pdb 2win ) is depicted and the localization of the disulphide bridge is shown.fig . 3details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the arrow points the disulphide bridge formed by both cysteine residues details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . at the age of 16 years , a male turkish patient born to consanguineous parents ( first - degree cousins ) as the third of eight children , was admitted to hospital with a history of fever , cough and respiratory distress for 48 h. physical examination revealed weight and height within normal range , a wound scar resulting from a thoracotomy performed at the age of 6 months , and crackles and bronchial respiratory sounds . a chest x - ray revealed pneumonia ( fig 1lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung lung radiography of the patient at admission showing pneumonia in the left lung and bronchial wall thickening on the right lung with regards to the past medical history , the patient had needed a tube thoracostomy due to pleural effusion at the age of 6 months and had suffered from recurrent lower respiratory infections with a frequency of 4 to 5 times per year . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . a perfect segregation of the c3 mutation ( c. c4554 g , p. cys1518trp ) is shown in the patient and parents . immunoglobulin m , g and e levels were within the normal range as well as the igg subclasses , while serum iga levels were reduced ( table 1 ) . as there were no signs or symptoms of autoimmunity , autoantibody levels were not evaluated.table 1description of the laboratory findings of the patient and parentspatientfathermotherreference valuesblood urea nitrogen ( mg / dl)262040creatinine ( mg / dl)0,80,61,2ch50 ( u / ml)07171>15c3 levels ( mg / dl)819637190180c4 levels ( mg / dl)201040iga ( mg / dl)<5.823034144244igg ( mg / dl)18116402010igg11220315855igg266864495igg312523196igg42511157igm ( mg / dl)8652297ige ( iu / ml)460100anti - a titer1/641/10anti - b titer1/1281/10tuberculin test ( mm)10510 in vitro pha ( % ) 6165.8 9.2unstimulated nbt ( % ) 50038stimulated nbt ( % ) 706090total lymphocyte counts ( cell / mm)350017005700 t lymphocytes ( cd3 + ) ( % ) 685579(cells / mm)204011004100 t helper cells ( cd4 + ) ( % ) 382851(cells / mm)11406002400cytotoxic t cells ( cd8 + ) ( % ) 261642(cells / mm)7804001500natural killer cells ( cd16 + 56 + ) ( % ) 7528(cells / mm)2102001000b lymphocytes ( cd19 + ) ( % ) 201028(cells / mm)6002001400total hemolytic complement activity test . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activitypatient examination at the age of 16 yearsnormal values in age - matched turkish subjects description of the laboratory findings of the patient and parents total hemolytic complement activity test . nitroblue tetrazolium semiquantitative test for evaluating neutrophil oxidative burst , values refer to the percentages of activated neutrophils presenting with respiratory burst activity patient examination at the age of 16 years normal values in age - matched turkish subjects six weeks after vaccination for s. pneumoniae ( 20 years of age ) , the patient demonstrated a positive antibody response ( table 2).table 2specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / mlserotypes36b1419f23f7fbefore vaccination2216321916186 weeks after vaccination29>10046656820 specific igg antibody levels in the patient before and after s. pneumoniae vaccine ( u / ml ) . reference values : 20 u / ml c3 levels were severely decreased in the patient , varying from 8 to 19 mg / dl at 6 different measurements ( table 1 ; reference values : 90180 mg / dl ) , with absent complement hemolytic activity measured using the ch50 test . the other complement levels ( c1 , c2 , c4 , c5 , c6 , c7 , c8 , c9 , factor h and factor i ) were found to be normal ( see table 1 for c4 ; other data not shown ) . the parents showed normal levels of iga and normal ch50 , but reduced c3 levels ( 63 mg / dl and 71 mg / dl ) , respectively . details of the laboratory findings of the patient and his parents are shown in table 1 . sanger sequencing identified a homozygous missense mutation in exon 38 ( c. c4554 g , p. cys1518trp ) . molecular segregation analysis showed perfect segregation , with both parents and three siblings being carriers of one affected allele ( fig . 3 , a detail of the c345c domain in complex with factor b ( model based on the pdb 2win ) is depicted and the localization of the disulphide bridge is shown.fig . 3details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the arrow points the disulphide bridge formed by both cysteine residues details of the structure of c345c domain of c3 and factor b based on the pdb model 2win . the latter is an important product for opsonization of bacteria including encapsulated bacteria such as s. pneumoniae [ 18 , 19 ] , for amplification of complement activation through the alternative pathway , where the association of c3b with complement factor b is essential , and for cell lysis through the formation of c5 convertase ( reviewed in ) . clinically , these patients present at an early age with overwhelming infections caused by s. pneumoniae [ 4 , 5 ] . since the parents of the patient described here were first - degree cousins and the index patient s severe clinical manifestations were unlikely to be explained by the diagnosis of isolated iga deficiency , an autosomal recessive disorder was suspected . we discovered and here describe a novel , homozygous missense mutation in c3 , altering a highly conserved amino acid found in the first position of the c345c domain of the c3 protein which is hypothesized to function as a binding site for factor b , as required for c3 convertase formation [ 2022 ] . furthermore , this cysteine residue forms one of the disulfide bonds in the c3 protein [ 20 , 25 ] , thus its loss will likely affect protein folding and/or stability . after vaccination for s. pneumoniae , our patient showed a marked clinical improvement . in line with this observation , it has been shown that mice depleted for c3 by intraperitoneal injection of cobra venom factor which are immunized against s. pneumoniae have reduced sepsis when colonized with this bacterium compared to control or neutrophil - depleted mice . as mentioned , in addition to c3 deficiency the patient showed selective iga deficiency ( sigad ) . besides c3 deficiency , other deficiencies of adaptive or innate immunity can also lead to increased susceptibility to infections caused by s. pneumoniae , albeit with differences in the clinical and laboratory findings ( reviewed in [ 4 , 12 ] ) . taken together , the following observations support our hypothesis that the clinical phenotype of our patient was - at least predominantly - caused by the underlying deficiency in c3 rather than associated with sigad : 1 ) sinusitis or gastrointestinal disorders are absent in the patient ; 2 ) bronchiectasis is observed although our patient presented with normal igg2 levels and normal antibody responses to polysaccharide antigens ; 3 ) the patient displays a marked and relatively specific susceptibility to infections with encapsulated bacteria such as s. pneumoniae ; and 4 ) there was a marked clinical amelioration upon vaccination against s. pneumoniae . we here report a novel homozygous mutation in c3 in a patient with recurrent and severe infections caused by streptococcus pneumoniae and associated iga deficiency . in case of severe infections caused by encapsulated agents such as s. pneumoniae , our data lend further support to the concept that vaccination against this microorganism is critical for immunodeficient patients , in particular for inherited complement deficiencies . the c3 mutation ( c. c4554 g , p. cys1518trp ) shows perfect segregation with the disease . note that three siblings , one nephew and one niece of the affected subject are also heterozygous for the c3 mutation . the c3 mutation ( c. c4554 g , p. cys1518trp ) shows perfect segregation with the disease . note that three siblings , one nephew and one niece of the affected subject are also heterozygous for the c3 mutation .

non - st - segment elevation acute coronary syndromes ( nste acs ) are highly prevalent in the united states and globally , and are associated with significant morbidity and mortality . the key role of platelet - mediated thrombosis in the pathogenesis of nste acs is confirmed by the proven clinical benefits of antiplatelet agents ( aspirin and a p2y12 adenosine diphosphate [ adp ] receptor antagonist ) in this setting . despite the documented advantages and broad use of antiplatelet therapy , the long - term morbidity and mortality rates remain significant , and residual risk can be attributed , at least in part , to the fact that thrombosis continues in the presence of current treatments because aspirin and p2y12 adp receptor antagonists each block only one of multiple platelet activation pathways , and thus do not impact other platelet activation pathways , such as the one triggered by interaction of thrombin with protease - activated receptor ( par)-1 , thereby exposing patients to continued accumulation of thrombotic events . these considerations suggest that novel therapies with a different mechanism of action , when used in combination with current antiplatelet agents , may provide more comprehensive inhibition of platelet activation and additional reductions in morbidity and mortality , potentially without incremental bleeding risk . non - st - segment elevation acute coronary syndromes ( nste acs ) , which comprise unstable angina ( ua ) and non - st - segment elevation myocardial infarction ( nstemi ) , are associated with significant morbidity , mortality and economic burden in the united states . of the over 1.3 million unique annual hospitalizations for acs in the united states in 2006 , over 800,000 were for myocardial infarction ( mi ) ( approximately two - thirds of these were nstemi ) and almost 540,000 were for ua . the primary pathophysiological mechanism responsible for clinical manifestations of nste acs involves occlusion of coronary arteries by platelet - rich thrombi , whose generation was triggered in response to injury to vascular endothelium , such as a rupture or erosion of an atherosclerotic plaque . platelet activation , a key step in platelet thrombus formation , can be initiated by multiple agonists , such as thrombin , thromboxane a2 , adenosine diphosphate ( adp ) and collagen ; the goal of medical therapy is to prevent platelet - mediated thrombosis and the resulting acute ischemic events . the key role of platelet - mediated thrombosis in the pathogenesis of nste acs is confirmed by the proven clinical benefits of antiplatelet agents in these patients [ 46 ] . however , despite the documented clinical efficacy of antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist , the long - term morbidity and mortality associated with nste acs remains significant [ 5 , 6 ] , as these agents each block only one of the multiple platelet activation pathways leading to thrombotic events ; they do not interfere with the pathways stimulated by other platelet activators , including thrombin , the most potent platelet agonist [ 7 , 8 ] . the stimulatory effect of thrombin on platelet - mediated thrombosis continues even in the presence of aspirin and a p2y12 adp receptor antagonist , thereby potentially leading to thrombotic events . in clinical practice , the residual ischemic risk in patients with nste acs is often further exacerbated by the underuse of antiplatelet agents in spite of their well - documented benefits [ 911 ] . apart from the substantial residual risk for ischemic events , current oral antiplatelet agents are also associated with increased bleeding risk . these considerations underscore the need for more comprehensive prevention of platelet - mediated thrombosis and associated ischemic events , ideally without an incremental bleeding risk . the aims of this review are to discuss the results and clinical implications of key clinical trials with oral antiplatelet agents in patients with nste acs , as well as to review the emerging evidence regarding the net clinical benefit or the balance between bleeding and ischemic events . in addition , this review will also address the current management of nste acs in us clinical practice , based on the findings from the action ( nrmi / crusade ) and grace registries , and the opportunities for improvements in patient care . aspirin aspirin inhibits the activity of the cyclooxygenase ( cox)-1 enzyme , thereby limiting the production of thromboxane a2 , an important platelet agonist implicated in both pathologic thrombus formation and protective hemostasis . the efficacy of aspirin for treatment of patients presenting with nste acs and for secondary prevention in patients with established atherothrombotic disease has been demonstrated in several clinical trials [ 1214 ] and meta - analyses . aspirin is the foundation of treatment recommendations for both acute and chronic antiplatelet therapy in patients with nste acs [ 15 , 16 ] . however , residual morbidity and mortality and bleeding risk remain substantial with aspirin . an observed inverse relationship between aspirin dose and clinical benefit may be attributed to a dose - dependent increase in bleeding risk ( mainly gastrointestinal bleeding ) . it should be noted that even low aspirin doses ( 100 mg ) are associated with increased bleeding rates compared with placebo . however , the inverse relationship between aspirin dose and clinical benefit may not rely solely on higher bleeding risk , but also on a trend toward a lower anti - thrombotic benefit at doses higher than 75100 mg / day . the lack of additional cardiovascular benefit by increasing doses of aspirin has been recently confirmed by results of the current - oasis 7 trial ( discussed below ) and may be attributed to a dose - dependent inhibition of prostacyclin at higher doses of aspirin . p2y12 adp receptor inhibitors these agents , including clopidogrel , prasugrel and ticagrelor , bind to and inhibit the activation of the platelet p2y12 receptor by its physiological ligand adp , which is released from activated platelets , and amplifies platelet activation and aggregation . clopidogrel is a thienopyridine prodrug that is converted into an active compound by cytochrome p450 isoenzymes in the liver . addition of clopidogrel to aspirin in patients with nste acs was demonstrated to reduce the risk of adverse ischemic outcomes and to increase bleeding risk versus aspirin alone in the cure trial ( table 1 ) , as well as in its substudy pci - cure in patients with nste acs who have undergone percutaneous coronary intervention ( pci ) . on the basis of these findings , the addition of clopidogrel to aspirin has become the standard of care for patients with nste acs , and its use in these patients is recommended by both the american college of cardiology ( acc)/american heart association ( aha ) and european guidelines . furthermore , the benefits of adding clopidogrel to aspirin have also been demonstrated in patients with st - segment elevation mi and patients undergoing elective pci , as well as in patients with atrial fibrillation who do not wish to or can not take anticoagulant warfarin . the charisma trial did not demonstrate a significant benefit of clopidogrel plus aspirin vs aspirin alone in the overall population of patients with clinically documented atherothrombotic disease or multiple risk factors ; however , a significant reduction in the incidence of ischemic events with the combination of clopidogrel and aspirin was evident in the secondary prevention cohort ( ie , in patients with prior mi , stroke or symptomatic peripheral arterial disease ) . most recently , the current - oasis 7 trial evaluated the efficacy and safety of high - dose versus standard - dose clopidogrel in patients with acs . patients ( n = 25,087 ) assigned to high - dose clopidogrel received a 600-mg loading dose and 150 mg once daily for seven days , followed by 75 mg once daily until day 30 . patients in the standard clopidogrel arm received a 300-mg loading dose , followed by 75 mg once daily until 30 days . after day 30 , all patients received clopidogrel 75 mg once - daily . patients were also randomized to receive low - dose ( 75 to 100 mg per day ) or high - dose ( 300 to 325 mg per day ) aspirin . in the overall study population , there was no significant difference in the primary endpoint ( combined rate of death from cv causes , mi , and stroke ) between patients receiving the high - dose and the standard - dose clopidogrel therapy ( 4.2% vs 4.4% ; p = 0.37 ) in patients who underwent pci , however , the high - dose clopidogrel regimen was associated with a 15% relative reduction in risk of death from cv causes , mi , or stroke at 30 days versus the standard - dose regimen ( 3.9% versus 4.5% ; p = 0.036 ) . no significant differences in efficacy were observed between the high - dose and the standard - dose clopidogrel arms in patients who did not undergo pci . in patients who underwent pci , treatment with the high - dose clopidogrel regimen was also associated with significant reductions in stent thrombosis and mi at 30 days no significant differences in timi major bleeding , fatal bleeding , intracranial hemorrhage , or cabg - related bleeding were observed between the two clopidogrel treatment arms . as alluded to above , there was no significant difference in the primary outcome in patients allocated high - dose versus low - dose aspirin ( 4.2% vs 4.4% ; p = 0.47 ) . of note , rates of timi major bleeding were also comparable between the high - dose and low - dose aspirin groups ( 0.97% vs 1.03% ; p = 0.71 ) . table 1summaries of key results of phase 3 trials of p2y12 adp receptor antagonists in the treatment of patients with acstrial namepatientsactive treatment versus controlprimary end pointevent rate ( active treatment versus control)p valuemajor bleeding ( active treatment versus control)p valuecure nste acs ( n = 12,652)clopidogrel + asa vs placebo + asacv death , nonfatal mi , or stroke9.3% vs 11.4% ( 20% relative reduction , 80% residual risk with clopidogrel + asa)<0.0013.7% vs 2.7% ( 37% relative increase vs placebo + asa)0.001triton - timi 38 nste acs and stemi undergoing pci ( n = 13,608)prasugrel + asa vs clopidogrel + asacv death , non - fatal mi , or non - fatal stroke9.9% vs 12.1% ( 20% relative reduction , 80% residual risk with prasugrel + asa)<0.0012.4% vs 1.8% ( 33% relative increase vs clopidogrel + asa)0.03plato nste acs and stemi ( n = 18,624)ticagrelor + asa vs clopidogrel + asacv death , mi , or stroke9.8% vs 11.7% ( 16% relative reduction ; 84% residual risk with ticagrelor + asa<0.00111.6% vs 11.2%0.43acs acute coronary syndromes , asa aspirin , cv cardiovascular , mi myocardial infarction , nste non - st - segment elevation , pci percutaneous coronary intervention , timi thrombolysis in myocardial infarction clopidogrel loading dose = 300 mg ; maintenance dose = 75 mg / dmajor bleeding was defined as substantially disabling bleeding , intraocular bleeding leading to the loss of vision , or bleeding necessitating the transfusion of at least 2 units of bloodprasugrel loading dose = 60 mg ; maintenance dose = 10 mg / d . clopidogrel loading dose = 300 mg maintenance dose = 75 mg / dtimi major bleedingticagrelor loading dose = 180 mg ; maintenance dose = 90 mg twice daily . clopidogrel loading dose = 300600 mg ; maintenance dose = 75 mg / daymajor bleeding was defined as bleeding that led to clinically significant disability ( e.g. , intraocular bleeding with permanent vision loss ) or bleeding either associated with a drop in the hemoglobin level of at least 3.0 g per deciliter but less than 5.0 g per deciliter or requiring transfusion of 2 to 3 units of red cellseveral studies have documented variable responsiveness of platelets to therapy with clopidogrel . although a standardized definition and methodology for assessment of responsiveness to antiplatelet therapy has not been established , sufficient evidence supports the concept that persistence of enhanced platelet reactivity despite the use of clopidogrel is clinically relevant [ 3033 ] . a correlation between low level of inhibition of adp - induced platelet aggregation in response to clopidogrel and recurrence of ischemic events has been documented in several studies in patients with acs and those undergoing pci [ 3133].although the mechanisms responsible for the variability and low responsiveness to clopidogrel have not been fully elucidated , recent analyses suggest that genetic polymorphisms of the cytochrome p ( cyp ) 450 enzymes can significantly modulate individual response to clopidogrel and are important determinants of prognosis [ 3436 ] . a study of patients with acute mi treated with clopidogrel demonstrated that the carriers of the cyp2c19 * 2 allelic variant ( cyp2c19 ) had a significantly higher rate of ischemic events ( death , non - fatal mi , or urgent revascularization ) than non - carriers ( 10.9 events per 100 patient - years vs 2.9 events per 100 patient - years , respectively ; adjusted hazard ratio : 5.38 , p < 0.0001 ) . similarly , in a french registry of patients with acute mi treated with clopidogrel , patients with any two loss - of - function cyp2c19 variants had a significantly higher rate of death , non - fatal mi , or stroke versus patients no loss - of - function alleles ( 21.5% vs. 13.3% ; adjusted hr 1.98 ) . additionally , in the triton trial ( discussed below ) , patients treated with clopidogrel who were carriers of at least one reduced - function cyp2c19 allele had a significantly higher rate of cardiovascular events than non - carriers ( 12.1% vs. 8.0% , respectively ; p = 0.01 ) . other studies have suggested that co - administration of clopidogrel with a proton pump inhibitor , particularly omeprazole , decreases the antiplatelet effects and clinical benefit of clopidogrel [ 37 , 38 ] , but the data supporting this association are mixed .the delayed onset of action and variable inhibition of platelet aggregation with clopidogrel have prompted the search for potentially more effective p2y12 adp receptor antagonists . the first among these newer p2y12 adp receptor antagonists is prasugrel , which is characterized by a faster onset of action and more potent inhibition of adp - induced platelet aggregation as compared with clopidogrel [ 40 , 41 ] . the triton - timi 38 trial evaluated the efficacy and safety of the combination of prasugrel plus aspirin versus clopidogrel ( 300-mg loading dose plus 75-mg once - daily maintenance dose ) plus aspirin in patients with acs scheduled for pci . in triton , treatment with prasugrel plus aspirin was associated with a significantly lower rate of the combined end point of cardiovascular death , non - fatal mi and non - fatal stroke versus treatment with clopidogrel plus aspirin ( table 1 ) . the net clinical benefit ( combined incidence of cardiovascular death , non - fatal mi , non - fatal stroke and non - fatal thrombolyis in myocardial infarction [ timi ] major bleeding not related to coronary artery bypass grafting [ cabg ] surgery ) of prasugrel was particularly pronounced in patients with diabetes ( hazard ratio [ hr ] : 0.74 ; p = 0.001 ) and those presenting with st - segment elevation mi ( hr : 0.81 ; p = 0.02 ) , largely because these patient groups did not experience an increase in timi major bleeding [ 42 , 43 ] . the incidence of stent thrombosis was also substantially lower in the prasugrel - plus - aspirin arm ( fig . 1 ) . however , the residual risk for ischemic events with prasugrel plus aspirin in triton - timi 38 remained substantial ( 10% at 15 months ; table 1 ) , and the risk of bleeding ( timi major , life - threatening , fatal , timi major and minor , requiring transfusion and cabg surgery - related ) in the overall population was significantly higher than with clopidogrel plus aspirin ( table 1 ) . furthermore , no net benefit of prasugrel plus aspirin over clopidogrel plus aspirin was observed in patients aged 75 years with acs ( hr : 0.99 ; p = 0.92 ) or those weighing < 60 kg ( hr : 1.03 ; p = 0.89 ) , and a net clinical harm was apparent in patients with prior stroke or transient ischemic attack ( tia ) ( hr : 1.54 ; p = 0.04 ) , primarily due to higher rates of bleeding with prasugrel plus aspirin than with clopidogrel plus aspirin in these patient groups . these results suggest that the selection of a p2y12 adp receptor antagonist should take into account not only the greater pharmacological potency of prasugrel versus clopidogrel , but also the increased risk for bleeding with prasugrel as compared with clopidogrel . the net harm observed in patients with stroke or tia suggests that prasugrel should be avoided in this patient group . likewise , patients at higher bleeding risk , such as the elderly or those with low body weight , may not derive a net benefit from prasugrel . it is important to note that consideration of the use of the 5-mg maintenance dose of prasugrel in patients weighing < 60 kg , as described in the us prescribing information for prasugrel , is entirely based on pharmacokinetic modeling and is not supported by any clinical evidence . in selected patients ( ie , patients with diabetes or those with stemi ) with acs undergoing pci , the combination of prasugrel plus aspirin may provide greater protection against ischemic events than the combination of clopidogrel plus aspirin , although even in these patients the residual risk for ischemic events with prasugrel plus aspirin remained substantial [ 42 , 43 ] . on the basis of these results , prasugrel has recently been approved in europe and in the united states , although the pattern of its use in clinical practice remains to be seen . 1incidence of stent thrombosis over 15 months in patients receiving prasugrel plus aspirin versus clopidogrel plus aspirin in the triton - timi 38 trial . reproduced with permissionin addition to prasugrel , ticagrelor ( azd 6140 ) , a novel non - thienopyridine p2y12 adp receptor antagonist , has also demonstrated a faster onset of action , more potent inhibition of adp - induced platelet aggregation than clopidogrel , as well as more rapid reversal of inhibition of adp - induced platelet activation and aggregation than clopidogrel . unlike clopidogrel or prasugrel , ticagrelor is not a prodrug and does not require metabolic conversion into an active metabolite . these properties suggest that ticagrelor may be an attractive alternative to clopidogrel , especially in clinical situations where rapid inhibition of platelet aggregation or its quick reversal may be required . the efficacy and safety of 1-year treatment with ticagrelor ( plus aspirin versus clopidogrel plus aspirin was recently reported in patients with acs in the phase 3 plato trial ( table 1 ) . patients receiving ticagrelor plus aspirin experienced a significant reduction in the incidence of the composite endpoint of first occurrence of cv death , mi , or stroke versus patients receiving clopidogrel plus aspirin ( table 1 ) . of note , the rate of all - cause death was 22% lower with ticagrelor versus clopidogrel ( p < 0.001 ) . there was no significant difference in rates of major bleeding between the treatment arms ( table 1 ) . rates of other adverse events were higher with ticagrelor versus clopidogrel : dyspnea ( 13.8% versus 7.8% ; p < 0.001 ) , syncope ( 1.1% versus 0.8% ; p = 0.08 ) , ventricular pauses 3 s during the first week of treatment ( 5.8% versus 3.6% ; p = 0.01 ) and increase in serum uric acid and serum creatinine at 1 month and 1 year ( p < 0.001 for each comparison ] ) . it is important to note that ticagrelor is administered twice daily ; in clinical practice , the need for twice - daily dosing may increase the risk for ischemic events in patients who are not fully compliant with the prescribed therapy.even in the presence of dual antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist ( clopidogrel , prasugrel or ticagrelor ) , the risk for morbidity and mortality remains substantial in patients with nste acs , as well as in those with st - segment - elevation mi , atrial fibrillation , or a history of prior atherothrombotic disease , and in patients scheduled for pci . this residual risk can be attributed to the fact that multiple pathways contribute to platelet activation , and aspirin and p2y12 adp receptor antagonists each inhibit only one of these pathways ( the thromboxane a2 and adp pathways , respectively ) . the lack of an inhibitory effect of current therapies on other platelet activation pathways allows continued platelet reactivity in the presence of potent agonists , such as thrombin , thereby increasing the risk for recurrent thrombotic / ischemic events , including death . when used in combination with the current standard - of - care therapies , new agents that target pathways that are not affected by aspirin or p2y12 adp receptor antagonists may provide complementary and more comprehensive inhibition of platelet activation , and thereby contribute to greater inhibition of platelet - mediated thrombosis and incremental reductions in ischemic events . apart from the residual ischemic risk , dual antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist is also associated with an increased risk of bleeding , because these agents interfere with the thromboxane a2 and adp platelet activation pathways that are essential for normal hemostasis . these considerations underscore the need for novel antiplatelet agents that provide more comprehensive platelet inhibition without interfering with platelet activation pathways critical for hemostasis , for greater protection against thrombotic events without an incremental bleeding risk . summaries of key results of phase 3 trials of p2y12 adp receptor antagonists in the treatment of patients with acs acs acute coronary syndromes , asa aspirin , cv cardiovascular , mi myocardial infarction , nste non - st - segment elevation , pci percutaneous coronary intervention , timi thrombolysis in myocardial infarction clopidogrel loading dose = 300 mg ; maintenance dose = 75 mg / d major bleeding was defined as substantially disabling bleeding , intraocular bleeding leading to the loss of vision , or bleeding necessitating the transfusion of at least 2 units of blood prasugrel loading dose = 60 mg ; maintenance dose = 10 mg / d . clopidogrel loading dose = 300 mg maintenance dose = 75 mg / d ticagrelor loading dose = 180 mg ; maintenance dose = 90 mg twice daily . clopidogrel loading dose = 300600 mg ; maintenance dose = 75 mg / day major bleeding was defined as bleeding that led to clinically significant disability ( e.g. , intraocular bleeding with permanent vision loss ) or bleeding either associated with a drop in the hemoglobin level of at least 3.0 g per deciliter but less than 5.0 g per deciliter or requiring transfusion of 2 to 3 units of red cell incidence of stent thrombosis over 15 months in patients receiving prasugrel plus aspirin versus clopidogrel plus aspirin in the triton - timi 38 trial . reproduced with permission par-1 ( thrombin ) receptor antagonists par-1 is the principal receptor for thrombin on human platelets . interaction of thrombin , the most potent platelet agonist , with par-1 promotes platelet shape change and granule secretion , as well as other processes leading to platelet activation . preclinical observations indicate that inhibition or genetic inactivation of par-1 selectively interferes with platelet activation mediated by thrombin and with platelet deposition into an occlusive thrombus , but not with thrombin - mediated fibrin generation or initial platelet deposition that is important for healing in response to vascular injury [ 4850 ] . these results suggest that platelet activation mediated by par-1 may be critical for thrombosis but may not be necessary for hemostasis . par-1 inhibitors ( or thrombin receptor antagonists [ tras ] ) represent a novel class of antiplatelet agents . currently , two par-1 antagonists are in clinical development : e-5555 and sch 530348 . when used in combination with the current standard - of - care antiplatelet therapy ( aspirin alone or dual therapy with aspirin and a p2y12 adp receptor antagonist ) , a par-1 inhibitor offers more comprehensive platelet inhibition and potentially an incremental reduction in ischemic events , possibly without a risk of increased bleeding.e-5555 is an orally active , potent par-1 antagonist that has demonstrated antiplatelet effects without increasing bleeding times in preclinical studies [ 51 , 52 ] . evaluated the in vitro effects of e-5555 on platelet aggregation and biomarker expression in blood from healthy volunteers ( n = 10 ) , patients with documented coronary artery disease ( cad ) treated with aspirin ( n = 10 ) , and patients with documented cad who were treated with aspirin plus clopidogrel ( n = 10 ) . complete inhibition of trap - induced platelet aggregation was observed at all concentrations of e-5555 evaluated ( 20 , 50 , and 100 ng / ml ) , including the lowest dose . four ongoing phase 2 trials are evaluating the safety and tolerability of daily , oral administration of e-5555 ( 50 mg , 100 mg or 200 mg ) in patients with cad or nste acs in the us and japan [ 5457].sch 530348 is the first oral par-1 inhibitor in phase 3 clinical development . the phase 2 tra - pci trial evaluated the safety and efficacy of sch 530348 ( administered as either a 10- , 20- , or 40-mg loading dose followed by a maintenance dose of 0.5 mg qd , 1 mg qd , or 2.5 mg qd for 59 days ) used in combination with standard oral antiplatelet therapy ( aspirin and clopidogrel ) and an antithrombin agent ( heparin or bivalirudin ) versus placebo in patients with planned non - urgent pci . based on the recommendation of the safety review committee , enrollment of the prespecified 1,600 patients was reduced because of low timi major and minor bleeding rates . tra - pci randomized a total of 1,030 patients , of which 573 subsequently underwent pci as planned ( primary pci cohort ) , whereas the remaining patients either underwent cabg surgery ( n = 76 ) or were managed medically ( n = 381 ) . although the study may be underpowered for safety results , rates of clinically significant bleeding were low overall in tra - pci . administration of sch 530348 in combination with standard therapy was not associated with increased rates of timi major or minor bleeding , the primary endpoint , compared with standard therapy alone in the primary pci cohort ( 2.8% vs 3.3% ; p = 0.77 ) . none of the patients treated with the combination of the highest loading and maintenance doses of sch 530348 ( 40-mg loading dose plus 2.5-mg maintenance dose ) experienced timi major bleeding , although rates of overall bleeding , timi minor bleeding and non - timi bleeding were numerically higher with sch 530348 . the rate of discontinuations due to non - timi bleeding in the primary cohort were similar in the combined sch 530348 and placebo arms ( 1.4% and 1.3% , respectively ) . the rate of timi major or minor bleeding did not differ significantly between patients allocated sch 530348 versus placebo who underwent cabg surgery ( 90% vs 79% ) . no clear dose - response relationship for post - operative bleeding was evident in surgical patients . more patients who underwent cabg and received sch 530348 versus placebo needed transfusions ; however the proportion of patients who needed 2 units of packed red blood cells did not differ between groups . among patients who were managed medically , timi major / minor bleeding was observed in 3 patients allocated sch 530348 and zero patients allocated placebo . although not powered to detect differences in efficacy endpoints , a lower incidence of ischemic events , specifically mi , was observed in patients allocated sch 530348 . in this study , the most rapid onset of action ( as measured by inhibition of thrombin receptor agonist peptide [ trap]-induced platelet aggregation ) was provided by the 40-mg loading dose of sch 530348 , whereas the 2.5-mg once - daily maintenance dose of sch 530348 subsequently selected for phase 3 trials sustained complete ( > 80% ) inhibition of trap - induced platelet aggregation over the 60-day treatment period . importantly , sch 530348 selectively inhibits platelet aggregation induced by trap but does not interfere with the aggregation induced by other platelet agonists , such as arachidonic acid , adp , or collagen . a separate phase 2 trial in japanese patients with nste acs scheduled for pci also demonstrated that the addition of sch 530348 to the standard - of - care therapy ( aspirin plus ticlopidine ) did not result in increased bleeding risk and these results suggest that the use of sch 530348 in combination with standard antiplatelet therapy ( aspirin with or without a p2y12 adp receptor antagonist ) may provide incremental reductions in ischemic events , potentially without an increased risk of bleeding . two large ongoing phase 3 trials are evaluating the benefits and bleeding risks associated with the addition of sch 530348 to the standard - of - care therapy ( aspirin alone or aspirin plus a p2y12 adp receptor inhibitor ) in patients presenting with nste acs ( n 10,000 ; clinicaltrials.gov identifier : nct00527943 ) and in secondary prevention ( n 25,000 patients ; clinicaltrials.gov identifier : nct00526474 ) . an increase in bleeding risk in patients treated with aspirin and p2y12 adp receptor antagonists has been well recognized , and several recent studies suggest that bleeding and blood transfusions may represent independent predictors of short- and long - term mortality in patients with acs and in those undergoing pci [ 6266 ] . for example , a 2004 report by rao and colleagues evaluated the correlation of blood transfusions and morbidity and mortality in over 24,000 patients with acs , demonstrating that transfusions were a powerful predictor of 30-day mortality ( hr : 3.94 ; 95% confidence interval [ ci ] 3.264.75 ; p < 0.001 ) and 30-day death / mi ( hr : 2.92 ; 95% ci 2.553.35 ; p < 0.001 ) . similarly , the risk for 30-day and 6-month mortality among over 26,000 patients with nste acs was shown to increase proportionally with greater severity of bleeding . these observations were further confirmed by a pooled analysis of the oasis registry and the oasis-2 and cure trials , which involved over 34,000 patients with nste acs . this study demonstrated that major bleeding was associated with a fivefold increase in the risk of mortality at 30 days ( fig . 2a ) , and that the risk of death over the 6-month follow - up increased in proportion to the bleeding severity ( fig . in addition to the mortality increase , major bleeding was also associated with a significantly increased risk of mi ( hr : 4.44 ; 95% ci 3.166.24 ; p < 0.0001 ) and stroke ( hr : 6.46 ; 95% ci 3.5411.79 ; p < 0.0001 ) within 30 days . in the acuity trial , patients with nste acs who experienced major bleeding had significantly higher 30-day incidences of death , composite ischemia and stent thrombosis than patients without major bleeding ( p < 0.0001 for all 3 outcomes ) , and major bleeding was the strongest independent predictor of 30-day mortality ( hr : 7.55 ; 95% ci 4.6812.18 ; p < 0.0001 ) . additionally , a pooled analysis of 4 isar trials in patients undergoing pci ( isar - react , isar - sweet , isar - smart 2 and isar - react 2 ) demonstrated that bleeding within 30 days was an independent predictor of 1-year mortality ( hr : 2.96 ; 95% ci 1.964.48 ; p < 0.001 ) . of note , the predictive value of bleeding within 30 days for 1-year mortality was comparable to that of mi within 30 days ( hr : 2.29 ; 95% ci 1.523.46 ; p < 0.001 ) or urgent revascularization within 30 days ( hr : 2.49 ; 95% ci 1.165.35 ; p = 0.019 ) . collectively , these results suggest that bleeding is a significant risk factor for death and ischemic events . the mechanisms linking bleeding and blood transfusions to increased mortality in patients with acs and patients undergoing pci are incompletely understood but likely involve multiple factors , including the location and intensity of bleeding , impaired oxygen delivery , as well as discontinuation of antiplatelet therapy . regardless of the mechanisms and factors that may contribute to the increased risk of mortality associated with bleeding and blood transfusions , it is clear that the approaches designed to minimize the bleeding risk may also result in lower rates of ischemic events and thereby improve overall patient outcomes . 2a relationship between major bleeding and mortality in a meta - analysis of 34,146 patients with nste acs in the oasis-1 , oasis-2 and cure trials during the first 30 days ( hr : 5.37 ; 95% ci 3.977.26 ; p < 0.0001 ) . b relationship between bleeding severity and the risk of death : kaplan - meier estimates of mortality among patients who developed no , minor , major ( excluding life - threatening ) or life - threatening bleeding in the cure trial ( p for trend = 0.0009 ) . reproduced with permission a relationship between major bleeding and mortality in a meta - analysis of 34,146 patients with nste acs in the oasis-1 , oasis-2 and cure trials during the first 30 days ( hr : 5.37 ; 95% ci 3.977.26 ; p < 0.0001 ) . b relationship between bleeding severity and the risk of death : kaplan - meier estimates of mortality among patients who developed no , minor , major ( excluding life - threatening ) or life - threatening bleeding in the cure trial ( p for trend = 0.0009 ) . reproduced with permission continuous evaluations of management of patients with nste acs in the united states in the crusade registry from 2002 to 2004 have demonstrated significant improvements in use of medications both in the acute setting ( antiplatelet agents , anticoagulants , glycoprotein iib / iiia receptor inhibitors and beta - blockers ) and in the discharge setting ( antiplatelet agents , lipid - lowering agents , angiotensin - converting enzyme inhibitors ) . however , use of many therapies was suboptimal , and there was a clear need for greater implementation of the acc / aha guidelines recommendations . crusade has also documented significantly lower use of evidence - based therapies in the elderly , women , minority populations , and patients without private insurance [ 6870 ] . more recently , the action registry reported that clopidogrel was used in only 60% of patients with nstemi in the acute setting and in 74% of patients with nstemi at discharge during 2008 . differences in clopidogrel utilization have also been noted based on management strategy [ 70 , 71 ] . the latest available data from action for the year 2008 document that clopidogrel was used among patients with nstemi at the time of hospital discharge in 97% of those who underwent pci , but in only 55% of those who were medically managed , and in only 28% of those who underwent cabg , even though they were admitted to the hospital with an acs . importantly , lack of early clopidogrel use was associated with significantly higher in - hospital mortality and other adverse outcomes compared with early initiation of clopidogrel in crusade ( fig . 3in - hospital outcomes in 93,045 patients with nstemi not undergoing pci by pattern of clopidogrel use in crusade . p < 0.01 for all comparisons in - hospital outcomes in 93,045 patients with nstemi not undergoing pci by pattern of clopidogrel use in crusade . p < 0.01 for all comparisons the global grace registry of over 27,000 patients with nste acs in 14 countries has reported significant reductions in clinical events and increased utilization of medical therapies and pci between 19992000 and 2005 . these improvements included significant decreases in the rates of in - hospital death ( 2.9% in 19992000 versus 2.2% in 2005 ; p = 0.02 ) and 6-month mortality ( 4.9% in 19992000 versus 3.3% in 2005 ; p = 0.04 ) . the rates of in - hospital congestive heart failure or pulmonary edema , mi , cardiogenic shock and 6-month stroke were also significantly lower ( all p < 0.05 ) in 2005 than in 19992000 , while the in - hospital rates of stroke and 6-month rates of mi did not differ significantly . these reductions in clinical events may be attributed , at least in part , to considerable increases in adherence to guidelines - based use of thienopyridines and lipid - lowering agents , as recommended by the us and european guidelines . nevertheless , in - hospital mortality rates remain substantial in the united states ( 4.1% in patients with nstemi in action ) and globally ( 2.2% in patients with nste acs in grace ) [ 10 , 71 ] . despite the acc / aha guidelines recommendations , clopidogrel initiation is often postponed until after diagnostic catheterization and withheld at the time of discharge in patients who are managed medically and those who undergo surgical revascularization . thus , there is considerable room for greater adherence to the guidelines recommendations for the use of oral antiplatelet therapy among patients with nste acs in the acute and particularly in the chronic setting , which can lead to improved patient outcomes . at the same time , bleeding and blood transfusions , which are associated with increased mortality risk , remain a frequent complication in patients with nste acs . for example , major bleeding and red blood cell transfusion occurred during 2008 in the action registry in 12% and 15% of patients with nstemi , respectively , while grace reported major bleeding in 4.7% of patients with nstemi and 2.3% of patients with ua . unadjusted in - hospital death rates in grace in patients with nstemi with major bleeding versus no bleeding were 15.3% versus 5.3% ( p these considerations suggest that the clinical benefits of greater use of current oral antiplatelet therapies recommended by the guidelines may be partially offset by the increased risk for bleeding or blood transfusions , and thereby underscore a critical need for novel therapies that reduce the risk of ischemic events without exacerbating the risk of bleeding complications . clinical trials and registry data have documented clear beneficial effects of current oral antiplatelet agents ( aspirin and p2y12 adp receptor inhibitors ) on ischemic outcomes in patients with nste acs . however , despite the proven clinical efficacy of these agents , residual morbidity and mortality remain substantial even in patients receiving dual antiplatelet therapy . this residual ischemic risk may be explained by the fact that these agents interfere only with the thromboxane a2 and adp platelet activation pathways and do not block platelet - mediated thrombosis stimulated by other platelet activators ( such as thrombin ) , allowing the accumulation of ischemic events . in addition to a high residual risk for ischemic events , aspirin and p2y12 adp receptor antagonists are associated with increased bleeding risk , which can be attributed to the inhibitory effect of these agents on pathways essential for hemostasis . novel therapeutic approaches such as the par-1 inhibitors , targeting platelet activation pathways not affected by current antiplatelet agents , represent an attractive strategy to reduce the residual ischemic risk in patients with nste acs , possibly without exacerbating the risk of bleeding . in addition to novel therapeutic approaches , improvements in clinical outcomes in standard practice can also be achieved through the greater use of currently available antiplatelet agents with proven efficacy , such as aspirin and clopidogrel , particularly in patients who are managed without pci . the benefits of increased use of currently recommended oral antiplatelet agents , though , should be balanced against the increased risk for bleeding complications . the availability of oral antiplatelet agents with a more favorable benefit - to - risk profile than the currently available therapies would represent a major advance in the treatment of patients with nste acs .

introductionnon - st - segment elevation acute coronary syndromes ( nste acs ) are highly prevalent in the united states and globally , and are associated with significant morbidity and mortality.discussionthe key role of platelet - mediated thrombosis in the pathogenesis of nste acs is confirmed by the proven clinical benefits of antiplatelet agents ( aspirin and a p2y12 adenosine diphosphate [ adp ] receptor antagonist ) in this setting . despite the documented advantages and broad use of antiplatelet therapy , the long - term morbidity and mortality rates remain significant , and the bleeding risk remains substantial . residual risk can be attributed , at least in part , to the fact that thrombosis continues in the presence of current treatments because aspirin and p2y12 adp receptor antagonists each block only one of multiple platelet activation pathways , and thus do not impact other platelet activation pathways , such as the one triggered by interaction of thrombin with protease - activated receptor ( par)-1 , thereby exposing patients to continued accumulation of thrombotic events.conclusionthese considerations suggest that novel therapies with a different mechanism of action , when used in combination with current antiplatelet agents , may provide more comprehensive inhibition of platelet activation and additional reductions in morbidity and mortality , potentially without incremental bleeding risk .

Introduction Discussion Conclusion Introduction Oral antiplatelet therapy: benefits and risks Relationship between bleeding and ischemic events Use of antiplatelet therapy in clinical practice: insights from registries Conclusions

non - st - segment elevation acute coronary syndromes ( nste acs ) are highly prevalent in the united states and globally , and are associated with significant morbidity and mortality . the key role of platelet - mediated thrombosis in the pathogenesis of nste acs is confirmed by the proven clinical benefits of antiplatelet agents ( aspirin and a p2y12 adenosine diphosphate [ adp ] receptor antagonist ) in this setting . despite the documented advantages and broad use of antiplatelet therapy , the long - term morbidity and mortality rates remain significant , and residual risk can be attributed , at least in part , to the fact that thrombosis continues in the presence of current treatments because aspirin and p2y12 adp receptor antagonists each block only one of multiple platelet activation pathways , and thus do not impact other platelet activation pathways , such as the one triggered by interaction of thrombin with protease - activated receptor ( par)-1 , thereby exposing patients to continued accumulation of thrombotic events . these considerations suggest that novel therapies with a different mechanism of action , when used in combination with current antiplatelet agents , may provide more comprehensive inhibition of platelet activation and additional reductions in morbidity and mortality , potentially without incremental bleeding risk . non - st - segment elevation acute coronary syndromes ( nste acs ) , which comprise unstable angina ( ua ) and non - st - segment elevation myocardial infarction ( nstemi ) , are associated with significant morbidity , mortality and economic burden in the united states . the primary pathophysiological mechanism responsible for clinical manifestations of nste acs involves occlusion of coronary arteries by platelet - rich thrombi , whose generation was triggered in response to injury to vascular endothelium , such as a rupture or erosion of an atherosclerotic plaque . platelet activation , a key step in platelet thrombus formation , can be initiated by multiple agonists , such as thrombin , thromboxane a2 , adenosine diphosphate ( adp ) and collagen ; the goal of medical therapy is to prevent platelet - mediated thrombosis and the resulting acute ischemic events . the key role of platelet - mediated thrombosis in the pathogenesis of nste acs is confirmed by the proven clinical benefits of antiplatelet agents in these patients [ 46 ] . however , despite the documented clinical efficacy of antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist , the long - term morbidity and mortality associated with nste acs remains significant [ 5 , 6 ] , as these agents each block only one of the multiple platelet activation pathways leading to thrombotic events ; they do not interfere with the pathways stimulated by other platelet activators , including thrombin , the most potent platelet agonist [ 7 , 8 ] . the stimulatory effect of thrombin on platelet - mediated thrombosis continues even in the presence of aspirin and a p2y12 adp receptor antagonist , thereby potentially leading to thrombotic events . in clinical practice , the residual ischemic risk in patients with nste acs is often further exacerbated by the underuse of antiplatelet agents in spite of their well - documented benefits [ 911 ] . apart from the substantial residual risk for ischemic events , current oral antiplatelet agents are also associated with increased bleeding risk . these considerations underscore the need for more comprehensive prevention of platelet - mediated thrombosis and associated ischemic events , ideally without an incremental bleeding risk . in addition , this review will also address the current management of nste acs in us clinical practice , based on the findings from the action ( nrmi / crusade ) and grace registries , and the opportunities for improvements in patient care . p2y12 adp receptor inhibitors these agents , including clopidogrel , prasugrel and ticagrelor , bind to and inhibit the activation of the platelet p2y12 receptor by its physiological ligand adp , which is released from activated platelets , and amplifies platelet activation and aggregation . furthermore , the benefits of adding clopidogrel to aspirin have also been demonstrated in patients with st - segment elevation mi and patients undergoing elective pci , as well as in patients with atrial fibrillation who do not wish to or can not take anticoagulant warfarin . in the overall study population , there was no significant difference in the primary endpoint ( combined rate of death from cv causes , mi , and stroke ) between patients receiving the high - dose and the standard - dose clopidogrel therapy ( 4.2% vs 4.4% ; p = 0.37 ) in patients who underwent pci , however , the high - dose clopidogrel regimen was associated with a 15% relative reduction in risk of death from cv causes , mi , or stroke at 30 days versus the standard - dose regimen ( 3.9% versus 4.5% ; p = 0.036 ) . table 1summaries of key results of phase 3 trials of p2y12 adp receptor antagonists in the treatment of patients with acstrial namepatientsactive treatment versus controlprimary end pointevent rate ( active treatment versus control)p valuemajor bleeding ( active treatment versus control)p valuecure nste acs ( n = 12,652)clopidogrel + asa vs placebo + asacv death , nonfatal mi , or stroke9.3% vs 11.4% ( 20% relative reduction , 80% residual risk with clopidogrel + asa)<0.0013.7% vs 2.7% ( 37% relative increase vs placebo + asa)0.001triton - timi 38 nste acs and stemi undergoing pci ( n = 13,608)prasugrel + asa vs clopidogrel + asacv death , non - fatal mi , or non - fatal stroke9.9% vs 12.1% ( 20% relative reduction , 80% residual risk with prasugrel + asa)<0.0012.4% vs 1.8% ( 33% relative increase vs clopidogrel + asa)0.03plato nste acs and stemi ( n = 18,624)ticagrelor + asa vs clopidogrel + asacv death , mi , or stroke9.8% vs 11.7% ( 16% relative reduction ; 84% residual risk with ticagrelor + asa<0.00111.6% vs 11.2%0.43acs acute coronary syndromes , asa aspirin , cv cardiovascular , mi myocardial infarction , nste non - st - segment elevation , pci percutaneous coronary intervention , timi thrombolysis in myocardial infarction clopidogrel loading dose = 300 mg ; maintenance dose = 75 mg / dmajor bleeding was defined as substantially disabling bleeding , intraocular bleeding leading to the loss of vision , or bleeding necessitating the transfusion of at least 2 units of bloodprasugrel loading dose = 60 mg ; maintenance dose = 10 mg / d . other studies have suggested that co - administration of clopidogrel with a proton pump inhibitor , particularly omeprazole , decreases the antiplatelet effects and clinical benefit of clopidogrel [ 37 , 38 ] , but the data supporting this association are mixed .the delayed onset of action and variable inhibition of platelet aggregation with clopidogrel have prompted the search for potentially more effective p2y12 adp receptor antagonists . the first among these newer p2y12 adp receptor antagonists is prasugrel , which is characterized by a faster onset of action and more potent inhibition of adp - induced platelet aggregation as compared with clopidogrel [ 40 , 41 ] . however , the residual risk for ischemic events with prasugrel plus aspirin in triton - timi 38 remained substantial ( 10% at 15 months ; table 1 ) , and the risk of bleeding ( timi major , life - threatening , fatal , timi major and minor , requiring transfusion and cabg surgery - related ) in the overall population was significantly higher than with clopidogrel plus aspirin ( table 1 ) . these results suggest that the selection of a p2y12 adp receptor antagonist should take into account not only the greater pharmacological potency of prasugrel versus clopidogrel , but also the increased risk for bleeding with prasugrel as compared with clopidogrel . likewise , patients at higher bleeding risk , such as the elderly or those with low body weight , may not derive a net benefit from prasugrel . reproduced with permissionin addition to prasugrel , ticagrelor ( azd 6140 ) , a novel non - thienopyridine p2y12 adp receptor antagonist , has also demonstrated a faster onset of action , more potent inhibition of adp - induced platelet aggregation than clopidogrel , as well as more rapid reversal of inhibition of adp - induced platelet activation and aggregation than clopidogrel . it is important to note that ticagrelor is administered twice daily ; in clinical practice , the need for twice - daily dosing may increase the risk for ischemic events in patients who are not fully compliant with the prescribed therapy.even in the presence of dual antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist ( clopidogrel , prasugrel or ticagrelor ) , the risk for morbidity and mortality remains substantial in patients with nste acs , as well as in those with st - segment - elevation mi , atrial fibrillation , or a history of prior atherothrombotic disease , and in patients scheduled for pci . this residual risk can be attributed to the fact that multiple pathways contribute to platelet activation , and aspirin and p2y12 adp receptor antagonists each inhibit only one of these pathways ( the thromboxane a2 and adp pathways , respectively ) . the lack of an inhibitory effect of current therapies on other platelet activation pathways allows continued platelet reactivity in the presence of potent agonists , such as thrombin , thereby increasing the risk for recurrent thrombotic / ischemic events , including death . when used in combination with the current standard - of - care therapies , new agents that target pathways that are not affected by aspirin or p2y12 adp receptor antagonists may provide complementary and more comprehensive inhibition of platelet activation , and thereby contribute to greater inhibition of platelet - mediated thrombosis and incremental reductions in ischemic events . apart from the residual ischemic risk , dual antiplatelet therapy with aspirin and a p2y12 adp receptor antagonist is also associated with an increased risk of bleeding , because these agents interfere with the thromboxane a2 and adp platelet activation pathways that are essential for normal hemostasis . these considerations underscore the need for novel antiplatelet agents that provide more comprehensive platelet inhibition without interfering with platelet activation pathways critical for hemostasis , for greater protection against thrombotic events without an incremental bleeding risk . summaries of key results of phase 3 trials of p2y12 adp receptor antagonists in the treatment of patients with acs acs acute coronary syndromes , asa aspirin , cv cardiovascular , mi myocardial infarction , nste non - st - segment elevation , pci percutaneous coronary intervention , timi thrombolysis in myocardial infarction clopidogrel loading dose = 300 mg ; maintenance dose = 75 mg / d major bleeding was defined as substantially disabling bleeding , intraocular bleeding leading to the loss of vision , or bleeding necessitating the transfusion of at least 2 units of blood prasugrel loading dose = 60 mg ; maintenance dose = 10 mg / d . , intraocular bleeding with permanent vision loss ) or bleeding either associated with a drop in the hemoglobin level of at least 3.0 g per deciliter but less than 5.0 g per deciliter or requiring transfusion of 2 to 3 units of red cell incidence of stent thrombosis over 15 months in patients receiving prasugrel plus aspirin versus clopidogrel plus aspirin in the triton - timi 38 trial . interaction of thrombin , the most potent platelet agonist , with par-1 promotes platelet shape change and granule secretion , as well as other processes leading to platelet activation . when used in combination with the current standard - of - care antiplatelet therapy ( aspirin alone or dual therapy with aspirin and a p2y12 adp receptor antagonist ) , a par-1 inhibitor offers more comprehensive platelet inhibition and potentially an incremental reduction in ischemic events , possibly without a risk of increased bleeding.e-5555 is an orally active , potent par-1 antagonist that has demonstrated antiplatelet effects without increasing bleeding times in preclinical studies [ 51 , 52 ] . the phase 2 tra - pci trial evaluated the safety and efficacy of sch 530348 ( administered as either a 10- , 20- , or 40-mg loading dose followed by a maintenance dose of 0.5 mg qd , 1 mg qd , or 2.5 mg qd for 59 days ) used in combination with standard oral antiplatelet therapy ( aspirin and clopidogrel ) and an antithrombin agent ( heparin or bivalirudin ) versus placebo in patients with planned non - urgent pci . administration of sch 530348 in combination with standard therapy was not associated with increased rates of timi major or minor bleeding , the primary endpoint , compared with standard therapy alone in the primary pci cohort ( 2.8% vs 3.3% ; p = 0.77 ) . in this study , the most rapid onset of action ( as measured by inhibition of thrombin receptor agonist peptide [ trap]-induced platelet aggregation ) was provided by the 40-mg loading dose of sch 530348 , whereas the 2.5-mg once - daily maintenance dose of sch 530348 subsequently selected for phase 3 trials sustained complete ( > 80% ) inhibition of trap - induced platelet aggregation over the 60-day treatment period . a separate phase 2 trial in japanese patients with nste acs scheduled for pci also demonstrated that the addition of sch 530348 to the standard - of - care therapy ( aspirin plus ticlopidine ) did not result in increased bleeding risk and these results suggest that the use of sch 530348 in combination with standard antiplatelet therapy ( aspirin with or without a p2y12 adp receptor antagonist ) may provide incremental reductions in ischemic events , potentially without an increased risk of bleeding . two large ongoing phase 3 trials are evaluating the benefits and bleeding risks associated with the addition of sch 530348 to the standard - of - care therapy ( aspirin alone or aspirin plus a p2y12 adp receptor inhibitor ) in patients presenting with nste acs ( n 10,000 ; clinicaltrials.gov identifier : nct00527943 ) and in secondary prevention ( n 25,000 patients ; clinicaltrials.gov identifier : nct00526474 ) . an increase in bleeding risk in patients treated with aspirin and p2y12 adp receptor antagonists has been well recognized , and several recent studies suggest that bleeding and blood transfusions may represent independent predictors of short- and long - term mortality in patients with acs and in those undergoing pci [ 6266 ] . regardless of the mechanisms and factors that may contribute to the increased risk of mortality associated with bleeding and blood transfusions , it is clear that the approaches designed to minimize the bleeding risk may also result in lower rates of ischemic events and thereby improve overall patient outcomes . reproduced with permission continuous evaluations of management of patients with nste acs in the united states in the crusade registry from 2002 to 2004 have demonstrated significant improvements in use of medications both in the acute setting ( antiplatelet agents , anticoagulants , glycoprotein iib / iiia receptor inhibitors and beta - blockers ) and in the discharge setting ( antiplatelet agents , lipid - lowering agents , angiotensin - converting enzyme inhibitors ) . these reductions in clinical events may be attributed , at least in part , to considerable increases in adherence to guidelines - based use of thienopyridines and lipid - lowering agents , as recommended by the us and european guidelines . nevertheless , in - hospital mortality rates remain substantial in the united states ( 4.1% in patients with nstemi in action ) and globally ( 2.2% in patients with nste acs in grace ) [ 10 , 71 ] . thus , there is considerable room for greater adherence to the guidelines recommendations for the use of oral antiplatelet therapy among patients with nste acs in the acute and particularly in the chronic setting , which can lead to improved patient outcomes . unadjusted in - hospital death rates in grace in patients with nstemi with major bleeding versus no bleeding were 15.3% versus 5.3% ( p these considerations suggest that the clinical benefits of greater use of current oral antiplatelet therapies recommended by the guidelines may be partially offset by the increased risk for bleeding or blood transfusions , and thereby underscore a critical need for novel therapies that reduce the risk of ischemic events without exacerbating the risk of bleeding complications . clinical trials and registry data have documented clear beneficial effects of current oral antiplatelet agents ( aspirin and p2y12 adp receptor inhibitors ) on ischemic outcomes in patients with nste acs . however , despite the proven clinical efficacy of these agents , residual morbidity and mortality remain substantial even in patients receiving dual antiplatelet therapy . this residual ischemic risk may be explained by the fact that these agents interfere only with the thromboxane a2 and adp platelet activation pathways and do not block platelet - mediated thrombosis stimulated by other platelet activators ( such as thrombin ) , allowing the accumulation of ischemic events . in addition to a high residual risk for ischemic events , aspirin and p2y12 adp receptor antagonists are associated with increased bleeding risk , which can be attributed to the inhibitory effect of these agents on pathways essential for hemostasis . novel therapeutic approaches such as the par-1 inhibitors , targeting platelet activation pathways not affected by current antiplatelet agents , represent an attractive strategy to reduce the residual ischemic risk in patients with nste acs , possibly without exacerbating the risk of bleeding . in addition to novel therapeutic approaches , improvements in clinical outcomes in standard practice can also be achieved through the greater use of currently available antiplatelet agents with proven efficacy , such as aspirin and clopidogrel , particularly in patients who are managed without pci . the benefits of increased use of currently recommended oral antiplatelet agents , though , should be balanced against the increased risk for bleeding complications . the availability of oral antiplatelet agents with a more favorable benefit - to - risk profile than the currently available therapies would represent a major advance in the treatment of patients with nste acs .

in january 2007 , the american college of obstetricians and gynecologists ( acog ) practice bulletin number 77 recommended screening and invasive diagnostic testing should be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age . several strategies for screening have been proposed based on the first trimester combined test ( nuchal translucency ( nt ) measurement and biochemical markers ) and second trimester maternal serum quadruple ( quad ) screen . the highest detection rates and lowest screen positive rates have been reported for integrated screening . this includes first trimester nuchal translucency , pregnancy - associated plasma protein a ( papp - a ) and human chorionic gonadotropin ( hcg ) plus maternal serum quad screening in the mid - trimester , with the results provided only after all tests are completed . patient anxiety during the delay between presentation for screening and results make this an unacceptable option for many patients . proposed alternative screening options include stepwise sequential screening and contingent sequential screening based on classifying women as high- or low - risk using a predetermined threshold ( american college of obstetricians and gynecologists acog 2007 ) . stepwise sequential screening is first trimester combined testing plus maternal serum quad screening with results provided after each test . contingent sequential screening is first trimester combined testing with only women having a risk between 1:30 and 1:1,500 for trisomy 21 ( t21 ) proceeding to maternal serum quad screening . prenatal aneuploidy screening results are often reported or framed as positive or negative based on a defined threshold . this is usually the risk of a 35-year old woman having a child with t21 or trisomy 18 ( t18 ) . however , there is little information about the effects of using verbal descriptions of risk versus numerical data . in a survey of 169 british antenatal clinics about the method of communicating down syndrome screen negative results , 44 % used a verbal phrase ( low risk , screen negative , within normal limits , not needing further action , among others ) , 16 % gave a numeric risk figure and 40 % used both a verbal phrase and numeric risk ( marteau 1999 ) . the goals of this study were to examine : 1- the uptake of stepwise sequential screening in our primarily urban , midwestern population , 2- the potential affects of ethnicity , education and proximity to the medical center on willingness to proceed with further testing , and 3- our method of risk reporting , which emphasized a woman s age - risk for t21 and adjusted risk after screening , rather than classifying a result as positive or negative , or high - risk , or low - risk . this is a retrospective cohort study of all patients who completed first trimester nt and biochemical screening at our institution between june 2007 and february 2010 . this study was approved by the washington university human research protection office , which waived informed consent . inclusion criteria included women with singleton gestations who completed combined first trimester screening with nt and the serum markers papp - a and hcg . for the first 3 months of the study period , the laboratory utilized at this institution used total beta - hcg . exclusion criteria included multiple gestations , blood testing not performed or analyzed and inability to obtain a nt measurement . all nt measurements were performed by physicians or sonographers certified in nt measurement by either the fetal medicine foundation or the nuchal translucency quality review ( ntqr ) program . patient s were categorized at the time of their first trimester screening visit into one of three groups as follows : u = undecided as to whether they wished to proceed with stepwise sequential screening ; y = yes , they planned to pursue stepwise sequential screening ; or n = no , they do not plan to have stepwise sequential screening . results of first trimester screening were not reported as positive or negative . the phone call and/or letter informing patients of their first trimester screen results indicated the patient s age - risk for ( t21 ) and trisomy 18 ( t18 ) and their revised risks for same based on the nt and biochemical markers . the determination of whether a patient was of high enough risk to warrant further screening or testing was at the patient s discretion . all phone calls and/or result letters also again reviewed the patient s subsequent options , including no further screening , maternal serum quad screening , mid - trimester ultrasound and invasive testing via chorionic villus sampling ( cvs ) or amniocentesis . the patient s decision about whether to proceed with stepwise sequential screening ( both before and after their first trimester screening results ) were recorded in a computerized database for subsequent correlation with variables including ethnicity , level of education , distance from our medical center , nt measurement , crown rump length , age- and screen - risks for t21 , results of invasive testing and pregnancy outcomes . in order to assess the impact of non - invasive first trimester screening and stepwise sequential screening ( first trimester screening plus maternal serum quad screening ) on the utilization of amniocentesis and cvs , the number of these procedures was compared as follows : for a 2-year period prior to the initiation of first trimester screening ( january 2000december 2001 ) , for a 2-year period when first trimester screening was utilized but prior to the availability of stepwise sequential screening ( june 2005may 2007 ) and for the 2-year period following initiation of stepwise sequential screening ( july 2007june 2009 ) . relationship between decision status ( yes , no , undecided ) and various characteristics were compared using the chi - square test or fisher s exact test , as appropriate . differences in the counts of invasive procedures performed across time periods were evaluated using simple poisson regression models , where time period ( 20002001 , 20052007 , and 20072009 ) was included using indicator variables . the study population consists of 2,643 patients who met inclusion criteria during the study period ( june 2007february 2010 ) . during this time , an acceptable nt measurement was unable to be obtained in 8 patients ( 0.3 % of the initial study population ) . at the time of the first trimester examination , 1,926 ( 72.8 % ) were undecided regarding stepwise sequential screening ( u group ) pending their first trimester results . 434 patients ( 16.4 % ) indicated they planned to return for stepwise sequential screening , regardless of their results ( y group ) and 283 patients ( 10.7 % ) declined stepwise sequential screening at the time of their first trimester screening ( n group ) . the numbers of patients who actually pursued stepwise sequential screening are listed in table 1 . this included a minority of the undecided and no groups ( 14.9 % and 1.4 % , respectively ) and only 56.4 % of those who originally intended to have stepwise sequential screening . overall , 79.8 % of the study population elected not to pursue follow - up screening.table 1stepwise sequential screening following first trimester combined screengroupnno ss screeningfollow - up ss screening p undecided1,926 ( 72.9 % ) 1,639 ( 85.1 % ) 287 ( 14.9 % ) < 0.0001yes434 ( 16.4 % ) 190 ( 43.8 % ) 244 ( 56.2 % ) no283 ( 10.7 % ) 279 ( 98.6 % ) 4 ( 1.4 % ) 2,643 ( 100 % ) 2,108 ( 79.8 % ) 535 ( 20.2 % ) u vs y<0.0001u vs n<0.0001y vs n<0.0001 stepwise sequential screening following first trimester combined screen the relation between education level and choice of screening is presented in table 2 . in the group of women who did not plan to pursue stepwise sequential screening there were a significantly greater number who were not high school graduates and significantly fewer who were college graduates compared to women in the other two groups . table 3 demonstrates that women who declined stepwise sequential screening were more likely to be black and hispanic and less likely to be white , than women in the other groups . table 4 indicates that distance from the medical center did not correlate with the decision to pursue or decline stepwise sequential screening.table 2eduction level and decision to pursue stepwise sequential screeninggroupnsome high schoolhs grad some collegecollege grad post grad p < 0.0001undecided1,92368 ( 3.5 % ) 501 ( 26.1 % ) 1,354 ( 70.4 % ) yes43411 ( 2.5 % ) 115 ( 26.5 % ) 308 ( 71.0 % ) no28222 ( 7.8 % ) 111 ( 39.4 % ) 149 ( 52.8 % ) 2,639101 ( 3.8 % ) 727 ( 27.5 % ) 1,811 ( 68.6 % ) u vs. y0.58u vs. n<0.0001y vs n<0.0001table 3ethnicity and decision to pursue stepwise sequential screeningethnicitygroupnwhiteblackasianhispother p < 0.0001undecided1,9261,395 ( 72.4 % ) 293 ( 15.2 % ) 106 ( 5.5 % ) 49 ( 2.5 % ) 83 ( 4.3 % ) yes434326 ( 75.1 % ) 50 ( 11.5 % ) 18 ( 4.1 % ) 8 ( 1.8 % ) 32 ( 7.4 % ) no283165 ( 58.3 % ) 71 ( 25.1 % ) 10 ( 3.5 % ) 17 ( 6.0 % ) 20 ( 7.1 % ) 2,6431,886 ( 71.4 % ) 414 ( 15.7 % ) 134 ( 5.1 % ) 74 ( 2.8 % ) 135 ( 5.1 % ) u vs. n0.01u vs. n<0.0001y vs. n<0.001table 4distance from medical center and decision to pursue stepwise sequential screeningmiles p groupn0202150>500.14undecided1,9261,293 ( 67.1 % ) 507 ( 26.3 % ) 126 ( 6.5 % ) yes434284 ( 65.4 % ) 114 ( 26.4 % ) 36 ( 8.3 % ) no283170 ( 60.1 % ) 88 ( 31.1 % ) 25 ( 8.8 % ) 2,6431,747 ( 66.1 % ) 709 ( 26.8 % ) 187 ( 7.1 % ) u vs. y0.42u vs. n0.05y vs. n0.32 eduction level and decision to pursue stepwise sequential screening ethnicity and decision to pursue stepwise sequential screening distance from medical center and decision to pursue stepwise sequential screening tables 5 and 6 demonstrate that the percentages of women at risk for t21 were similar between the groups.table 5age risk for t21 and decision to pursue stepwise sequential screeninggroup1:501:511:270>1:270 p*undecided ( n-1,926)44 ( 2.3 % ) 896 ( 46.5 % ) 986 ( 51.2 % ) 0.26yes ( n-434)17 ( 3.9 % ) 204 ( 47.0 % ) 213 ( 49.1 % ) no ( n-283)9 ( 2.6 % ) 140 ( 49.5 % ) 134 ( 47.3 % ) u vs. y0.14u vs. n0.37y vs. n0.75*fishers exact testtable 6screen risk for t21 and decision to pursue stepwise sequential screeninggroup1:501:511:270>1:270 p * 0.63undecided ( n-1,926)26 ( 1.3 % ) 86 ( 4.5 % ) 1,814 ( 94.2 % ) yes ( n-434)7 ( 1.6 % ) 21 ( 4.8 % ) 406 ( 93.5 % ) no ( n-283)7 ( 2.5 % ) 13 ( 4.6 % ) 263 ( 92.9 % ) uvs . y0.79u vs. n0.30yvsn.0.74*fishers exact test age risk for t21 and decision to pursue stepwise sequential screening screen risk for t21 and decision to pursue stepwise sequential screening the number of patients whose first trimester screen - risk for t21 exceeded their age - risk , and their choice of follow - up , is depicted in table 7 . overall , 126 patients ( 4.8 % of the study population ) had a first trimester screen - risk for t21 which was greater than their age - risk . of these , 73(57.9 % ) women elected not to have follow - up stepwise sequential screening or diagnostic testing , 24(19.8 % ) had stepwise sequential screening only and 29(23.0 % ) had invasive testing via cvs ( n = 11 ) or amniocentesis ( n = 18 ) . there was one case of 47 , xy , + 21 and one apparently balanced robertsonian translocation between chromosomes 13 and 14 . women who originally declined stepwise sequential screening were significantly less likely to pursue invasive testing than women in the other two groups.table 7screen risk for t21 greater than age risk and choice of follow - upgroupsr > ar p no further testingss screen onlyinvasive testing p*undecided ( n-1,926)91 ( 4.7 % ) 0.9852 ( 57.9 % ) 15 ( 19.0 % ) 24 ( 26.4 % ) yes ( n-434)21 ( 4.8 % ) 8 ( 38.1 % ) 9 ( 42.9 % ) 4 ( 19.0 % ) no ( n-283)14 ( 4.6 % ) 13 ( 92.9 % ) 01 ( 7.1 % ) 2,643126 ( 4.8 % ) 73 ( 57.9 % ) 24 ( 19.0 % ) 29 ( 23.0 % ) u vs. y0.92u vs. y0.05u vs. n0.87u vs n0.04y vs. n0.99y vs. n0.002*fisher 's exact test screen risk for t21 greater than age risk and choice of follow - up a total of 91(3.4 % of the study population ) women had invasive testing by amniocentesis ( n = 76 ) or cvs ( n = 15 ) during the study period . in 59(64.8 % ) , the age - risk for t21 was greater than the screen - risk . there were three cases of t21 and the aforementioned translocation . in two of the three t21 cases , the maternal age risk was greater than the screen risk ( the age risks were 1:43 and 1:235 versus screen risks of 1:96 and 1:246 , respectively ) . the number of invasive procedures performed prior to the availability of first trimester screening , following initiation of first trimester screening and following the availability of stepwise sequential screening is indicated in table 8 . the indications for invasive testing were varied and included ama , abnormal ultrasound findings , abnormal screening , and family history of aneuploidy . the number of amniocenteses in our center declined by 51.4 % following the availability of first trimester screening with a further 17.4 % reduction upon initiation of stepwise sequential screening . the number of cvs procedures declined by 19.2 % following availability of first trimester screening . there was a significant reduction in the number of cvs procedures from pre - first trimester screening and its utilization . however , there was no significant difference in the number of cvs procedures between availability of first trimester screening and stepwise sequential screening.table 8number of invasive procedures before and after first trimester and stepwise sequential screening availabilityprocedurepre - ftsfts onlyss availp , pre - fts vs fts onlyp , fts only vs. ss availamniocentesis1,632793655 p < 0.0001 p < 0.0001cvs494399371 p < 0.001 p = 0.31 first trimester screening sequential screening number of invasive procedures before and after first trimester and stepwise sequential screening availability first trimester screening sequential screening in our population , only 20 % of women having first trimester screening , elected to pursue stepwise sequential screening with 3.4 % undergoing invasive testing . proximity to the medical center women of lower education level and black or hispanic ancestry were least likely to desire further screening or testing following their first trimester screen results . it is possible this represents lower health literacy in these groups with less of an appreciation or even misinterpretation of the consequences and benefits of stepwise sequential screening . the amount of information to be collected and conveyed at the initial prenatal visit , in addition to decisions regarding aneuploidy screening , can be overwhelming even for relatively medically sophisticated women . perhaps women of lower education and minorities should be offered pre - conception counseling so that they have more time to consider the benefits and risks of genetic screening and testing . it may also be that women in these groups are more conservative in their approach to prenatal screening overall , and consider themselves less likely to act on the basis of a positive result . additionally , it is possible that their perception and tolerance of risk may be different than women in other groups due to differences in their own frame of reference . how results are reported , and its potential impact on how a woman interprets this information was a focus of this work . a woman whose screen - risk of t21 is 1:310 will be considered to have a negative result and may well be reassured even if this is higher than her age - related risk . by the same token , a woman with a screen - risk of 1:260 will have a result reported as positive or elevated risk even if it is a significant reduction compared to her age - related risk . the difference between 0.32 % ( 1:310 ) and 0.38 % ( 1:260 ) is not great but the implications for a woman s attitude , anxiety and subsequent plans for screening and testing may be significant . for many patients , interpretation of risk the manner in which risk information is presented or framed assumes special importance when it involves communication of genetic testing results . negative framing , in this case the designation of a test result as indicating elevated risk , may have a greater impact on the perceived need for follow - up testing , than the numerical description of risk , which is more understandable to many patients . this is consistent with evidence that numeric communication of risk is often preferred by individuals when making major medical decisions ( lipkus 2007 ) . the intent of our approach was that the women s perceptions of their risks would be based on their screen - risk for t21 relative to their age - risk , and not necessarily relative to the risk of a 35-year old woman . with respect to implications for genetic counselors , an appreciation of the potential impact of framing as it relates to prenatal screening results is appropriate . it is important that the magnitude of the increase in risk , whether increased or decreased , be presented as simply and effectively as possible . classifying a result as positive because the t21 risk exceeds that of a 35-year old , may be less useful and more frightening than expressing the screen - risk relative to a woman s age - risk . verbal communication of risk is commonly used in other areas of life ( it is likely to rain , a certain condition is common , etc . ) . the limitations of this approach when applied to medical screening is that it lacks precision due to variability in interpretation of what represents increased risk . describing a result as negative has the effect of providing maximum reassurance , even is cases where the screen related - risk is similar to a woman s age - related risk . conversely , informing a woman her result is positive is often associated with great stress , which may be ameliorated when the numeric risk is placed in the context of a woman s age - related risk . because of the imprecision associated with terms like positive , negative , high - risk , and low - risk , it is our recommendation that when screen results are discussed , numeric risks should be stressed over verbal description of risks . additionally , an absolute number as expressed by the screen - risk provides the patient with an estimate not only of the risk for t21 , but also the chance the pregnancy is not at risk . a screen - risk of 1:1,000 or less , not an unusual result , predicts not only a 1:1,000 chance for t21 , but roughly a 999:1,000 a fetus does not have t21 . the numerical expression of risk allows not only a precise estimate of the risk of a problem , but also the likelihood of normalcy or absence of a condition . in this way , risks can be presented in a balanced manner , not merely focusing on whether a result is positive or negative . unlike other forms of medical screening , which are designed to predict or prevent disease , prenatal screening provides women with information allowing them to make informed choices regarding their prenatal care as well as continuation or interruption of their pregnancy . in our approach , positive test can be defined practically as one which prompts further screening and/or diagnostic testing . similarly , a negative test provides sufficient reassurance to obviate further testing or to allow diagnostic testing via amniocentesis rather than cvs . as demonstrated by the first and second trimester evaluation of risk ( faster ) trial , detection rates and screen positive rates are modestly improved with the addition of maternal serum quad screening ( ball , et al . however , the majority of our population did not require or desire this information to make informed choices regarding their pregnancy . while this may reflect the generally conservative nature of our population , it may also indicate the information , when presented as described , facilitates women s understanding of the change in absolute risk for fetal trisomy relative to their baseline , age - related risk . this is consistent with evidence that responses to risk often depend on that to which the calculated risk is being compared ( johnson 2004 ) . it also reflects the contextualized nature of prenatal risk assessment and the observation that objective risk and perceived risk are often dissimilar ( marteau 1999 ) . the influence of other factors , including religious , economic , and the perceived effects of a child with aneuploidy on the existing family structure also play an important role in the patient interpretation of and response to screening results ( garcia et al . the availability of first trimester screening and stepwise sequential screening were each associated with a significant decline in the number of amniocenteses performed at our institution . the smaller decrease between first trimester and stepwise sequential screening can be at least partially attributed to the fact only 20 % of our population elected sequential screening . first trimester screening was also associated with a significant reduction in the number of cvs procedures performed . the option of stepwise sequential screening did not further reduce the number of cvs s . this is not unexpected as most pregnancies at increased risk for fetal aneuploidy are identified earlier with the option for first trimester diagnostic testing , obviating the need for mid - trimester testing . this also reflects some cases where the screen risk was lower than the maternal age risk but the reduction was not adequately reassuring , resulting in the decision to proceed with earlier diagnostic testing . with respect to the utilization of invasive procedures , our findings are similar to those described by others . wray , et al . reported that availability of first trimester screening coincided with more women of advanced maternal age ( ama ) referred for early genetic counseling . in 2001 , 68 women were seen for early genetic counseling versus 172 in 2003 . the rates of invasive testing dropped significantly from pre- to post - first trimester screening availability ( 71 % vs 26 % , p < 0.01 ) . the types of invasive testing were not specified ( wray et al . 2005 ) . benn , et al . reviewed a series of nearly 2000 amniocentesis and cvs samples from 1991 to 2002 . they reported a 50 % reduction in the number of invasive procedures over this time . however , this was before the widespread application of first trimester screening and it was not stipulated whether nuchal translucency or first trimester serum markers were included in this analysis . additionally , cvs specimens comprised only 4.3 % of their samples ( benn , et al . it is felt that first trimester screening is the major reason for the decrease in invasive procedures . in our experience , prenatal care providers have embraced this technology and routinely offer this option even to at - risk women , ahead of invasive testing . it is possible this predisposes women to have confidence in first trimester screening sufficient to obviate the need for absolute reassurance via invasive testing with its attendant risks . it is also possible the risks of invasive testing are exaggerated in many womens minds . if you have a result you like , why seek additional information which may detract from your peace of mind ? in our opinion , receiving a very low risk early in pregnancy gives many women the reassurance that their pregnancies are normal . it may be that this makes it easier for some to believe that further testing is unnecessary . similar reassurance can be provided by maternal serum quad screening , but not until later in the pregnancy . the main limitation of our study is that it reflects a local population whose attitudes regarding screening and testing and thresholds for pursuing follow - up may not be reflective of women in other parts of the country . additionally , while most cases of t21 and t18 are reliably diagnosed postnatally , the majority of women did not have cytogenetic testing and postnatal follow - up was unavailable for 64 patients . other limitations include the fact that there is no control population of women who did not receive results reported as positive or negative . it is also plausible that both health care providers and patients are used to considering medical test results as normal or abnormal , and may be less familiar with the notion of comparative quantitative risk assessment . lastly , while noninvasive prenatal testing with cell free fetal dna is presently limited , the anticipated expanded application of such technology will likely limit the utilization of current screening paradigms . therefore , other risk communication and counseling strategies will ultimately need to be developed to suit different circumstances . in summary , communication of risk for aneuploidy is especially important and challenging in the prenatal setting . following the performance of first trimester screening , 80 % of our study population ultimately declined follow - up testing , including nearly one - half of those who initially planned to pursue stepwise sequential screening . the policy of providing patients with their results by specifying their age - related risk , their revised screen - related risk and further options , without classifying results as positive or negative , seemed to communicate risk in a manner which was understandable and clinically useful , with minimal bias . this also resulted in far fewer stepwise sequential screens being performed than would be predicted using most current sequential screening paradigms . the introduction and increasing utilization of first trimester combined screening has been accompanied by a steady decrease in the number of invasive diagnostic procedures , which is more pronounced for amniocentesis than cvs .

the types , interpretation , and use of first- and second - trimester aneuploidy screening are often unclear for many women . this impairs appropriate decision making and understanding of the implications of prenatal genetic testing options . the purpose of this study was to examine the utilization of stepwise sequential screening in our midwestern population , demographic factors associated with choice of screening and method of risk reporting and it s potential impact on women s choices . first trimester screening was performed for 2,634 women during the study period . results were not reported or framed as positive or negative . rather , the specific age - risk and screen - risk for t21 were relayed , along with options for follow - up stepwise sequential screening and invasive testing . nearly 80 % of women declined stepwise sequential screening . minorities and women of lower education were least likely to pursue further screening . less than 4 % of the study population elected invasive testing . first trimester screening was associated with a 53 % reduction in amniocenteses and 20 % fewer cvs s compared to pre - first trimester screening availability . reporting age - and screen - risks for t21 , rather than classifying results as positive or negative based on a pre - determined threshold , was associated with a low uptake of further testing .

Introduction Methods Results Discussion Conclusion

in january 2007 , the american college of obstetricians and gynecologists ( acog ) practice bulletin number 77 recommended screening and invasive diagnostic testing should be available to all women who present for prenatal care before 20 weeks of gestation regardless of maternal age . this includes first trimester nuchal translucency , pregnancy - associated plasma protein a ( papp - a ) and human chorionic gonadotropin ( hcg ) plus maternal serum quad screening in the mid - trimester , with the results provided only after all tests are completed . patient anxiety during the delay between presentation for screening and results make this an unacceptable option for many patients . proposed alternative screening options include stepwise sequential screening and contingent sequential screening based on classifying women as high- or low - risk using a predetermined threshold ( american college of obstetricians and gynecologists acog 2007 ) . stepwise sequential screening is first trimester combined testing plus maternal serum quad screening with results provided after each test . prenatal aneuploidy screening results are often reported or framed as positive or negative based on a defined threshold . the goals of this study were to examine : 1- the uptake of stepwise sequential screening in our primarily urban , midwestern population , 2- the potential affects of ethnicity , education and proximity to the medical center on willingness to proceed with further testing , and 3- our method of risk reporting , which emphasized a woman s age - risk for t21 and adjusted risk after screening , rather than classifying a result as positive or negative , or high - risk , or low - risk . for the first 3 months of the study period , the laboratory utilized at this institution used total beta - hcg . patient s were categorized at the time of their first trimester screening visit into one of three groups as follows : u = undecided as to whether they wished to proceed with stepwise sequential screening ; y = yes , they planned to pursue stepwise sequential screening ; or n = no , they do not plan to have stepwise sequential screening . results of first trimester screening were not reported as positive or negative . the phone call and/or letter informing patients of their first trimester screen results indicated the patient s age - risk for ( t21 ) and trisomy 18 ( t18 ) and their revised risks for same based on the nt and biochemical markers . all phone calls and/or result letters also again reviewed the patient s subsequent options , including no further screening , maternal serum quad screening , mid - trimester ultrasound and invasive testing via chorionic villus sampling ( cvs ) or amniocentesis . the patient s decision about whether to proceed with stepwise sequential screening ( both before and after their first trimester screening results ) were recorded in a computerized database for subsequent correlation with variables including ethnicity , level of education , distance from our medical center , nt measurement , crown rump length , age- and screen - risks for t21 , results of invasive testing and pregnancy outcomes . in order to assess the impact of non - invasive first trimester screening and stepwise sequential screening ( first trimester screening plus maternal serum quad screening ) on the utilization of amniocentesis and cvs , the number of these procedures was compared as follows : for a 2-year period prior to the initiation of first trimester screening ( january 2000december 2001 ) , for a 2-year period when first trimester screening was utilized but prior to the availability of stepwise sequential screening ( june 2005may 2007 ) and for the 2-year period following initiation of stepwise sequential screening ( july 2007june 2009 ) . the study population consists of 2,643 patients who met inclusion criteria during the study period ( june 2007february 2010 ) . at the time of the first trimester examination , 1,926 ( 72.8 % ) were undecided regarding stepwise sequential screening ( u group ) pending their first trimester results . 434 patients ( 16.4 % ) indicated they planned to return for stepwise sequential screening , regardless of their results ( y group ) and 283 patients ( 10.7 % ) declined stepwise sequential screening at the time of their first trimester screening ( n group ) . this included a minority of the undecided and no groups ( 14.9 % and 1.4 % , respectively ) and only 56.4 % of those who originally intended to have stepwise sequential screening . overall , 79.8 % of the study population elected not to pursue follow - up screening.table 1stepwise sequential screening following first trimester combined screengroupnno ss screeningfollow - up ss screening p undecided1,926 ( 72.9 % ) 1,639 ( 85.1 % ) 287 ( 14.9 % ) < 0.0001yes434 ( 16.4 % ) 190 ( 43.8 % ) 244 ( 56.2 % ) no283 ( 10.7 % ) 279 ( 98.6 % ) 4 ( 1.4 % ) 2,643 ( 100 % ) 2,108 ( 79.8 % ) 535 ( 20.2 % ) u vs y<0.0001u vs n<0.0001y vs n<0.0001 stepwise sequential screening following first trimester combined screen the relation between education level and choice of screening is presented in table 2 . in the group of women who did not plan to pursue stepwise sequential screening there were a significantly greater number who were not high school graduates and significantly fewer who were college graduates compared to women in the other two groups . table 3 demonstrates that women who declined stepwise sequential screening were more likely to be black and hispanic and less likely to be white , than women in the other groups . table 4 indicates that distance from the medical center did not correlate with the decision to pursue or decline stepwise sequential screening.table 2eduction level and decision to pursue stepwise sequential screeninggroupnsome high schoolhs grad some collegecollege grad post grad p < 0.0001undecided1,92368 ( 3.5 % ) 501 ( 26.1 % ) 1,354 ( 70.4 % ) yes43411 ( 2.5 % ) 115 ( 26.5 % ) 308 ( 71.0 % ) no28222 ( 7.8 % ) 111 ( 39.4 % ) 149 ( 52.8 % ) 2,639101 ( 3.8 % ) 727 ( 27.5 % ) 1,811 ( 68.6 % ) u vs. y0.58u vs. n<0.0001y vs n<0.0001table 3ethnicity and decision to pursue stepwise sequential screeningethnicitygroupnwhiteblackasianhispother p < 0.0001undecided1,9261,395 ( 72.4 % ) 293 ( 15.2 % ) 106 ( 5.5 % ) 49 ( 2.5 % ) 83 ( 4.3 % ) yes434326 ( 75.1 % ) 50 ( 11.5 % ) 18 ( 4.1 % ) 8 ( 1.8 % ) 32 ( 7.4 % ) no283165 ( 58.3 % ) 71 ( 25.1 % ) 10 ( 3.5 % ) 17 ( 6.0 % ) 20 ( 7.1 % ) 2,6431,886 ( 71.4 % ) 414 ( 15.7 % ) 134 ( 5.1 % ) 74 ( 2.8 % ) 135 ( 5.1 % ) u vs. n0.01u vs. n<0.0001y vs. n<0.001table 4distance from medical center and decision to pursue stepwise sequential screeningmiles p groupn0202150>500.14undecided1,9261,293 ( 67.1 % ) 507 ( 26.3 % ) 126 ( 6.5 % ) yes434284 ( 65.4 % ) 114 ( 26.4 % ) 36 ( 8.3 % ) no283170 ( 60.1 % ) 88 ( 31.1 % ) 25 ( 8.8 % ) 2,6431,747 ( 66.1 % ) 709 ( 26.8 % ) 187 ( 7.1 % ) u vs. y0.42u vs. n0.05y vs. n0.32 eduction level and decision to pursue stepwise sequential screening ethnicity and decision to pursue stepwise sequential screening distance from medical center and decision to pursue stepwise sequential screening tables 5 and 6 demonstrate that the percentages of women at risk for t21 were similar between the groups.table 5age risk for t21 and decision to pursue stepwise sequential screeninggroup1:501:511:270>1:270 p*undecided ( n-1,926)44 ( 2.3 % ) 896 ( 46.5 % ) 986 ( 51.2 % ) 0.26yes ( n-434)17 ( 3.9 % ) 204 ( 47.0 % ) 213 ( 49.1 % ) no ( n-283)9 ( 2.6 % ) 140 ( 49.5 % ) 134 ( 47.3 % ) u vs. y0.14u vs. n0.37y vs. n0.75*fishers exact testtable 6screen risk for t21 and decision to pursue stepwise sequential screeninggroup1:501:511:270>1:270 p * 0.63undecided ( n-1,926)26 ( 1.3 % ) 86 ( 4.5 % ) 1,814 ( 94.2 % ) yes ( n-434)7 ( 1.6 % ) 21 ( 4.8 % ) 406 ( 93.5 % ) no ( n-283)7 ( 2.5 % ) 13 ( 4.6 % ) 263 ( 92.9 % ) uvs . y0.79u vs. n0.30yvsn.0.74*fishers exact test age risk for t21 and decision to pursue stepwise sequential screening screen risk for t21 and decision to pursue stepwise sequential screening the number of patients whose first trimester screen - risk for t21 exceeded their age - risk , and their choice of follow - up , is depicted in table 7 . overall , 126 patients ( 4.8 % of the study population ) had a first trimester screen - risk for t21 which was greater than their age - risk . of these , 73(57.9 % ) women elected not to have follow - up stepwise sequential screening or diagnostic testing , 24(19.8 % ) had stepwise sequential screening only and 29(23.0 % ) had invasive testing via cvs ( n = 11 ) or amniocentesis ( n = 18 ) . women who originally declined stepwise sequential screening were significantly less likely to pursue invasive testing than women in the other two groups.table 7screen risk for t21 greater than age risk and choice of follow - upgroupsr > ar p no further testingss screen onlyinvasive testing p*undecided ( n-1,926)91 ( 4.7 % ) 0.9852 ( 57.9 % ) 15 ( 19.0 % ) 24 ( 26.4 % ) yes ( n-434)21 ( 4.8 % ) 8 ( 38.1 % ) 9 ( 42.9 % ) 4 ( 19.0 % ) no ( n-283)14 ( 4.6 % ) 13 ( 92.9 % ) 01 ( 7.1 % ) 2,643126 ( 4.8 % ) 73 ( 57.9 % ) 24 ( 19.0 % ) 29 ( 23.0 % ) u vs. y0.92u vs. y0.05u vs. n0.87u vs n0.04y vs. n0.99y vs. n0.002*fisher 's exact test screen risk for t21 greater than age risk and choice of follow - up a total of 91(3.4 % of the study population ) women had invasive testing by amniocentesis ( n = 76 ) or cvs ( n = 15 ) during the study period . in 59(64.8 % ) , the age - risk for t21 was greater than the screen - risk . the number of invasive procedures performed prior to the availability of first trimester screening , following initiation of first trimester screening and following the availability of stepwise sequential screening is indicated in table 8 . the number of amniocenteses in our center declined by 51.4 % following the availability of first trimester screening with a further 17.4 % reduction upon initiation of stepwise sequential screening . there was a significant reduction in the number of cvs procedures from pre - first trimester screening and its utilization . however , there was no significant difference in the number of cvs procedures between availability of first trimester screening and stepwise sequential screening.table 8number of invasive procedures before and after first trimester and stepwise sequential screening availabilityprocedurepre - ftsfts onlyss availp , pre - fts vs fts onlyp , fts only vs. ss availamniocentesis1,632793655 p < 0.0001 p < 0.0001cvs494399371 p < 0.001 p = 0.31 first trimester screening sequential screening number of invasive procedures before and after first trimester and stepwise sequential screening availability first trimester screening sequential screening in our population , only 20 % of women having first trimester screening , elected to pursue stepwise sequential screening with 3.4 % undergoing invasive testing . proximity to the medical center women of lower education level and black or hispanic ancestry were least likely to desire further screening or testing following their first trimester screen results . it is possible this represents lower health literacy in these groups with less of an appreciation or even misinterpretation of the consequences and benefits of stepwise sequential screening . perhaps women of lower education and minorities should be offered pre - conception counseling so that they have more time to consider the benefits and risks of genetic screening and testing . how results are reported , and its potential impact on how a woman interprets this information was a focus of this work . by the same token , a woman with a screen - risk of 1:260 will have a result reported as positive or elevated risk even if it is a significant reduction compared to her age - related risk . for many patients , interpretation of risk the manner in which risk information is presented or framed assumes special importance when it involves communication of genetic testing results . negative framing , in this case the designation of a test result as indicating elevated risk , may have a greater impact on the perceived need for follow - up testing , than the numerical description of risk , which is more understandable to many patients . the intent of our approach was that the women s perceptions of their risks would be based on their screen - risk for t21 relative to their age - risk , and not necessarily relative to the risk of a 35-year old woman . classifying a result as positive because the t21 risk exceeds that of a 35-year old , may be less useful and more frightening than expressing the screen - risk relative to a woman s age - risk . because of the imprecision associated with terms like positive , negative , high - risk , and low - risk , it is our recommendation that when screen results are discussed , numeric risks should be stressed over verbal description of risks . additionally , an absolute number as expressed by the screen - risk provides the patient with an estimate not only of the risk for t21 , but also the chance the pregnancy is not at risk . a screen - risk of 1:1,000 or less , not an unusual result , predicts not only a 1:1,000 chance for t21 , but roughly a 999:1,000 a fetus does not have t21 . as demonstrated by the first and second trimester evaluation of risk ( faster ) trial , detection rates and screen positive rates are modestly improved with the addition of maternal serum quad screening ( ball , et al . while this may reflect the generally conservative nature of our population , it may also indicate the information , when presented as described , facilitates women s understanding of the change in absolute risk for fetal trisomy relative to their baseline , age - related risk . the availability of first trimester screening and stepwise sequential screening were each associated with a significant decline in the number of amniocenteses performed at our institution . the smaller decrease between first trimester and stepwise sequential screening can be at least partially attributed to the fact only 20 % of our population elected sequential screening . first trimester screening was also associated with a significant reduction in the number of cvs procedures performed . the option of stepwise sequential screening did not further reduce the number of cvs s . this is not unexpected as most pregnancies at increased risk for fetal aneuploidy are identified earlier with the option for first trimester diagnostic testing , obviating the need for mid - trimester testing . reported that availability of first trimester screening coincided with more women of advanced maternal age ( ama ) referred for early genetic counseling . the rates of invasive testing dropped significantly from pre- to post - first trimester screening availability ( 71 % vs 26 % , p < 0.01 ) . the types of invasive testing were not specified ( wray et al . however , this was before the widespread application of first trimester screening and it was not stipulated whether nuchal translucency or first trimester serum markers were included in this analysis . in our experience , prenatal care providers have embraced this technology and routinely offer this option even to at - risk women , ahead of invasive testing . it is possible this predisposes women to have confidence in first trimester screening sufficient to obviate the need for absolute reassurance via invasive testing with its attendant risks . the main limitation of our study is that it reflects a local population whose attitudes regarding screening and testing and thresholds for pursuing follow - up may not be reflective of women in other parts of the country . additionally , while most cases of t21 and t18 are reliably diagnosed postnatally , the majority of women did not have cytogenetic testing and postnatal follow - up was unavailable for 64 patients . other limitations include the fact that there is no control population of women who did not receive results reported as positive or negative . lastly , while noninvasive prenatal testing with cell free fetal dna is presently limited , the anticipated expanded application of such technology will likely limit the utilization of current screening paradigms . following the performance of first trimester screening , 80 % of our study population ultimately declined follow - up testing , including nearly one - half of those who initially planned to pursue stepwise sequential screening . the policy of providing patients with their results by specifying their age - related risk , their revised screen - related risk and further options , without classifying results as positive or negative , seemed to communicate risk in a manner which was understandable and clinically useful , with minimal bias .

the institutional review board and ethics committee of our hospital approved this study . from a consecutive series of images of suspicious masses in 183 patients ( age range , 17 - 80 years ; mean age , 44 years ) , 97 benign and 86 malignant cases were identified . the masses had undergone fine needle aspiration cytology , a core biopsy or / and an excisional biopsy and patients were subjected to an us examination with the use of both non - harmonic and thi . the largest tumor was selected in the study if more than one lesion was detected in one patient and a tumor size larger than 3.1 cm was excluded because of probe limitations . malignant masses included infiltrating ductal carcinoma ( n = 75 ) , apocrine carcinoma ( n = 3 ) , ductal carcinoma in situ ( n = 5 ) , papillary carcinoma ( n = 1 ) , mucinous carcinoma ( n = 1 ) and intracystic carcinoma ( n = 1 ) . benign lesions included fibroadenoma ( n = 81 ) , papilloma ( n = 1 ) , phylloid ( n = 1 ) , sclerosing adenosis ( n = 2 ) and other benign tumors ( n = 12 ) . non - harmonic and thi scans were obtained using a voluson 730 scanner ( ge healthcare , zipf , austria ) with a linear - array broadband 6 - 12 mhz transducer . the transducer had a relative stopping power ( rsp ) index of 6 - 12 . a fixed installed 3d power doppler setting for all us examinations was used as follows : a sweep angle of 5 to 29 , ' low 1 ' wall motion filter , 0.9 khz pulse repetition frequency , -0.6 gain and mid frequency . patients were examined in the supine position and were asked to hold their breath while the scanner generated the 3d volume . all of the acquired 3d power doppler non - harmonic and thi scans were stored on a disc and no compression of the data was used at any time . acquired 3d volumes were transferred to a personal computer using a digital imaging and communications in medicine ( dicom ) connection for later offline imaging analysis . the virtual organ computer - aided analysis ( vocal)-imaging program ( version 2.1 ) was used to analyze the stored volume with a personal computer ( m5 , asus , taipei , taiwan ) . detailed measurements and the application of the vocal - imaging program have been reported previously ( 19 - 21 ) . in brief , the program can be used to calculate the histogram indices of the vascularity and blood flow obtained from quantitative 3d power doppler us scanning . both gray - scale and color scale voxels are graded from the lowest value ( intensity , 0 ) and the highest value ( intensity , 100 ) . the stored us volume is defined by the smallest unit of volume , also a voxel . three histogram indices , the vascularization index ( vi ) , flow index ( fi ) and vascularization flow index ( vfi ) were calculated using these values . in a previous study , 19 ) have described these indices and detailed formulas . in brief , vi represents the vessels in tissue and vi is expressed as a percentage ; fi represents the average intensity of flow ( i.e. , the mean color value of the color voxels ) ; vfi represents both vascularization and flow ( i.e. , the mean color value of all of the voxels in the obtained volume ) . mean grayness ( mg ) is a representation of the average grayness in the gray voxels of a sphere . during measurement for all non - harmonic and harmonic 3d power doppler image analyses , we used the vocal - program with the manual model with a rotation step of 30 to perform contour defining . the volume was obtained , since contours in the six image planes were determined ; the histogram indices mg , vi , fi and vfi were determined once the contour was accepted ( fig . one signal was the shell - off contour ( i.e. , the contour around the tumor margin ) and the other signal was the outside shell with a thickness of 3 mm . we labeled the histogram indices vi , fi and vfi for the intra - tumor ( invi , infi and invfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( out3mmvi , out3mmfi and out3mmvfi ) . in addition , two vascular signals were obtained for the thi scans , the histogram indices of vi , fi and vfi for the intra - tumor ( hinvi , hinfi and hinvfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( hout3mmvi , hout3mmfi and hout3mmvfi ) ( fig . 2 ) . briefly , histogram indices representing the mg and vascularity characteristics ( vi , fi and vfi ) of a mass were measured quantitatively from sonograms for both 3d power doppler non - harmonic and thi . vascularity indices were recorded as 0 in scans with no vascularization as determined with the use of 3d power doppler us . imaging analysis was performed by one physician using the virtual organ computer - aided analysis ( vocal tm ) imaging program ( version 2.1 , ge medical systems ) who was blinded to the histology results . were employed in a comparative analysis for the characterization of benign and malignant solid breast masses using 3d power doppler imaging with the use of both non - harmonic us and thi . the performance of each statistical method included the overall parameters of invi , infi , invfi , out3mmvi , out3mmfi and out3mmvfi . the performance measurements , including diagnostic accuracy , sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and the area ( az ) under the roc curve were calculated to estimate the performance of the diagnostic system . all p values < 0.05 were considered to be statistically significant . for a pool of n samples , n-1 was fitted to an lra model defined by log ( p ) = + 1x1 + 2x2 + + kxk , where p is the probability of a malignancy , is a constant , 1 , 2 , , k are logistic regression coefficients and x1 , x2 , , xk are parameters ; and determine the shape of the logistic curve . we employed the statistical package for the social sciences ( spss 15.0 statistical software , spss , chicago , il ) to calculate the multivariate lra of patient age , tumor volume and the vascularization indices of the benign and malignant breast tumors . an svm is a machine learning system developed using statistical learning theories to classify data points into two classes . notably , svm models have been applied extensively for classification , image recognition and bioinformatics . the svm has been shown to be an effective tool in sonography for the diagnosis of breast cancer ( 16 , 17 , 22 ) . of the possible hyperplanes , only one hyperplane maximizes the margin ( distance between the hyperplane ) and the nearest data point of each class . figure 3 shows an optimal separating hyperplane with the largest margin . the value produced by the output node is used to decide whether a tumor is benign or malignant . for an output value 0 , the svm system will classify the tumor as malignant ; conversely , for an output value < 0 , the tumor will be diagnosed as benign . neural networks have been successfully applied for aided diagnoses of solid breast nodules in us examinations . chen et al . ( 9 ) have described in detail the application of cad to us of solid breast nodules by the use of nns . the function of neurons in the hidden layer is to arbitrate between the input and output of the nn . the input vector is first supplied to the source nodes in the input layer . the neurons of the input layer constitute the signals applied to the neurons of the hidden layer . the output results of the hidden layer are employed as inputs to the next hidden layer . the output layer eventually generates the results and terminates the nn computing procedure . among the learning algorithms used to train an nn the learning method iteratively executes the back - propagation algorithm for the training set and then produces the final synaptic weight vectors . the nn model has been used as a classifier by applying the final synaptic weight vectors to identify the tumor as benign or malignant . the input signals of the nn classifier contain a feature vector that comprises the vascularity indices of each breast tumor . the tumor is classified as benign if the output value is close to 0 ; the tumor is classified as malignant if the output value is near to 1 . the institutional review board and ethics committee of our hospital approved this study . from a consecutive series of images of suspicious masses in 183 patients ( age range , 17 - 80 years ; mean age , 44 years ) , 97 benign and 86 malignant cases were identified . the masses had undergone fine needle aspiration cytology , a core biopsy or / and an excisional biopsy and patients were subjected to an us examination with the use of both non - harmonic and thi . the largest tumor was selected in the study if more than one lesion was detected in one patient and a tumor size larger than 3.1 cm was excluded because of probe limitations . malignant masses included infiltrating ductal carcinoma ( n = 75 ) , apocrine carcinoma ( n = 3 ) , ductal carcinoma in situ ( n = 5 ) , papillary carcinoma ( n = 1 ) , mucinous carcinoma ( n = 1 ) and intracystic carcinoma ( n = 1 ) . benign lesions included fibroadenoma ( n = 81 ) , papilloma ( n = 1 ) , phylloid ( n = 1 ) , sclerosing adenosis ( n = 2 ) and other benign tumors ( n = 12 ) . non - harmonic and thi scans were obtained using a voluson 730 scanner ( ge healthcare , zipf , austria ) with a linear - array broadband 6 - 12 mhz transducer . the transducer had a relative stopping power ( rsp ) index of 6 - 12 . a fixed installed 3d power doppler setting for all us examinations was used as follows : a sweep angle of 5 to 29 , ' low 1 ' wall motion filter , 0.9 khz pulse repetition frequency , -0.6 gain and mid frequency . patients were examined in the supine position and were asked to hold their breath while the scanner generated the 3d volume . all of the acquired 3d power doppler non - harmonic and thi scans were stored on a disc and no compression of the data was used at any time . acquired 3d volumes were transferred to a personal computer using a digital imaging and communications in medicine ( dicom ) connection for later offline imaging analysis . the virtual organ computer - aided analysis ( vocal)-imaging program ( version 2.1 ) was used to analyze the stored volume with a personal computer ( m5 , asus , taipei , taiwan ) . detailed measurements and the application of the vocal - imaging program have been reported previously ( 19 - 21 ) . in brief , the program can be used to calculate the histogram indices of the vascularity and blood flow obtained from quantitative 3d power doppler us scanning . both gray - scale and color scale voxels are graded from the lowest value ( intensity , 0 ) and the highest value ( intensity , 100 ) . the stored us volume is defined by the smallest unit of volume , also a voxel . three histogram indices , the vascularization index ( vi ) , flow index ( fi ) and vascularization flow index ( vfi ) were calculated using these values . in a previous study , 19 ) have described these indices and detailed formulas . in brief , vi represents the vessels in tissue and vi is expressed as a percentage ; fi represents the average intensity of flow ( i.e. , the mean color value of the color voxels ) ; vfi represents both vascularization and flow ( i.e. , the mean color value of all of the voxels in the obtained volume ) . mean grayness ( mg ) is a representation of the average grayness in the gray voxels of a sphere . during measurement for all non - harmonic and harmonic 3d power doppler image analyses , we used the vocal - program with the manual model with a rotation step of 30 to perform contour defining . the volume was obtained , since contours in the six image planes were determined ; the histogram indices mg , vi , fi and vfi were determined once the contour was accepted ( fig . one signal was the shell - off contour ( i.e. , the contour around the tumor margin ) and the other signal was the outside shell with a thickness of 3 mm . we labeled the histogram indices vi , fi and vfi for the intra - tumor ( invi , infi and invfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( out3mmvi , out3mmfi and out3mmvfi ) . in addition , two vascular signals were obtained for the thi scans , the histogram indices of vi , fi and vfi for the intra - tumor ( hinvi , hinfi and hinvfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( hout3mmvi , hout3mmfi and hout3mmvfi ) ( fig . 2 ) . briefly , histogram indices representing the mg and vascularity characteristics ( vi , fi and vfi ) of a mass were measured quantitatively from sonograms for both 3d power doppler non - harmonic and thi . vascularity indices were recorded as 0 in scans with no vascularization as determined with the use of 3d power doppler us . imaging analysis was performed by one physician using the virtual organ computer - aided analysis ( vocal tm ) imaging program ( version 2.1 , ge medical systems ) who was blinded to the histology results . logistic regression analysis , svm and nn were employed in a comparative analysis for the characterization of benign and malignant solid breast masses using 3d power doppler imaging with the use of both non - harmonic us and thi . the performance of each statistical method included the overall parameters of invi , infi , invfi , out3mmvi , out3mmfi and out3mmvfi . the performance measurements , including diagnostic accuracy , sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and the area ( az ) under the roc curve were calculated to estimate the performance of the diagnostic system . all p values < 0.05 were considered to be statistically significant . for a pool of n samples , n-1 was fitted to an lra model defined by log ( p ) = + 1x1 + 2x2 + + kxk , where p is the probability of a malignancy , is a constant , 1 , 2 , , k are logistic regression coefficients and x1 , x2 , , xk are parameters ; and determine the shape of the logistic curve . we employed the statistical package for the social sciences ( spss 15.0 statistical software , spss , chicago , il ) to calculate the multivariate lra of patient age , tumor volume and the vascularization indices of the benign and malignant breast tumors . an svm is a machine learning system developed using statistical learning theories to classify data points into two classes . notably , svm models have been applied extensively for classification , image recognition and bioinformatics . the svm has been shown to be an effective tool in sonography for the diagnosis of breast cancer ( 16 , 17 , 22 ) . of the possible hyperplanes , only one hyperplane maximizes the margin ( distance between the hyperplane ) and the nearest data point of each class . figure 3 shows an optimal separating hyperplane with the largest margin . the value produced by the output node is used to decide whether a tumor is benign or malignant . for an output value 0 , the svm system will classify the tumor as malignant ; conversely , for an output value < 0 , the tumor will be diagnosed as benign . neural networks have been successfully applied for aided diagnoses of solid breast nodules in us examinations . chen et al . ( 9 ) have described in detail the application of cad to us of solid breast nodules by the use of nns . the function of neurons in the hidden layer is to arbitrate between the input and output of the nn . the input vector is first supplied to the source nodes in the input layer . the neurons of the input layer constitute the signals applied to the neurons of the hidden layer . the output results of the hidden layer are employed as inputs to the next hidden layer . the output layer eventually generates the results and terminates the nn computing procedure . among the learning algorithms used to train an nn the learning method iteratively executes the back - propagation algorithm for the training set and then produces the final synaptic weight vectors . the nn model has been used as a classifier by applying the final synaptic weight vectors to identify the tumor as benign or malignant . the input signals of the nn classifier contain a feature vector that comprises the vascularity indices of each breast tumor . the tumor is classified as benign if the output value is close to 0 ; the tumor is classified as malignant if the output value is near to 1 . a total of 183 solid breast image pairs ( as measured with non - harmonic and harmonic 3d power doppler imaging ) were analyzed . the sizes of the lesions ranged from 6.00 - 31.00 mm ( mean size , 18.97 mm ) for malignant tumors and 5.00 - 30.20 mm ( mean size , 14.73 mm ) for benign lesions . histogram indices for the use of 3d power doppler non - harmonic us scans were calculated . twenty - three benign and two malignant cases were detected without intra - tumor vascularity ; 14 benign cases and one malignant case were detected without vascular signals in shells with a thickness of 3 mm surrounding the breast lesions as determined by use of the vocal program . table 1 summarizes the accuracy , sensitivity , specificity , ppv and npv for the use of 3d power doppler us non - harmonic and thi for the differentiation of benign from malignant breast tumors by the use of the different statistical methods . table 2 shows the az value of roc curve analysis for the diagnostic performance of lra , svm and nn for 3d power doppler non - harmonic and thi of solid breast tumors . the statistical significance of the difference comparing two roc curves ( lra versus svm , lra versus nn and svm versus nn , respectively ) is also presented in table 2 . the computation time of the cads using the svm and nn classifiers is displayed in table 3 . the training time was evaluated by the use of the us image database containing 183 images . analysis of az by lra , svm and nn for non - harmonic and harmonic imaging using 3d power doppler us is shown in figures 4 and 5 . in our study , satisfactory results were shown by the use of both harmonic and non - harmonic 3d power doppler sonographic imaging to classify benign and malignant breast tumors by vascularization , and lra , an svm or nn were applied to assess diagnostic performance . an nn and svm have been shown to be acceptable diagnostic models for cad systems . vascularity scoring with 3d power doppler us can predict the malignant potential of breast tumors , as has been recently demonstrated ( 7 ) . our study demonstrated that the areas under the six roc curves of lra , the svm and nn for nonharmonic or harmonic 3d power doppler imaging were similar . we conclude that there was no difference in diagnostic ability among these three statistical models by applying the models to both non - harmonic and harmonic 3d power doppler images . in a study by song et al . ( 23 ) that compared lra with an nn for 24 malignant and 30 benign masses , no difference in performance for the masses as measured by the area under the roc curve was demonstrated ; however , the nn had better specificity than lra for a fixed sensitivity . however , it is difficult to assess whether the advantage of an nn as compared to lra in a local region of an roc curve is statistically significant . in several previous studies ( 11 , 14 , 15 ) , a diagnostic model based on an nn showed promise for breast tumor diagnosis . in our study , the lra approach for non - harmonic imaging had the highest az value ( 0.9341 ) and the nn method for non - harmonic imaging had the highest level of accuracy ( 88% ) . in a study by huang and chen ( 17 ) that compared multilayer perception neural networks ( mlps ) with an svm for 140 us images of solid breast nodules , the investigators demonstrated that the training and diagnosis procedures for the proposed svm model were 700-fold and 2380-fold faster as compared to the mlps , respectively . furthermore , the results from the use of the svm model revealed better classification performance by the use of texture analysis . the time consumed for training and diagnosis procedures for the nn classifier were 39.97-fold and 58.75-fold that of the use of the svm cad in our study . the diagnostic performances as determined by the az values for the use of lra , the svm and nn were nearly identical , and the shapes of the six curves were similar ( figs . 4 , 5 ) . ( 23 ) reported that the use of an nn had a higher specificity as compared with lra at a fixed 95% sensitivity . according to our results , the left portion of the roc curve of ' non - harmonic'_lra was extended more to the left side as compared with ' non - harmonic ' _ svm or ' non - harmonic'_nn ( fig . this finding implies that lra for non - harmonic 3d power doppler imaging has better specificity as compared to the svm or nn for a fixed sensitivity . for harmonic imaging , ' harmonic'_lra is extended more to the left side as compared with ' harmonic ' svm or ' harmonic'_nn ( fig . it is difficult to appraise whether the advantage of the use of lra as compared to the other models for the local region of an roc curve reaches statistical significance . furthermore , it is difficult to compare differences among investigations , as the application of different parameters ( such as texture analysis or vascular indices ) to the same model does not necessarily indicate identical diagnostic performance . in summary , moreover , lra for both non - harmonic and harmonic 3d power doppler imaging had better specificity as compared to the svm or nn for a fixed sensitivity .

objectivelogistic regression analysis ( lra ) , support vector machine ( svm ) and a neural network ( nn ) are commonly used statistical models in computer - aided diagnostic ( cad ) systems for breast ultrasonography ( us ) . the aim of this study was to clarify the diagnostic ability of the use of these statistical models for future applications of cad systems , such as three - dimensional ( 3d ) power doppler imaging , vascularity evaluation and the differentiation of a solid mass.materials and methodsa database that contained 3d power doppler imaging pairs of non - harmonic and tissue harmonic images for 97 benign and 86 malignant solid tumors was utilized . the virtual organ computer - aided analysis - imaging program was used to analyze the stored volumes of the 183 solid breast tumors . lra , an svm and nn were employed in comparative analyses for the characterization of benign and malignant solid breast masses from the database.resultsthe values of area under receiver operating characteristic ( roc ) curve , referred to as az values for the use of non - harmonic 3d power doppler us with lra , svm and nn were 0.9341 , 0.9185 and 0.9086 , respectively . the az values for the use of harmonic 3d power doppler us with lra , svm and nn were 0.9286 , 0.8979 and 0.9009 , respectively . the az values of six roc curves for the use of lra , svm and nn for non - harmonic or harmonic 3d power doppler imaging were similar.conclusionthe diagnostic performances of these three models ( lra , svm and nn ) are not different as demonstrated by roc curve analysis . depending on user emphasis for the use of roc curve findings , the use of lra appears to provide better sensitivity as compared to the other statistical models .

MATERIALS AND METHODS Patients 3D Power Doppler US Examinations Imaging Analysis Statistical Analysis Logistic Regression Analysis Support Vector Machine Neural Network RESULTS DISCUSSION

the institutional review board and ethics committee of our hospital approved this study . from a consecutive series of images of suspicious masses in 183 patients ( age range , 17 - 80 years ; mean age , 44 years ) , 97 benign and 86 malignant cases were identified . the masses had undergone fine needle aspiration cytology , a core biopsy or / and an excisional biopsy and patients were subjected to an us examination with the use of both non - harmonic and thi . the largest tumor was selected in the study if more than one lesion was detected in one patient and a tumor size larger than 3.1 cm was excluded because of probe limitations . malignant masses included infiltrating ductal carcinoma ( n = 75 ) , apocrine carcinoma ( n = 3 ) , ductal carcinoma in situ ( n = 5 ) , papillary carcinoma ( n = 1 ) , mucinous carcinoma ( n = 1 ) and intracystic carcinoma ( n = 1 ) . benign lesions included fibroadenoma ( n = 81 ) , papilloma ( n = 1 ) , phylloid ( n = 1 ) , sclerosing adenosis ( n = 2 ) and other benign tumors ( n = 12 ) . non - harmonic and thi scans were obtained using a voluson 730 scanner ( ge healthcare , zipf , austria ) with a linear - array broadband 6 - 12 mhz transducer . a fixed installed 3d power doppler setting for all us examinations was used as follows : a sweep angle of 5 to 29 , ' low 1 ' wall motion filter , 0.9 khz pulse repetition frequency , -0.6 gain and mid frequency . all of the acquired 3d power doppler non - harmonic and thi scans were stored on a disc and no compression of the data was used at any time . the virtual organ computer - aided analysis ( vocal)-imaging program ( version 2.1 ) was used to analyze the stored volume with a personal computer ( m5 , asus , taipei , taiwan ) . detailed measurements and the application of the vocal - imaging program have been reported previously ( 19 - 21 ) . in brief , the program can be used to calculate the histogram indices of the vascularity and blood flow obtained from quantitative 3d power doppler us scanning . both gray - scale and color scale voxels are graded from the lowest value ( intensity , 0 ) and the highest value ( intensity , 100 ) . the stored us volume is defined by the smallest unit of volume , also a voxel . three histogram indices , the vascularization index ( vi ) , flow index ( fi ) and vascularization flow index ( vfi ) were calculated using these values . , the mean color value of the color voxels ) ; vfi represents both vascularization and flow ( i.e. , the mean color value of all of the voxels in the obtained volume ) . mean grayness ( mg ) is a representation of the average grayness in the gray voxels of a sphere . during measurement for all non - harmonic and harmonic 3d power doppler image analyses , we used the vocal - program with the manual model with a rotation step of 30 to perform contour defining . , the contour around the tumor margin ) and the other signal was the outside shell with a thickness of 3 mm . we labeled the histogram indices vi , fi and vfi for the intra - tumor ( invi , infi and invfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( out3mmvi , out3mmfi and out3mmvfi ) . in addition , two vascular signals were obtained for the thi scans , the histogram indices of vi , fi and vfi for the intra - tumor ( hinvi , hinfi and hinvfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( hout3mmvi , hout3mmfi and hout3mmvfi ) ( fig . briefly , histogram indices representing the mg and vascularity characteristics ( vi , fi and vfi ) of a mass were measured quantitatively from sonograms for both 3d power doppler non - harmonic and thi . vascularity indices were recorded as 0 in scans with no vascularization as determined with the use of 3d power doppler us . imaging analysis was performed by one physician using the virtual organ computer - aided analysis ( vocal tm ) imaging program ( version 2.1 , ge medical systems ) who was blinded to the histology results . were employed in a comparative analysis for the characterization of benign and malignant solid breast masses using 3d power doppler imaging with the use of both non - harmonic us and thi . the performance measurements , including diagnostic accuracy , sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and the area ( az ) under the roc curve were calculated to estimate the performance of the diagnostic system . for a pool of n samples , n-1 was fitted to an lra model defined by log ( p ) = + 1x1 + 2x2 + + kxk , where p is the probability of a malignancy , is a constant , 1 , 2 , , k are logistic regression coefficients and x1 , x2 , , xk are parameters ; and determine the shape of the logistic curve . we employed the statistical package for the social sciences ( spss 15.0 statistical software , spss , chicago , il ) to calculate the multivariate lra of patient age , tumor volume and the vascularization indices of the benign and malignant breast tumors . an svm is a machine learning system developed using statistical learning theories to classify data points into two classes . notably , svm models have been applied extensively for classification , image recognition and bioinformatics . the svm has been shown to be an effective tool in sonography for the diagnosis of breast cancer ( 16 , 17 , 22 ) . of the possible hyperplanes , only one hyperplane maximizes the margin ( distance between the hyperplane ) and the nearest data point of each class . the value produced by the output node is used to decide whether a tumor is benign or malignant . for an output value 0 , the svm system will classify the tumor as malignant ; conversely , for an output value < 0 , the tumor will be diagnosed as benign . neural networks have been successfully applied for aided diagnoses of solid breast nodules in us examinations . ( 9 ) have described in detail the application of cad to us of solid breast nodules by the use of nns . the input vector is first supplied to the source nodes in the input layer . the neurons of the input layer constitute the signals applied to the neurons of the hidden layer . the output results of the hidden layer are employed as inputs to the next hidden layer . among the learning algorithms used to train an nn the learning method iteratively executes the back - propagation algorithm for the training set and then produces the final synaptic weight vectors . the input signals of the nn classifier contain a feature vector that comprises the vascularity indices of each breast tumor . the institutional review board and ethics committee of our hospital approved this study . from a consecutive series of images of suspicious masses in 183 patients ( age range , 17 - 80 years ; mean age , 44 years ) , 97 benign and 86 malignant cases were identified . the masses had undergone fine needle aspiration cytology , a core biopsy or / and an excisional biopsy and patients were subjected to an us examination with the use of both non - harmonic and thi . the largest tumor was selected in the study if more than one lesion was detected in one patient and a tumor size larger than 3.1 cm was excluded because of probe limitations . malignant masses included infiltrating ductal carcinoma ( n = 75 ) , apocrine carcinoma ( n = 3 ) , ductal carcinoma in situ ( n = 5 ) , papillary carcinoma ( n = 1 ) , mucinous carcinoma ( n = 1 ) and intracystic carcinoma ( n = 1 ) . benign lesions included fibroadenoma ( n = 81 ) , papilloma ( n = 1 ) , phylloid ( n = 1 ) , sclerosing adenosis ( n = 2 ) and other benign tumors ( n = 12 ) . non - harmonic and thi scans were obtained using a voluson 730 scanner ( ge healthcare , zipf , austria ) with a linear - array broadband 6 - 12 mhz transducer . a fixed installed 3d power doppler setting for all us examinations was used as follows : a sweep angle of 5 to 29 , ' low 1 ' wall motion filter , 0.9 khz pulse repetition frequency , -0.6 gain and mid frequency . all of the acquired 3d power doppler non - harmonic and thi scans were stored on a disc and no compression of the data was used at any time . the virtual organ computer - aided analysis ( vocal)-imaging program ( version 2.1 ) was used to analyze the stored volume with a personal computer ( m5 , asus , taipei , taiwan ) . detailed measurements and the application of the vocal - imaging program have been reported previously ( 19 - 21 ) . in brief , the program can be used to calculate the histogram indices of the vascularity and blood flow obtained from quantitative 3d power doppler us scanning . both gray - scale and color scale voxels are graded from the lowest value ( intensity , 0 ) and the highest value ( intensity , 100 ) . three histogram indices , the vascularization index ( vi ) , flow index ( fi ) and vascularization flow index ( vfi ) were calculated using these values . , the mean color value of the color voxels ) ; vfi represents both vascularization and flow ( i.e. , the mean color value of all of the voxels in the obtained volume ) . mean grayness ( mg ) is a representation of the average grayness in the gray voxels of a sphere . during measurement for all non - harmonic and harmonic 3d power doppler image analyses , we used the vocal - program with the manual model with a rotation step of 30 to perform contour defining . , the contour around the tumor margin ) and the other signal was the outside shell with a thickness of 3 mm . we labeled the histogram indices vi , fi and vfi for the intra - tumor ( invi , infi and invfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( out3mmvi , out3mmfi and out3mmvfi ) . in addition , two vascular signals were obtained for the thi scans , the histogram indices of vi , fi and vfi for the intra - tumor ( hinvi , hinfi and hinvfi ) and for shells with a thickness of 3 mm surrounding the breast lesion ( hout3mmvi , hout3mmfi and hout3mmvfi ) ( fig . briefly , histogram indices representing the mg and vascularity characteristics ( vi , fi and vfi ) of a mass were measured quantitatively from sonograms for both 3d power doppler non - harmonic and thi . vascularity indices were recorded as 0 in scans with no vascularization as determined with the use of 3d power doppler us . imaging analysis was performed by one physician using the virtual organ computer - aided analysis ( vocal tm ) imaging program ( version 2.1 , ge medical systems ) who was blinded to the histology results . logistic regression analysis , svm and nn were employed in a comparative analysis for the characterization of benign and malignant solid breast masses using 3d power doppler imaging with the use of both non - harmonic us and thi . the performance measurements , including diagnostic accuracy , sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and the area ( az ) under the roc curve were calculated to estimate the performance of the diagnostic system . for a pool of n samples , n-1 was fitted to an lra model defined by log ( p ) = + 1x1 + 2x2 + + kxk , where p is the probability of a malignancy , is a constant , 1 , 2 , , k are logistic regression coefficients and x1 , x2 , , xk are parameters ; and determine the shape of the logistic curve . we employed the statistical package for the social sciences ( spss 15.0 statistical software , spss , chicago , il ) to calculate the multivariate lra of patient age , tumor volume and the vascularization indices of the benign and malignant breast tumors . an svm is a machine learning system developed using statistical learning theories to classify data points into two classes . notably , svm models have been applied extensively for classification , image recognition and bioinformatics . the svm has been shown to be an effective tool in sonography for the diagnosis of breast cancer ( 16 , 17 , 22 ) . of the possible hyperplanes , only one hyperplane maximizes the margin ( distance between the hyperplane ) and the nearest data point of each class . the value produced by the output node is used to decide whether a tumor is benign or malignant . neural networks have been successfully applied for aided diagnoses of solid breast nodules in us examinations . ( 9 ) have described in detail the application of cad to us of solid breast nodules by the use of nns . the function of neurons in the hidden layer is to arbitrate between the input and output of the nn . the input vector is first supplied to the source nodes in the input layer . the neurons of the input layer constitute the signals applied to the neurons of the hidden layer . the output results of the hidden layer are employed as inputs to the next hidden layer . among the learning algorithms used to train an nn the learning method iteratively executes the back - propagation algorithm for the training set and then produces the final synaptic weight vectors . the input signals of the nn classifier contain a feature vector that comprises the vascularity indices of each breast tumor . a total of 183 solid breast image pairs ( as measured with non - harmonic and harmonic 3d power doppler imaging ) were analyzed . the sizes of the lesions ranged from 6.00 - 31.00 mm ( mean size , 18.97 mm ) for malignant tumors and 5.00 - 30.20 mm ( mean size , 14.73 mm ) for benign lesions . histogram indices for the use of 3d power doppler non - harmonic us scans were calculated . twenty - three benign and two malignant cases were detected without intra - tumor vascularity ; 14 benign cases and one malignant case were detected without vascular signals in shells with a thickness of 3 mm surrounding the breast lesions as determined by use of the vocal program . table 1 summarizes the accuracy , sensitivity , specificity , ppv and npv for the use of 3d power doppler us non - harmonic and thi for the differentiation of benign from malignant breast tumors by the use of the different statistical methods . table 2 shows the az value of roc curve analysis for the diagnostic performance of lra , svm and nn for 3d power doppler non - harmonic and thi of solid breast tumors . the statistical significance of the difference comparing two roc curves ( lra versus svm , lra versus nn and svm versus nn , respectively ) is also presented in table 2 . the computation time of the cads using the svm and nn classifiers is displayed in table 3 . the training time was evaluated by the use of the us image database containing 183 images . analysis of az by lra , svm and nn for non - harmonic and harmonic imaging using 3d power doppler us is shown in figures 4 and 5 . in our study , satisfactory results were shown by the use of both harmonic and non - harmonic 3d power doppler sonographic imaging to classify benign and malignant breast tumors by vascularization , and lra , an svm or nn were applied to assess diagnostic performance . an nn and svm have been shown to be acceptable diagnostic models for cad systems . vascularity scoring with 3d power doppler us can predict the malignant potential of breast tumors , as has been recently demonstrated ( 7 ) . our study demonstrated that the areas under the six roc curves of lra , the svm and nn for nonharmonic or harmonic 3d power doppler imaging were similar . we conclude that there was no difference in diagnostic ability among these three statistical models by applying the models to both non - harmonic and harmonic 3d power doppler images . ( 23 ) that compared lra with an nn for 24 malignant and 30 benign masses , no difference in performance for the masses as measured by the area under the roc curve was demonstrated ; however , the nn had better specificity than lra for a fixed sensitivity . however , it is difficult to assess whether the advantage of an nn as compared to lra in a local region of an roc curve is statistically significant . in several previous studies ( 11 , 14 , 15 ) , a diagnostic model based on an nn showed promise for breast tumor diagnosis . in our study , the lra approach for non - harmonic imaging had the highest az value ( 0.9341 ) and the nn method for non - harmonic imaging had the highest level of accuracy ( 88% ) . in a study by huang and chen ( 17 ) that compared multilayer perception neural networks ( mlps ) with an svm for 140 us images of solid breast nodules , the investigators demonstrated that the training and diagnosis procedures for the proposed svm model were 700-fold and 2380-fold faster as compared to the mlps , respectively . furthermore , the results from the use of the svm model revealed better classification performance by the use of texture analysis . the time consumed for training and diagnosis procedures for the nn classifier were 39.97-fold and 58.75-fold that of the use of the svm cad in our study . the diagnostic performances as determined by the az values for the use of lra , the svm and nn were nearly identical , and the shapes of the six curves were similar ( figs . ( 23 ) reported that the use of an nn had a higher specificity as compared with lra at a fixed 95% sensitivity . according to our results , the left portion of the roc curve of ' non - harmonic'_lra was extended more to the left side as compared with ' non - harmonic ' _ svm or ' non - harmonic'_nn ( fig . this finding implies that lra for non - harmonic 3d power doppler imaging has better specificity as compared to the svm or nn for a fixed sensitivity . for harmonic imaging , ' harmonic'_lra is extended more to the left side as compared with ' harmonic ' svm or ' harmonic'_nn ( fig . it is difficult to appraise whether the advantage of the use of lra as compared to the other models for the local region of an roc curve reaches statistical significance . furthermore , it is difficult to compare differences among investigations , as the application of different parameters ( such as texture analysis or vascular indices ) to the same model does not necessarily indicate identical diagnostic performance . in summary , moreover , lra for both non - harmonic and harmonic 3d power doppler imaging had better specificity as compared to the svm or nn for a fixed sensitivity .

stem cells have two defining properties : the ability to differentiate to different lineages and the capacity for self - renewal . these cells have two general types : embryonic stem cells ( esc ) and adult stem cells ( 1 ) . esc are acquired from the inner cell mass of the blastocyst and are related to tumor genesis ; therefore , their application involves ethical and safety concerns . msc are stromal cells that have the capacity for self - renewal and also exhibit multilineage differentiation . msc can be isolated from two cell types , i.e. , adult sources ( bone marrow , peripheral blood , adipose , etc . ) and fetal sources ( placenta , amniotic fluid , umbilical cord and umbilical cord blood ) ( 2 - 5 ) . msc have been generally used in experimental and clinical research because of their unique biological characteristics and their advantages , especially ( 3 - 8 ) in bone marrow transplantation ( 6 , 9 ) , tissue engineering ( 9 , 10 ) and cell therapy ( 9 , 11 , 12 ) . adipose tissue is a main source of adult stem cells , called adipose stem cells ( adsc ) ( 13 , 14 ) . adscs are fairly easy to obtain from adipose tissue and unlike bone marrow tissue , can be grown in cell cultures . ( 15 ) . additionally , it has many of the same properties as bone marrow , including wide proliferation , the ability to undertake multilineage differentiation and evident plasticity both in vitro and in vivo ( 16 - 18 ) . adsc can represent the biochemical profile in vitro of adipocytes , chondrocytes and osteoblasts under proper culture conditions ( 19 , 20 ) . therefore , human adipose - derived msc are today viewed as potential sources for stem cell banks and in tissue engineering . from fetal sources , placenta due to its easy access without invasive procedures ( contrary to bone marrow harvest ) , its pluripotency potential ( as adipose tissue ) ( 21 , 22 ) and its immunomodulatory properties is defined as a good source of msc for use in medical applications ( 4 , 23 - 25 ) . therefore , the aim of this study was to isolate msc from adipose tissue and placenta and then to differentiate them into the adipocyte and osteocyte lineages . in addition , we compared morphological and immunophenotypic characteristics and the success rates of stem cells isolated from these two derived sources . this study was performed at the bu - ali research institute , mashhad university of medical sciences , mashhad , iran in 2012 . after receiving approval from the ethics committee ( no 900886 ) and obtaining informed consent from participants , samples were obtained from adipose tissues of 10 healthy women and one placenta . for the isolation of adsc , subcutaneous adipose tissues ( 50 - 100 g ) were obtained from the abdomen region of healthy women aged 25 to 40 undergoing liposuction surgery ( samples were collected by a surgeon in qaem hospital , mashhad , iran . ) . all samples outside the stated age parameters or those weighing less than 50 g , or samples with a particular disease especially cancer and cardiovascular disorders were excluded from the study . the tissues were transferred in a sterile solution of phosphate - buffered saline ( pbs ) , a 2% fetal bovine serum ( fbs ; stem cell technology inc . , london , uk ) , 100 units / ml penicillin ( gibco - invitrogen ) and 100 g /ml streptomycin ( gibco - invitrogen ) . a fresh term placenta ( 38 to 40 weeks gestation ) the samples were transferred to the bu - ali research institute s tissue culture department . after settling the adipose tissue above the bloody portion of the solution , the blood was removed using a sterile pipette and the sample was washed three times by way of a sterile pbs solution containing penicillin and streptomycin . then , the adipose tissue was cut carefully into 1 mm pieces to remove the connective tissue and blood vessels . in the next step , the extracellular matrix was digested by adding 0.1% collagenase type i at 37c , and shaken vigorously for 60 min to detach the stromal cells from primary adipocytes . then , by adding an equivalent volume of low glucose - dulbecco s modified eagle s medium ( l - dmem ) containing 10% fetal bovine serum ( fbs ) , the collagenase was inactivated and the supernatant was centrifuged for 10 min at 1000 rpm . the cellular pellet was re - suspended in dmem/10% fbs and filtered through 100 , 70 and 40 m filters to remove debris . the filtrate was centrifuged at 600 g for 10 min and was incubated with a lysis buffer ( 155 mm nh4cl , 10 mm khco3 , 0.1 mm edta ) for 10 min at 22c to 25c , then centrifuged at 300 g for 10 min before finally discarding the lysis buffer . by placing the cells for one hr on a glassy surface ( e.g. , a petri dish ) , hematopoietic cells were attached to the surface and isolated ; then , floating cells were transferred onto a six - well plate to culture at the final concentration of 110/m / in a complete medium ( dmem , 10% fbs , 100 units / ml penicillin , 100 mg /ml streptomycin ) . finally , msc , upon reaching 80% confluence , were detached using trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) and were cultured as the primary culture . initially , the placenta tissue was washed with pbs , ph 7.2 ; blood clots and vessels were mechanically removed and the tissue was minced into small pieces and washed again with pbs . then , tissues were incubated with 0.01% dnase i ( roche diagnostics australia pty . ltd . , australia ) and 0.25% trypsin ( gibco - invitrogen ) for one hr at 37c for removing trophoblasts . the remnants were collected and digested with collagenase i 0.1 % ( 1 hr at 37c ) . digested tissue was passed through a 250 m metal sieve and 100 m cell nylon membranes , to eliminate undigested fragments . following filtrate centrifugation at 300 g for 10 min , cells were collected ; red cells were lysed in a buffer containing 155 mmol / l nh4cl and 20 mmol / l tris for five min and cells were centrifuged at 300 g for 10 min . g / l ) dmem/20% fbs plus 100 u / ml penicillin , 100 g / ml streptomycin , 5 ng / ml basic fibroblast growth factor ( b - fgf , promega ) and 1mm / l - glutamine . cultures were incubated in humidified 5% co2 incubators , at 37c and plated into two 75 cm flasks . non adherent cells were removed ( after 24 hr ) and replaced with fresh medium every three days , and adherent msc were trypsinized upon reaching 80% confluence with trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) after 14 days of culture . for immunophenotyping , cells were initially washed twice with ice - cold 20 mm pbs , ph 7.2 and trypsinized with 0.25% trypsin , 0.02%edta , then 10 l of secondary conjugated ab ( abd serotec company , usa ) per 200 - 50010 cells ( up to 10 cells ) in 100 l pbs to which cd45 , cd34 , cd29 , cd105 , cd31 , cd166 , cd44 and hla - dr were added . following on , the final volumes of all the tubes were enhanced to 1 ml with pbs . in the next step , cells suspended with specific antibodies were incubated for 45 min at 4c . finally , after washing the samples in pbs , they were analyzed by flow cytometer ( bd facscalibur flow cytometer ; becton dickinson . all markers were stained with secondary conjugated ab ( mouse anti - human ab ) with fluorescein isothiocyanate ( fitc ) , except hla - dr , which was conjugated with phycoerythrin ( pe ) . then , cells were transferred into replicate 24-well plates and were incubated for 24 hr to adhere cells to plates . cells were plated in an osteocytogenic differentiation medium containing l - dmem , 10% fbs , 0.1 m dexamethasone ( sigma - aldrich ) , 200 m l - ascorbic acid-2-phosphate ( sigma - aldrich ) and 10 mm -glycerol phosphate ( sigma - aldrich ) for 21 days and induction was confirmed by alizarin red s staining ( 26 ) . alizarin red stain positive cells ( differentiated msc with calcium deposits ) under microscope were stained bright orange - red , while negative cells ( undifferentiated msc without calcium deposits ) were stained as faintly reddish cells . cells were seeded onto a medium consisting of dmem/10% fbs , 50 mol / l indomethacin , 10 m insulin , 1 mol / l dexamethasone and 0.5 mm 3-isobutyl-1-methyl - xanthine ( all from sigma - aldrich ) for two weeks and induction was confirmed by oil red o staining using a standard method ( 27 ) . the samples were transferred to the bu - ali research institute s tissue culture department . after settling the adipose tissue above the bloody portion of the solution , the blood was removed using a sterile pipette and the sample was washed three times by way of a sterile pbs solution containing penicillin and streptomycin . then , the adipose tissue was cut carefully into 1 mm pieces to remove the connective tissue and blood vessels . in the next step , the extracellular matrix was digested by adding 0.1% collagenase type i at 37c , and shaken vigorously for 60 min to detach the stromal cells from primary adipocytes . then , by adding an equivalent volume of low glucose - dulbecco s modified eagle s medium ( l - dmem ) containing 10% fetal bovine serum ( fbs ) , the collagenase was inactivated and the supernatant was centrifuged for 10 min at 1000 rpm . the cellular pellet was re - suspended in dmem/10% fbs and filtered through 100 , 70 and 40 m filters to remove debris . the filtrate was centrifuged at 600 g for 10 min and was incubated with a lysis buffer ( 155 mm nh4cl , 10 mm khco3 , 0.1 mm edta ) for 10 min at 22c to 25c , then centrifuged at 300 g for 10 min before finally discarding the lysis buffer . by placing the cells for one hr on a glassy surface ( e.g. , a petri dish ) , hematopoietic cells were attached to the surface and isolated ; then , floating cells were transferred onto a six - well plate to culture at the final concentration of 110/m / in a complete medium ( dmem , 10% fbs , 100 units / ml penicillin , 100 mg /ml streptomycin ) . finally , msc , upon reaching 80% confluence , were detached using trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) and were cultured as the primary culture . initially , the placenta tissue was washed with pbs , ph 7.2 ; blood clots and vessels were mechanically removed and the tissue was minced into small pieces and washed again with pbs . then , tissues were incubated with 0.01% dnase i ( roche diagnostics australia pty . ltd . , australia ) and 0.25% trypsin ( gibco - invitrogen ) for one hr at 37c for removing trophoblasts . the remnants were collected and digested with collagenase i 0.1 % ( 1 hr at 37c ) . digested tissue was passed through a 250 m metal sieve and 100 m cell nylon membranes , to eliminate undigested fragments . following filtrate centrifugation at 300 g for 10 min , cells were collected ; red cells were lysed in a buffer containing 155 mmol / l nh4cl and 20 mmol / l tris for five min and cells were centrifuged at 300 g for 10 min . g / l ) dmem/20% fbs plus 100 u / ml penicillin , 100 g / ml streptomycin , 5 ng / ml basic fibroblast growth factor ( b - fgf , promega ) and 1mm / l - glutamine . cultures were incubated in humidified 5% co2 incubators , at 37c and plated into two 75 cm flasks . non adherent cells were removed ( after 24 hr ) and replaced with fresh medium every three days , and adherent msc were trypsinized upon reaching 80% confluence with trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) after 14 days of culture . for immunophenotyping , cells were initially washed twice with ice - cold 20 mm pbs , ph 7.2 and trypsinized with 0.25% trypsin , 0.02%edta , then 10 l of secondary conjugated ab ( abd serotec company , usa ) per 200 - 50010 cells ( up to 10 cells ) in 100 l pbs to which cd45 , cd34 , cd29 , cd105 , cd31 , cd166 , cd44 and hla - dr were added . following on , the final volumes of all the tubes were enhanced to 1 ml with pbs . in the next step , cells suspended with specific antibodies were incubated for 45 min at 4c . finally , after washing the samples in pbs , they were analyzed by flow cytometer ( bd facscalibur flow cytometer ; becton dickinson . all markers were stained with secondary conjugated ab ( mouse anti - human ab ) with fluorescein isothiocyanate ( fitc ) , except hla - dr , which was conjugated with phycoerythrin ( pe ) . then , cells were transferred into replicate 24-well plates and were incubated for 24 hr to adhere cells to plates . cells were plated in an osteocytogenic differentiation medium containing l - dmem , 10% fbs , 0.1 m dexamethasone ( sigma - aldrich ) , 200 m l - ascorbic acid-2-phosphate ( sigma - aldrich ) and 10 mm -glycerol phosphate ( sigma - aldrich ) for 21 days and induction was confirmed by alizarin red s staining ( 26 ) . alizarin red stain positive cells ( differentiated msc with calcium deposits ) under microscope were stained bright orange - red , while negative cells ( undifferentiated msc without calcium deposits ) were stained as faintly reddish cells . cells were seeded onto a medium consisting of dmem/10% fbs , 50 mol / l indomethacin , 10 m insulin , 1 mol / l dexamethasone and 0.5 mm 3-isobutyl-1-methyl - xanthine ( all from sigma - aldrich ) for two weeks and induction was confirmed by oil red o staining using a standard method ( 27 ) . msc from both adipose and placenta were observed as fibroblast - like adherent cells at passage 0 , that at first appeared as small cells with two forms epithelial and fibroblast - like ; at later stages ( third passage ) the epithelial - like morphology was eliminated and the remaining cells were larger with fibroblast - like and cobblestone morphologies . with these morphologic findings , we were unable to differentiate msc from the differently - derived sources ( msc morphologies were imaged using a carl zeiss inverted axiovert 40 microscope , equipped with a camera ) ( figure 1a-1f ) . the isolation process for admsc ( adipose - derived mesenchymal stem cells ) were conducted with a 100% success rate for all the samples ( n=10 ) ; this ratio was not calculated for plc - msc , because of limitations in the number of evaluated placenta samples in this study . in all passages , human mesenchymal stem cells ( a - i ) , adipogenic and osteogenic differentiation ; ( g , h ) morphology and growth of fibroblastoid - adherent cells or placenta - derived mesenchymal stem cells at phase 0 on days 2 , 5 and 12 , respectively ( a - c ) . ( a ) small , round cells ( magnification : 100 ) . ( b , c ) long , thin cells ( magnification : 100 ) with spindle - shape , which were reached to high confluence and were formed in a colony , as seen in the picture . ( c ) with morphology and growth of adipose - derived mesenchymal stem cells at passage 0 on days 2 , 4 and 8 ( colony ) , respectively ( d - f ) . ( d ) a single long spindle - shaped adsc with thin processes ; ( magnification : 1000 ) . ( e , f ) adscs that were growing ( cells were created in a colony ( f ) ) ( magnification : 100 ) . adipogenic differentiation was evidenced by the formation of lipid vacuoles ( yellow ) by oil red o staining at passage three in adipose - derived msc ( g ) ( magnification : 1000 ) . osteogenic differentiation was evidenced by the formation of a mineralized matrix at passage three in adipose - derived msc ( magnification : 400 ) ( h ) undifferentiated adipose msc ( i ) scale bar is 50 m d , g , h ) and 100 m ( a , b , c , e , f , i ) the ability of msc to differentiate into mesodermal cells , such as adipocytes , osteocytes and myocytes was proven . these abilities were proved via oil red o staining and showed the presence of lipid droplets ( figure 1 g ) , while alizarin red s staining showed calcium deposits ( figure 1h ) . these results were verified by comparing the samples with control cultures ( no morphological changes were observed in control cultures ) . adsc was differentiated quicker than placenta ; however , both were differentiated within 21 days for osteocytes and 14 days for adipocyte differentiation ( all samples of the two sources were differentiated to adipocytes and osteocytes ) . about 90% of plc - msc and adsc were positive for cd 29 , cd44 , cd105 and cd166 , and negative for cd31 , cd34 , cd45 , hla - dr ( the two sources were similar ) ( figures 2 , 3 ) adipose msc : cd 105 : 75.98 % ; cd 29 : 82.93 % ; cd 44 : 86.34 % ; cd 166 : 27.65 % . placenta msc : cd 105 : 84.82% ; cd 29 : 83.52 % ; cd 44 : 88.59 % ; cd 166 : 77.67 % . adsc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hla - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 immunophenotyping analysis of placenta mesenchymal stem cells . plc - msc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hl - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 about 90% of plc - msc and adsc were positive for cd 29 , cd44 , cd105 and cd166 , and negative for cd31 , cd34 , cd45 , hla - dr ( the two sources were similar ) ( figures 2 , 3 ) . adipose msc : cd 105 : 75.98 % ; cd 29 : 82.93 % ; cd 44 : 86.34 % ; cd 166 : 27.65 % . placenta msc : cd 105 : 84.82% ; cd 29 : 83.52 % ; cd 44 : 88.59 % ; cd 166 : 77.67 % . adsc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hla - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 immunophenotyping analysis of placenta mesenchymal stem cells . plc - msc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hl - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 msc exists in many tissues in adults ( bone marrow , adipose , cartilage , peripheral blood ) and in fetal tissues ( placenta , cord blood , amniotic fluid ) . despite bone marrow being the main source of isolation for msc , its aspiration is an extremely invasive procedure and differentiation potential , life span and the number of msc derived from it decreases with increasing age . therefore , other sources of msc isolation such as adipose tissue and placenta are being studied . the collection of plc - msc is less invasive and poses no side - effects for the mother or the neonate . msc can also be obtained from adipose tissue ( these cells can be isolated from cosmetic liposuction procedures in large numbers and quantities and easily cultured under standard conditions ) ( 28 ) . in this study , msc from two sources , placenta and adipose , were isolated and after characterization successfully differentiated into two lineages . the results showed that both adipocyte msc and plc - msc had osteogenic and adipogenic differentiation potential , as have been shown in previous studies highlighting their pluripotency potential ( 3 , 21 , 29 , 30 ) . in this study , assessments were restricted to the mesodermal differentiation capacity ( adipose tissue and bone ) . based on recent reports , however , the range of differentiation for msc does not appear to be limited to this lineage . msc derived from both tissues have been shown able to differentiate to endo- and ectodermal lineages , too ( 14 , 31 - 34 ) . all msc derived from these two sources showed typical msc features with a fibroblastoid morphology , the formation of single separated fibroblastoid colonies , differentiation ability and the expression of classical markers of msc , but without the expression of hematopoietic markers . we were able to differentiate msc from two different sources to adipogenic and osteogenic tissues with similar capabilities . however , in a study by kern et al ( 2006 ) , which compared isolation of msc from bone marrow , umbilical cord blood and adipose tissue , the researchers were unable to differentiate cord blood msc to adipose tissue . neither msc derived from the two sources expressed hematopoietic stem cell markers ( cd45 , cd34 ) , but they expressed msc markers ( cd29 , cd44 , cd105 , cd166 ) with similar intensity . as in other studies , none of the msc expressed hla ii ( 28 ) . definitive cell markers of adsc or pl - msc will help not only to separate them from other cell populations in cultures , but will also enable their purification from stromal vascular fraction . however , we have not distinguished these markers in the current study ; this separation can be done by functional assay ( 35 ) . the reproduction rate of adipose - derived msc was much faster than placenta derived msc . it took seven days for adipocyte- derived msc to reach confluence of 80% , whereas this took 12 to 15 days for plc - msc ( at passage 0 ) . however , this difference was minimized in the following passages ( plc - msc reached similar duplication rate in the third passage ) . the study showed that adipogenesis differentiation was relatively quick , occurring in nearly two weeks , but that chondrogenic and osteogenic differentiation took notably longer ( roughly 21 days ) ( 36 ) . according to some studies , bone marrow msc has been recognized as the most important source of msc for medical applications ( 37 ) ; however due to limitations in obtaining bone marrow , as we have mentioned previously , and its reduced differentiation with age ( 38 , 39 ) , other sources of msc such as adipose tissue and placenta , with easy isolation and sufficient msc numbers , should be considered for clinical application ( 4 , 28 , 40 - 42 ) . addition to the regenerative function of adsc and pl - msc , is their important potential immunomodulatory effects . cp - msc may be more effective than other msc in relation to immunomodulation and have been suggested as useful sources for cell therapy ( 25 ) . according to some studies , isolated msc from different sources , including peripheral blood , adipose and wharton s jelly have different immunomodulatory characteristics ( 43 , 44 ) . studies have also shown that these characteristics result in membrane or secretory msc molecules ( 45 ) . of course , these studies demonstrate that msc could identify environmental signals and through understanding these signals , as well as the cell s needs , stimulatory or inhibitory functions can be achieved ( 45 ) . our study , as in a number of others , showed similar immunophenotypic and differentiation properties for neonatal ( e.g. , placenta ) and adult msc sources ( e.g. , adipose ) ( 46 , 47 ) . these results differ from some studies that indicate differentiated potential for neonatal msc sources ( 48 ) . the current study showed that plc - msc and adsc show similar mesenchymal cell surface markers , morphology and differentiation potential , and due to their multipotency for differentiation into another lineage , like osteocytes and adipocytes , they can be considered an attractive source of msc for use in regenerative medicine .

objective(s):mesenchymal stem cells ( msc ) can be isolated from adult tissues such as adipose tissue and other sources . among these sources , adipose tissue ( because of easy access ) and placenta ( due to its immunomodulatory properties , in addition to other useful properties ) , have attracted more attention in terms of research . the isolation and comparison of msc from these two sources provides a proper source for clinical experimentation . the aim of this study was to compare the characteristics of msc isolated from human adipose tissue and placenta.materials and methods : adipose and placental msc were isolated from the subcutaneous adipose tissues of 10 healthy women ( 25 to 40 years ) and from a fresh term placenta ( n= 1 ) , respectively . stem cells were characterized and compared by flow cytometry using cd29 , cd31 , cd34 , cd44 , cd45 , cd105 , cd166 and hla - dr markers . osteocytes and adipocytes were differentiated from isolated human mesenchymal stem cells ( hmsc).results : adipose and placenta - derived msc exhibited the same morphological features . adsc differentiated faster than placenta ; however , both were differentiated , taking up to 21 days for osteocyte and 14 days for adipocyte differentiation . about 90% of plc - msc and adsc were positive for cd29 , cd44 , cd105 , and cd166 ; and negative for cd31 , cd34 , cd45 , and hla-dr.conclusion:the two sources of stem cells showed similar surface markers , morphology and differentiation potential and because of their multipotency for differentiating to adipocytes and osteocytes , they can be applied as attractive sources of msc for regenerative medicine .

Introduction Materials and Methods Isolation of ADSC Isolation of placenta mesenchymal stem cells (PLC-MSC) Flow cytometry analysis of ADSC and PLC-MSC Differentiation study Osteogenic differentiation Adipogenic differentiation Results Cell surface markers Discussion Conclusion

these cells have two general types : embryonic stem cells ( esc ) and adult stem cells ( 1 ) . msc can be isolated from two cell types , i.e. msc have been generally used in experimental and clinical research because of their unique biological characteristics and their advantages , especially ( 3 - 8 ) in bone marrow transplantation ( 6 , 9 ) , tissue engineering ( 9 , 10 ) and cell therapy ( 9 , 11 , 12 ) . adipose tissue is a main source of adult stem cells , called adipose stem cells ( adsc ) ( 13 , 14 ) . adscs are fairly easy to obtain from adipose tissue and unlike bone marrow tissue , can be grown in cell cultures . therefore , human adipose - derived msc are today viewed as potential sources for stem cell banks and in tissue engineering . from fetal sources , placenta due to its easy access without invasive procedures ( contrary to bone marrow harvest ) , its pluripotency potential ( as adipose tissue ) ( 21 , 22 ) and its immunomodulatory properties is defined as a good source of msc for use in medical applications ( 4 , 23 - 25 ) . therefore , the aim of this study was to isolate msc from adipose tissue and placenta and then to differentiate them into the adipocyte and osteocyte lineages . in addition , we compared morphological and immunophenotypic characteristics and the success rates of stem cells isolated from these two derived sources . this study was performed at the bu - ali research institute , mashhad university of medical sciences , mashhad , iran in 2012 . after receiving approval from the ethics committee ( no 900886 ) and obtaining informed consent from participants , samples were obtained from adipose tissues of 10 healthy women and one placenta . for the isolation of adsc , subcutaneous adipose tissues ( 50 - 100 g ) were obtained from the abdomen region of healthy women aged 25 to 40 undergoing liposuction surgery ( samples were collected by a surgeon in qaem hospital , mashhad , iran . ) all samples outside the stated age parameters or those weighing less than 50 g , or samples with a particular disease especially cancer and cardiovascular disorders were excluded from the study . , london , uk ) , 100 units / ml penicillin ( gibco - invitrogen ) and 100 g /ml streptomycin ( gibco - invitrogen ) . a fresh term placenta ( 38 to 40 weeks gestation ) the samples were transferred to the bu - ali research institute s tissue culture department . after settling the adipose tissue above the bloody portion of the solution , the blood was removed using a sterile pipette and the sample was washed three times by way of a sterile pbs solution containing penicillin and streptomycin . then , the adipose tissue was cut carefully into 1 mm pieces to remove the connective tissue and blood vessels . then , by adding an equivalent volume of low glucose - dulbecco s modified eagle s medium ( l - dmem ) containing 10% fetal bovine serum ( fbs ) , the collagenase was inactivated and the supernatant was centrifuged for 10 min at 1000 rpm . , a petri dish ) , hematopoietic cells were attached to the surface and isolated ; then , floating cells were transferred onto a six - well plate to culture at the final concentration of 110/m / in a complete medium ( dmem , 10% fbs , 100 units / ml penicillin , 100 mg /ml streptomycin ) . finally , msc , upon reaching 80% confluence , were detached using trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) and were cultured as the primary culture . , australia ) and 0.25% trypsin ( gibco - invitrogen ) for one hr at 37c for removing trophoblasts . following filtrate centrifugation at 300 g for 10 min , cells were collected ; red cells were lysed in a buffer containing 155 mmol / l nh4cl and 20 mmol / l tris for five min and cells were centrifuged at 300 g for 10 min . g / l ) dmem/20% fbs plus 100 u / ml penicillin , 100 g / ml streptomycin , 5 ng / ml basic fibroblast growth factor ( b - fgf , promega ) and 1mm / l - glutamine . non adherent cells were removed ( after 24 hr ) and replaced with fresh medium every three days , and adherent msc were trypsinized upon reaching 80% confluence with trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) after 14 days of culture . for immunophenotyping , cells were initially washed twice with ice - cold 20 mm pbs , ph 7.2 and trypsinized with 0.25% trypsin , 0.02%edta , then 10 l of secondary conjugated ab ( abd serotec company , usa ) per 200 - 50010 cells ( up to 10 cells ) in 100 l pbs to which cd45 , cd34 , cd29 , cd105 , cd31 , cd166 , cd44 and hla - dr were added . finally , after washing the samples in pbs , they were analyzed by flow cytometer ( bd facscalibur flow cytometer ; becton dickinson . all markers were stained with secondary conjugated ab ( mouse anti - human ab ) with fluorescein isothiocyanate ( fitc ) , except hla - dr , which was conjugated with phycoerythrin ( pe ) . cells were plated in an osteocytogenic differentiation medium containing l - dmem , 10% fbs , 0.1 m dexamethasone ( sigma - aldrich ) , 200 m l - ascorbic acid-2-phosphate ( sigma - aldrich ) and 10 mm -glycerol phosphate ( sigma - aldrich ) for 21 days and induction was confirmed by alizarin red s staining ( 26 ) . alizarin red stain positive cells ( differentiated msc with calcium deposits ) under microscope were stained bright orange - red , while negative cells ( undifferentiated msc without calcium deposits ) were stained as faintly reddish cells . cells were seeded onto a medium consisting of dmem/10% fbs , 50 mol / l indomethacin , 10 m insulin , 1 mol / l dexamethasone and 0.5 mm 3-isobutyl-1-methyl - xanthine ( all from sigma - aldrich ) for two weeks and induction was confirmed by oil red o staining using a standard method ( 27 ) . then , the adipose tissue was cut carefully into 1 mm pieces to remove the connective tissue and blood vessels . in the next step , the extracellular matrix was digested by adding 0.1% collagenase type i at 37c , and shaken vigorously for 60 min to detach the stromal cells from primary adipocytes . , a petri dish ) , hematopoietic cells were attached to the surface and isolated ; then , floating cells were transferred onto a six - well plate to culture at the final concentration of 110/m / in a complete medium ( dmem , 10% fbs , 100 units / ml penicillin , 100 mg /ml streptomycin ) . g / l ) dmem/20% fbs plus 100 u / ml penicillin , 100 g / ml streptomycin , 5 ng / ml basic fibroblast growth factor ( b - fgf , promega ) and 1mm / l - glutamine . non adherent cells were removed ( after 24 hr ) and replaced with fresh medium every three days , and adherent msc were trypsinized upon reaching 80% confluence with trypsin - edta ( 0.25% trypsin+0.02%edta , gibco - invitrogen ) after 14 days of culture . for immunophenotyping , cells were initially washed twice with ice - cold 20 mm pbs , ph 7.2 and trypsinized with 0.25% trypsin , 0.02%edta , then 10 l of secondary conjugated ab ( abd serotec company , usa ) per 200 - 50010 cells ( up to 10 cells ) in 100 l pbs to which cd45 , cd34 , cd29 , cd105 , cd31 , cd166 , cd44 and hla - dr were added . finally , after washing the samples in pbs , they were analyzed by flow cytometer ( bd facscalibur flow cytometer ; becton dickinson . all markers were stained with secondary conjugated ab ( mouse anti - human ab ) with fluorescein isothiocyanate ( fitc ) , except hla - dr , which was conjugated with phycoerythrin ( pe ) . cells were plated in an osteocytogenic differentiation medium containing l - dmem , 10% fbs , 0.1 m dexamethasone ( sigma - aldrich ) , 200 m l - ascorbic acid-2-phosphate ( sigma - aldrich ) and 10 mm -glycerol phosphate ( sigma - aldrich ) for 21 days and induction was confirmed by alizarin red s staining ( 26 ) . alizarin red stain positive cells ( differentiated msc with calcium deposits ) under microscope were stained bright orange - red , while negative cells ( undifferentiated msc without calcium deposits ) were stained as faintly reddish cells . msc from both adipose and placenta were observed as fibroblast - like adherent cells at passage 0 , that at first appeared as small cells with two forms epithelial and fibroblast - like ; at later stages ( third passage ) the epithelial - like morphology was eliminated and the remaining cells were larger with fibroblast - like and cobblestone morphologies . with these morphologic findings , we were unable to differentiate msc from the differently - derived sources ( msc morphologies were imaged using a carl zeiss inverted axiovert 40 microscope , equipped with a camera ) ( figure 1a-1f ) . the isolation process for admsc ( adipose - derived mesenchymal stem cells ) were conducted with a 100% success rate for all the samples ( n=10 ) ; this ratio was not calculated for plc - msc , because of limitations in the number of evaluated placenta samples in this study . in all passages , human mesenchymal stem cells ( a - i ) , adipogenic and osteogenic differentiation ; ( g , h ) morphology and growth of fibroblastoid - adherent cells or placenta - derived mesenchymal stem cells at phase 0 on days 2 , 5 and 12 , respectively ( a - c ) . ( a ) small , round cells ( magnification : 100 ) . ( c ) with morphology and growth of adipose - derived mesenchymal stem cells at passage 0 on days 2 , 4 and 8 ( colony ) , respectively ( d - f ) . ( e , f ) adscs that were growing ( cells were created in a colony ( f ) ) ( magnification : 100 ) . adipogenic differentiation was evidenced by the formation of lipid vacuoles ( yellow ) by oil red o staining at passage three in adipose - derived msc ( g ) ( magnification : 1000 ) . osteogenic differentiation was evidenced by the formation of a mineralized matrix at passage three in adipose - derived msc ( magnification : 400 ) ( h ) undifferentiated adipose msc ( i ) scale bar is 50 m d , g , h ) and 100 m ( a , b , c , e , f , i ) the ability of msc to differentiate into mesodermal cells , such as adipocytes , osteocytes and myocytes was proven . these abilities were proved via oil red o staining and showed the presence of lipid droplets ( figure 1 g ) , while alizarin red s staining showed calcium deposits ( figure 1h ) . adsc was differentiated quicker than placenta ; however , both were differentiated within 21 days for osteocytes and 14 days for adipocyte differentiation ( all samples of the two sources were differentiated to adipocytes and osteocytes ) . about 90% of plc - msc and adsc were positive for cd 29 , cd44 , cd105 and cd166 , and negative for cd31 , cd34 , cd45 , hla - dr ( the two sources were similar ) ( figures 2 , 3 ) adipose msc : cd 105 : 75.98 % ; cd 29 : 82.93 % ; cd 44 : 86.34 % ; cd 166 : 27.65 % . adsc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hla - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 immunophenotyping analysis of placenta mesenchymal stem cells . plc - msc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hl - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 about 90% of plc - msc and adsc were positive for cd 29 , cd44 , cd105 and cd166 , and negative for cd31 , cd34 , cd45 , hla - dr ( the two sources were similar ) ( figures 2 , 3 ) . adsc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hla - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 immunophenotyping analysis of placenta mesenchymal stem cells . plc - msc was positive for cd105 , cd29 , cd44 and cd166 , and negative for cd31 , hl - dr , cd45 and cd34 . a : control and cd105 ; b : control and cd29 ; c : control and cd44 ; d : control and cd166 ; e : control and cd31 ; f : control and hla dr ; g : control and cd45 ; h : control and cd34 msc exists in many tissues in adults ( bone marrow , adipose , cartilage , peripheral blood ) and in fetal tissues ( placenta , cord blood , amniotic fluid ) . despite bone marrow being the main source of isolation for msc , its aspiration is an extremely invasive procedure and differentiation potential , life span and the number of msc derived from it decreases with increasing age . therefore , other sources of msc isolation such as adipose tissue and placenta are being studied . the collection of plc - msc is less invasive and poses no side - effects for the mother or the neonate . msc can also be obtained from adipose tissue ( these cells can be isolated from cosmetic liposuction procedures in large numbers and quantities and easily cultured under standard conditions ) ( 28 ) . in this study , msc from two sources , placenta and adipose , were isolated and after characterization successfully differentiated into two lineages . the results showed that both adipocyte msc and plc - msc had osteogenic and adipogenic differentiation potential , as have been shown in previous studies highlighting their pluripotency potential ( 3 , 21 , 29 , 30 ) . in this study , assessments were restricted to the mesodermal differentiation capacity ( adipose tissue and bone ) . all msc derived from these two sources showed typical msc features with a fibroblastoid morphology , the formation of single separated fibroblastoid colonies , differentiation ability and the expression of classical markers of msc , but without the expression of hematopoietic markers . we were able to differentiate msc from two different sources to adipogenic and osteogenic tissues with similar capabilities . however , in a study by kern et al ( 2006 ) , which compared isolation of msc from bone marrow , umbilical cord blood and adipose tissue , the researchers were unable to differentiate cord blood msc to adipose tissue . neither msc derived from the two sources expressed hematopoietic stem cell markers ( cd45 , cd34 ) , but they expressed msc markers ( cd29 , cd44 , cd105 , cd166 ) with similar intensity . however , we have not distinguished these markers in the current study ; this separation can be done by functional assay ( 35 ) . the reproduction rate of adipose - derived msc was much faster than placenta derived msc . it took seven days for adipocyte- derived msc to reach confluence of 80% , whereas this took 12 to 15 days for plc - msc ( at passage 0 ) . however , this difference was minimized in the following passages ( plc - msc reached similar duplication rate in the third passage ) . the study showed that adipogenesis differentiation was relatively quick , occurring in nearly two weeks , but that chondrogenic and osteogenic differentiation took notably longer ( roughly 21 days ) ( 36 ) . according to some studies , bone marrow msc has been recognized as the most important source of msc for medical applications ( 37 ) ; however due to limitations in obtaining bone marrow , as we have mentioned previously , and its reduced differentiation with age ( 38 , 39 ) , other sources of msc such as adipose tissue and placenta , with easy isolation and sufficient msc numbers , should be considered for clinical application ( 4 , 28 , 40 - 42 ) . addition to the regenerative function of adsc and pl - msc , is their important potential immunomodulatory effects . cp - msc may be more effective than other msc in relation to immunomodulation and have been suggested as useful sources for cell therapy ( 25 ) . according to some studies , isolated msc from different sources , including peripheral blood , adipose and wharton s jelly have different immunomodulatory characteristics ( 43 , 44 ) . of course , these studies demonstrate that msc could identify environmental signals and through understanding these signals , as well as the cell s needs , stimulatory or inhibitory functions can be achieved ( 45 ) . our study , as in a number of others , showed similar immunophenotypic and differentiation properties for neonatal ( e.g. , placenta ) and adult msc sources ( e.g. the current study showed that plc - msc and adsc show similar mesenchymal cell surface markers , morphology and differentiation potential , and due to their multipotency for differentiation into another lineage , like osteocytes and adipocytes , they can be considered an attractive source of msc for use in regenerative medicine .

nk cells are effector lymphocytes of the innate immune system that control several types of tumors by limiting their growth and dissemination . in vitro studies using cells from humans and several other mammalian species , as well as in vivo studies in mice and rats , there are numerous studies in mice supporting the notion that nk cells are involved in the elimination of tumor cells . the in vivo analyses relied on antibody - mediated depletion of nk cells in mice , targeting either nk1.1 or the glycolipid asialo - gm1 [ 35 ] . however , nk cell depletion with antibodies to nk1.1 may also affect populations of invariant natural killer t cells and other nk1.1 t - cell populations . the selectivity of nk cell depletion with asialo - gm1 antibodies ( abs ) has also been hampered by the expression of asialo - gm1 by several cell types including myeloid cells , epithelial cells , and t - cell subsets . caution is therefore required when interpreting studies based upon ab depletion because of the lack of specificity of ab treatment against nk populations . nevertheless , many other independent studies advocate a role for nk cells in the control of tumor development in mice . particularly , mouse nk cells are involved in the in vivo rejection of several transplanted tumors , in a manner dependent upon the presence or absence of nk cell receptor ligands expressed by the tumor . more informative studies regarding tumor immunosurveillance are the experiments addressing the control of newly arising tumors . in this respect schreiber 's group demonstrated that frequencies of spontaneously arising tumors or induced by methylcholantrene ( mca ) were higher in mice deficient for key effector molecules of nk cells or the respective receptors [ 7 , 8 ] . nevertheless , in a further study from the same group , it was shown that although nk cells are important in the early elimination of mca - induced tumors , control of the dormant tumor state depends mainly on adaptive immunity . the lack of mhc class i expression or the upregulation of nkg2d ligands [ 11 , 12 ] can render tumor cells susceptible to nk cell - mediated lysis . in some of these experimental models , nk cell - mediated elimination of tumor cells induced the subsequent development of tumor - specific t - cell responses to the parental tumor cells [ 11 , 13 ] as a bridge between innate and adaptive immune responses . a role for nk cells in tumor immunosurveillance has also been implicated in controlling the growth of b - cell lymphomas that spontaneously arise in mice lacking both perforin and 2-microglobulin . moreover , the blocking of nk cell mhc class - i - specific inhibitory receptors increases nk cell effector function against tumor cells in mice . in addition to their endogenous protective role in tumor models , nk cells are also mediators of the antitumor effects of several recombinant cytokines , such as il-2 , il-12 , il-18 , and il-21 . so far , these studies in vitro and in animal models show the role of nk cells in cancer immunosurveillance . clinical and experimental evidence demonstrate that this important role of nk cells holds true in humans . although the paucity of nk cell - selective deficiencies has limited the characterization of nk cell biological function in vivo in general and in antitumor immunosurveillance in particular , there is an 11-year follow - up epidemiologic survey which has shown that the extent of nk cell activity in peripheral blood is associated with cancer risk in adults : low nk cell activity is associated with increased cancer risk . furthermore , intratumoral nk cell studies have been hampered because of the low numbers of these cells and the difficulty to obtain them from tumor samples . however , in the last few years , novel studies have revealed the phenotypic status and functionality of nk cells in tumor site and also in peripheral blood ( pb ) of cancer patients . first studies about tumor immune infiltration have shown that the presence of nk cells represents a positive prognostic marker in different carcinomas [ 1921 ] . further studies in established human tumors showed that there are often only a few infiltrating nk cells which are unlikely to greatly contribute to the elimination of tumor cells [ 22 , 23 ] . it has been suggested that despite of low nk cell numbers in tumors , due to their inefficient homing into malignant tissues , this situation may be overcome by cytokine - mediated activation in immunotherapeutical regimens . in this respect , all this evidence about nk importance in tumor immune control enhances the appeal of nk cell - based immunotherapeutic approaches . for instance , the beneficial role of nk cells in controlling human malignancies stems from clinical studies of leukemia patients who received alloreactive nk cells in the course of allogeneic hematopoietic stem cell transplantation [ 25 , 26 ] . likewise , several studies have established the importance of fc - fcr interactions for the in vivo antitumor effects of certain monoclonal antibodies [ 2730 ] . in the present work we will revise key aspects of nk cells role from molecular and cellular characteristics to therapeutic applications in cancer patients . primary tumor growth is a complex process , involving many interactions between the tumors and surrounding tissue . a developing tumor influences and is influenced by its stroma , initiates angiogenesis , and interacts with both the adaptive and innate immune systems . the clinicopathological significance of the tumor - infiltrating lymphocytes ( tils ) in various human cancers has been an issue of great interest . cd8 cytotoxic t cells ( ctl ) and nk cells are the most likely effectors for an effective antitumor immunity . several studies have shown that the infiltration of lymphocytes significantly correlates with a prolonged survival time of patients , at least in certain types of cancer [ 3234 ] . typically , nk cells are not found in large numbers in advanced human neoplasms , indicating that they do not normally home efficiently to malignant tissues . for instance , a low prevalence of gastric and colorectal ( crc ) tumor - infiltrating cd56 cells in livers with multiple metastases was detected , whereas in cases with solitary metastases a higher degree of lymphocyte infiltration was observed . moreover , the percentage of intrahepatic nk ( cd56 ) cells was also decreased in patients with metastases compared to those without , being almost twice lower than cd8 and cd4 . this suggests that low nk cell number could be a reason for the escape of metastatic cells from the mechanisms of liver immune control . in human nonsmall cell lung cancers ( nsclc ) nk cells percentage within cd45 mononuclear cells ( mcs ) isolated from lung tumors was consistently lower than in mc from the pb counterpart and was comparable to that in mc from peritumoral lung tissue . with regard to their localization , nk cells were found in tumor stroma , whereas they were not in direct contact with cancer cells . malignant and nonmalignant tissue areas in nsclc were selectively infiltrated by certain immune cell types with nk cells being displaced from the tumor tissue and displaying low cytotoxic activity . in another study , the cd56/cd16 cell ratio in renal cell carcinoma microenvironment was found generally lower than 1 , suggesting that a predominant number of cd16 cells were cd56 macrophages , and a low nk cell infiltration . the limited number of nk cells infiltrating tumors has hampered more extensive ex vivo analyses of such tumor - associated cells . however , novel studies of tumor - associated nk cells demonstrated a striking phenotype , supporting the notion that tumor - induced alterations of activating nk cell receptor expression may hamper immune surveillance and promote tumor progression . in this sense , several authors studied the balance between inhibitory and activating receptors of nk cells that infiltrate tumors , observing in some cases downregulation of activating receptors and in others , overexpression of inhibitory ones . in a study which analyzed the receptor repertoire and functional integrity of nk cells in peritoneal effusions from patients with ovarian carcinoma , tumor - associated nk cells expressed reduced levels of the activating dnam-1 , 2b4 , and cd16 receptors and were hyporesponsive to hla class i - deficient k562 cells and to coactivation via dnam-1 and 2b4 , relative to autologous peripheral blood nk cells . moreover , tumor - associated nk cells were also refractory to cd16 receptor stimulation , resulting in diminished ab - dependent cellular cytotoxicity ( adcc ) against autologous tumor cells . in another study , tongue cancer intraepithelial nk cells expressed nkg2a , an inhibitory receptor that recognizes hla - e , more frequently than those in the stroma or in lichen planus . collectively , the intraepithelial cd8 t cells and nk cells were phenotypically inactivated , whereas stromal counterparts were phenotypically just as active as those in lichen planus , suggesting the suppressed state of the intraepithelial nk cells . carrega and coworkers described both relevant molecule expression and function of nk cells infiltrating nsclc in comparison with autologous nk cells isolated from either peritumoral normal lung tissues or pb . the cd56cd16 nk cell subset was highly enriched in tumor infiltrate and displayed activation markers , including nkp44 , cd69 , and hla - dr . remarkably , the cytolytic potential of nk cells isolated from cancer tissues was lower than that of nk cells from pb or normal lung tissue , whereas no difference was observed regarding their capability of producing cytokines . in acute myeloid leukemia ( aml ) it was demonstrated that cd137 ligand ( cd137l ) was expressed on leukemic cells and interacts with cd137 on activated nk cells . bidirectional signaling following cd137-cd137l interaction induced the release of the immunomodulatory cytokines il-10 and tnf by aml cells and directly diminished granule mobilization , cytotoxicity , and ifn- production by human nk cells , demonstrating an immune evasion of aml cells by impairing nk - cell tumor surveillance . not only did nk infiltrating tumor cells show a different phenotype or reduced cytotoxicity , but in some kind of cancers this was also observed in nk cells from pb . in a study in metastatic melanoma ( mm ) patients , nk cells presented decreased activity and ifn- production and also had a redistribution of nk cell subsets . it was observed an increase in noncytotoxic cd16cd56 and a reduction in cytotoxic cd16cd56 nk cell subsets . moreover , there was a decreased cd161 and nkg2d activating receptors and an overexpressed cd158a inhibitory nk cell receptor , which correlated to lower nk cell cytotoxicity . these results are in agreement with another study in mm where a significantly lower percentage of nk cells expressed cd16 , nkp30 , and nkp46 activating receptors as compared to healthy donors . in addition , despite the importance of nkg2d in recognizing mm cells , no substantial differences were observed between stage iv mm patients and healthy donors . these observations imply that down - regulated nk litic receptor expression in mm patients may affect nk cells ability to recognize and eliminate tumor cells . likewise , the frequency of nk cells expressing the activating receptors nkp30 , nkp44 , nkp46 , nkg2d , and nkg2c was significantly decreased in aml patients compared to the nk cells of normal controls . although the molecular mechanisms responsible for the reduced receptor expression in pb nk cells remains elusive , elevated serum levels of soluble nk cell receptor ligands shed by tumor cells have been associated with downregulation of the nk cell receptors and might contribute to the decreased levels of nk cell activity . in this section , we cited works which showed some impaired nk cells characteristics in cancer patients which determine their inability to eliminate tumor cells ( depicted in figure 1 ) . on the other hand , it is a fact that nk cells are potentially active against tumor cells and it is feasible to manipulate them to restore and/or improve their antitumoral activity . many mechanisms have been proposed to explain the clinical antitumor activity of unconjugated tumor antigen - specific monoclonal abs ( mabs ) . the ability of some mabs to disrupt signalling pathways involved in the maintenance of the malignant phenotype has received widespread attention . in addition , abs exhibit various immunomodulatory properties and , by directly activating or inhibiting molecules of the immune system , they can promote the induction of antitumor immune responses . , we will describe nk cell - mediated adcc mechanism and discuss the potential use of mabs to manipulate the host immune response to tumors . abs are linked to immune effector functions by the fc fragment , which is capable of initiating adcc when binding to fc receptors , specially fcriii ( cd16 ) on nk cells , which initiates a sequence of cellular events culminating in the release of cytotoxic granzyme - containing granules and inf- secretion [ 4547 ] . several studies have established the importance of fc - fcr interactions for the antitumor effects of certain mabs in murine models and clinical trials . a seminal paper showed that the antitumor activities of the anti - her-2 mab , trastuzumab , and the anti - cd20 mab , rituximab , were lower in fcr - deficient mice than in wild - type mice . the role of fcr in the antitumor response has been further supported by the finding that polymorphisms in the gene encoding fcriii , which lead to higher binding of ab to fcriii , are associated with high response rates to rituximab in patients with follicular non - hodgkin 's lymphoma . a separate study that compared clinical responses to rituximab in patients with follicular lymphoma suggested that both fcriii and fcriib have a role in the response to rituximab . more recent findings show that polymorphisms in genes encoding fcrs are associated with clinical responses to other abs , including trastuzumab and the anti - egfr mab , cetuximab . patients with breast cancer who responded to trastuzumab with complete or partial remission have been found to have a higher capability to mediate in vitro adcc in response to trastuzumab than patients whose tumors failed to respond to therapy . adcc enhancement through fc domain modification has shown promise in the development of next generation mabs . for example , a cd19-specific mab with increased fcriiia binding affinity mediated significantly increased adcc compared to its parental mab and rituximab . the in vivo infusion of this high affinity mab efficiently cleared malignant b cells in cynomolgus macaques ( macaca fascicularis ) . in previous reports , we and others proved that nk cells produce cetuximab - mediated adcc of metastatic crc ( mcrc ) and that this activity is not affected by the mutational status of the downstream molecule k - ras [ 51 , 52 ] . this effect is expected because nk cells recognize the surface - bound abs and are able to kill tumor cells independently of the egfr pathway activation . nevertheless , it remains to be answered why in mcrc patients with k - ras mutated status cetuximab - mediated adcc does not induce clinical remissions . as we discussed , one of the possibilities is the low proportion of nk cells infiltrating crc tumors and the low functional capacity of these cells observed in cancer patients [ 54 , 55 ] . therefore , if it were possible to enhance the activity of nk cells , the efficacy of treatment with cetuximab could be increased . the anti - vegf - a mab , bevacizumab , is also active in mcrc , regardless of the k - ras mutation status ; nevertheless , it needs to be administered in combination with a cytotoxic agent . regarding adcc enhancer molecules , lenalidomide , an analog of thalidomide , is able to potentiate adcc in vitro . lenalidomide enhanced nk cell and monocyte - mediated adcc of rituximab against a variety of hematological cell lines in vitro . lenalidomide also enhanced nk cell - mediated lysis of cetuximab and trastuzumab - coated crc and breast cancer cells , respectively . the ability of lenalidomide to enhance cetuximab - mediated adcc of crc cells was not affected by the k - ras mutational status . because panitumumab , an igg2a anti - egfr mab , does not effectively interact with fc receptors on the nk cell surface , it was unable to initiate adcc and , as expected , lenalidomide had no effect because its activity is reliant on the augmentation of nk cell signaling downstream of fcr . an early clinical study exploring this effect of lenalidomide in k - ras mutant tumors has been initiated . as explained previously , many studies demonstrated the impairment of nk cell activity in cancer patients . in these cases , cytokines could restore and potentiate nk cell - mediated adcc . the works cited below explain how these treatments work when patients present nk cell dysfunction . cetuximab was tested in various in vitro studies in patients with esophageal squamous cell carcinoma ( escc ) to evaluate the possibility of treatment . authors first performed detailed analysis of adcc mediated by cetuximab against escc cell lines with various levels of egfr . the activities of cetuximab - mediated adcc by patients ' pbmc were impaired in comparison with those by healthy donors ' pbmc . moreover , the inhibition of transforming growth factor ( tgf)- could enhance cetuximab - mediated adcc against tgf--producing escc . in other work , downregulated cd16 and upregulated cd56 molecules on nk cells were observed in escc patients , resulting in nk cell dysfunction . after patients received curative resections of escc , the downregulated cd16 and upregulated cd56 were significantly restored to the levels of healthy donors . watanabe and coworkers evaluated whether il-21 could improve the impairment of adcc in patients with escc as il-21 was reported to have the ability to activate nk cells . trastuzumab- and cetuximab - mediated adcc of pbmc or of enriched nk cells was enhanced by the addition of il-21 in a dose - dependent manner and the levels of adcc enhanced by il-21 in patients were high enough in comparison with those in healthy donors , paralleling the upregulation of cd247 on nk cells . as in the previous example , nk cell response to cetuximab - coated tumor cells could be enhanced by the administration of nk cell stimulatory cytokines il-2 , il-12 , or il-21 and resulted in higher ifn- production than was observed with either agent alone . nk cell - derived ifn- significantly enhanced monocyte adcc against cetuximab - coated tumor cells . costimulated nk cells also secreted elevated levels of chemokines ( il-8 , macrophage inflammatory protein-1a , and rantes ) that could direct the migration of naive and activated t cells . furthermore , administration of il-21 enhanced the effects of cetuximab in a murine tumor model . in other experimental study , it was analyzed the correlation between egfr expression in lung cancer cell lines and the adcc activity of cetuximab as well as the influence of il-2 and chemotherapy on adcc activity . a logarithmic correlation was observed between the number of egfrs and adcc activity . in addition , nk cell - mediated adcc was enhanced by il-2 and such cells were also less susceptible to immunosuppression by chemotherapy than in lung cancer patients . the antitumor effect and mechanism of action of cetuximab using egfr high - expressing and egfr low - expressing gastric cancer cell lines without gene amplification was investigated . cetuximab showed neither significant growth inhibition nor induction of apoptosis in either cell line in vitro , and only slightly inhibited ligand - induced phosphorylation of protein kinase b and extracellular signal - regulated kinase . this antitumor activity was significantly enhanced and diminished by treatment with il-2 and antiasialo gm1 ab , respectively . in that study , her-2/neu - expressing gastric cancer cells could be killed by herceptin , the anti - her-2/neu mab . herceptin - mediated adcc correlated with the degree of her-2/neu expression on the gastric cancer cells . however , the herceptin - mediated adcc was significantly impaired in pbmc from advanced disease patients compared with that in early disease or healthy individuals . moreover , nk cells purified from patients with advanced disease indicated less herceptin - mediated adcc in comparison with that from healthy donors , whereas monocytes purified from the patients showed an almost equal amount of herceptin - mediated adcc in comparison with that from healthy individuals , indicating that nk cell dysfunction contributed to the impaired herceptin - mediated adcc in gastric cancer patients . furthermore , the nk - cell dysfunction on herceptin - mediated adcc correlated with the downregulation of cd16 expression in the patients , and il-2 ex vivo treatment of nk cells could restore the impairment of herceptin - mediated adcc , concomitant to the normalization of the expression of cd16 molecules . in our laboratory , we performed in vitro studies on human triple negative breast cancer ( tnbc ) k - ras - mutated cell lines . we found that egfr - expressing tnbc could be killed by cetuximab - mediated adcc at clinically achievable concentrations . furthermore , il-15 could replace il-2 in most of its immunologic activities , stimulating nk cells ability to produce ifn- , paralleling the upregulation of activating receptors . these results show that cetuximab - mediated nk cell activity can be significantly enhanced in the presence of nk cell stimulatory cytokines . adoptive cell transfer ( act ) therapy can be considered as a strategy aimed at replacing , repairing , or enhancing the biological function of the immune system by means of autologous or allogeneic cells . act therapy may include ( i ) removal or enrichment of various cell populations ; ( ii ) expansion of hematopoietic cell subsets ; ( iii ) activation of lymphocytes for immunotherapy ; ( iv ) genetic modification of lymphoid cells , when these cells are intended to engraft transiently in the recipient and/or be used in the treatment of cancer . this section contains extensive considerations on clinical and laboratory experience in immunologic targeting of malignant cell populations , and how lasting curative responses can be effectively generated . as we will discuss , the use of lymphocytes and/or nk cells as a strategic weapon in preventing or curing the neoplastic relapse after surgery / chemotherapy has been applied to the treatment of hematological malignancies and solid tumors . transfer of tumor - infiltrating lymphocytes has shown some remarkable responses in patients with advanced mm . nevertheless , problems with deriving sufficient numbers of these cells from patients , downregulation of mhc class i ligands and costimulatory ligands on tumor cells , combined with the lack of persistence of transferred cells have precluded broad utilization of these cells for the treatment of cancer patients . the transfer of other immune subsets , such as nk cells which do not require prior sensitization to respond to target cells and can potently induce a cytolytic response , represents a good alternate or auxiliary cell type for immunotherapy . one of the main hurdles involved in developing adoptive transfer of immune cells has been to define good manufacturing procedures ( gmps ) compliant methods to isolate defined subsets with the number of cells required that guarantee the safety of the injection to patients . in this respect , clinical - grade production of nk cells has proven efficient , and large - scale expansion method has been possible for human nk cells , using a cytokine - based culture system for ex vivo expansion of nk cells from hematopoietic progenitor cells from umbilical cord blood . systematic refinement of this two - step system using a novel clinical - grade medium resulted in a therapeutically applicable cell culture protocol . nevertheless , improved technologies for nk cells expansion lately have been tested . it was possible to partially attribute this to a higher expression level of nkp44 compared with nk cells expanded in flasks . these results demonstrate that large amounts of highly active nk cells for adoptive immunotherapy can be produced in a closed , automated , large - scale bioreactor under feeder - free current gmp conditions , facilitating clinical trials for the use of these cells . even when the first works were based on transplantation of autologous ex vivo expanded cells , and in addition to what we earlier discussed on cancer patients nk cells dysfunctions , it is now understood that the failure of autologous nk therapy is partially due to the downregulation of nk cell killing that occurs with recognition of self - class i mhc on tumor cells [ 7173 ] , making allogeneic cell transfer more attractive . during allogeneic hematopoietic stem cell transplantation , nk cells have been implicated in the suppression of graft versus host disease ( gvhd ) , the promotion of bone marrow engraftment , and mediation of a graft versus leukemia ( gvl ) effect . in the setting of allogeneic act in leukemias , the results of nk cells activity depend on the directionality of lysis [ 75 , 76 ] . when the nk cells are donor - derived and the recipient cells lack expression of the cognate kir ligand , the donor nk cell lysis of recipient target cells can result in gvl and/or gvhd , depending on the tissue origin of the nk cell target . however , if the target cell is of donor origin , and the nk effector cell is of recipient origin , nk cell lysis can result in graft rejection . recent studies have demonstrated that nk cell effector capacity is influenced by class and quantity of inhibitory receptors for self - hla - b and hla - c ligands [ 77 , 78 ] . it has been estimated that nk cell alloreactivity can be expected to occur in about 50% of unrelated donor transplants with velardi and coworkers announced a new era in the exploitation of nk cells for cancer immunotherapy with a pioneering study of hematopoietic stem cell transplantation that stratified patients according to kir - ligand mismatch between donor cells and recipient leukemic cells . this reveals that alloreactive nk cells , unrestrained by inhibitory signals from the recipient hla ligands , protects against disease relapse . this kir - ligand mismatch phenomenon has attracted researchers to study it , and similar observations have since been made in other trials of leukemia immunotherapy [ 7981 ] . on the other hand , further studies extended hla match analysis to the mechanisms involved in gvhd . in patients with hematologic malignancies who received transplants from unrelated donors , genotype analysis of six major hla loci identified 4 hla - c and 6 hladpb1 mismatch combinations responsible for a decreased risk of relapse and severe acute gvhd . donor selection made in consideration of these results might allow the separation of gvl from acute gvhd , especially in hla - dpb1 mismatch combinations . these findings might be crucial to elucidating the mechanism of the decreased risk of relapse on the basis of hla molecule haplotype ( figure 2 ) . the effectiveness of adoptive transfer of expanded nk cells for the treatment of relapsed leukemia has been demonstrated , and ongoing efforts are designed to evaluate this approach in the treatment of solid tumor malignancies as well [ 8385 ] . despite this therapy has been proven safe for patients and feasible , more effective strategies to augment in vivo nk cell persistence and expansion are needed to test the clinical benefit of nk cells against solid tumors , as literature supports the notion that there is a cell dose response in act . in a phase ii clinical trial in patients with ovarian and breast cancer , the adoptive transfer of haplo - identical nk cells after lymphodepleting chemotherapy was associated with transient donor chimerism that was not improved with the addition of low - dose total body irradiation . sustained in vivo nk cell expansion may be limited by host rejection , competition with host lymphocytes or suppression by recipient regulatory t cells ( treg ) or myelo - derived suppressor cells . to provide a greater number of nk cells with a differentiated activated phenotype for adoptive immunotherapy , some authors described cytokine - based methods useful for clinical - scale nk cell production . a phase i clinical study evaluated the safety of donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation to patients with progressive malignant disease . this study reported the safety of ex vivo - expanded nk and nk - like t cells with a feeder - free cgmp compatible expansion strategy administered to humans also in combination with il-2 . decot and coworkers described an immunomagnetic technique consisting in cd3/cd19 depletion effective to obtain highly t- and b - cell - depleted nk cell - enriched product with gmp - compliant reagents . il-2 or il-15 were equivalent to significantly enhance nk cell cytotoxicity after culture with mhc - class i negative erythroleukemia cell line . furthermore , they observed a modification in nk - cell receptor expression pattern with upregulation of the activating receptor nkg2d , but also of the inhibiting receptors kir2dl1 and kir2dl2 . in other work pbmc were cultured in serum - free medium and il-2 for 20 days . cells in the culture were also stimulated with anti - cd3 ab ( okt3 ) . safety and feasibility of administering ex vivo expanded nk and nk - like t - effector cells as donor lymphocytes infusion to five cancer patients after stem cell transplantation was evaluated . cell infusions , with or without il-2 injections , seemed to be safe as no gvhd was observed . not only do all these data support a role of nk cell during act therapy , but results also link nk cell recovery with a gvl rather than with a gvhd effect , as recipient 's nk cells have been shown to be the first of lymphoid lineage cells to reconstitute following allogeneic transplantation , and adequate recovery of nk cell number in the early posttransplant period has been associated with improved relapse - free outcome . these studies now provide the backdrop for the development of therapies that enhance the therapeutic benefit of allogeneic transplantation while minimizing the risks and toxicities associated with treatment ( figure 3 ) . in recent years there have been significant advances in the discovery of the molecular mechanisms that govern the reactivity of nk cells against tumors . in this paper we have reviewed how the expression and function of both inhibitory and activating nk receptors are involved in the molecular regulation of innate immunity in malignant settings . nk cells are also potential tools for cancer therapy , both due to cytotoxic ability enhanced by antibodies and the possibility of ex vivo expansion and adoptive transfer . . inasmuch as one scheme will not be suitable for all malignancies , different approaches are to be evaluated for each particular case .

natural killer ( nk ) cells are central components of the innate immunity . in murine models , it has been shown that nk cells can control both local tumor growth and metastasis due to their ability to exert direct cellular cytotoxicity without prior sensitization and to secrete immunostimulatory cytokines like ifn-. the latter participates in cancer elimination by inhibiting cellular proliferation and angiogenesis , promoting apoptosis , and stimulating the adaptive immune system , and it is instrumental for enhancing ag processing and presentation . nevertheless , nk cells display impaired functionality and capability to infiltrate tumors in cancer patients . also , nk cells are feasible targets of stimulation to participate in immunotherapeutic approaches like antibody - based strategies and adoptive cell transfer . thus , multiple attempts currently aim to manipulate nk for utilization in the immunotherapy of cancer .

1. Introduction 2. NK Cells in Cancer Patients 3. NK Cell Therapy Conflicts of Interests

nk cells are effector lymphocytes of the innate immune system that control several types of tumors by limiting their growth and dissemination . in vitro studies using cells from humans and several other mammalian species , as well as in vivo studies in mice and rats , there are numerous studies in mice supporting the notion that nk cells are involved in the elimination of tumor cells . the in vivo analyses relied on antibody - mediated depletion of nk cells in mice , targeting either nk1.1 or the glycolipid asialo - gm1 [ 35 ] . however , nk cell depletion with antibodies to nk1.1 may also affect populations of invariant natural killer t cells and other nk1.1 t - cell populations . caution is therefore required when interpreting studies based upon ab depletion because of the lack of specificity of ab treatment against nk populations . nevertheless , many other independent studies advocate a role for nk cells in the control of tumor development in mice . particularly , mouse nk cells are involved in the in vivo rejection of several transplanted tumors , in a manner dependent upon the presence or absence of nk cell receptor ligands expressed by the tumor . nevertheless , in a further study from the same group , it was shown that although nk cells are important in the early elimination of mca - induced tumors , control of the dormant tumor state depends mainly on adaptive immunity . in some of these experimental models , nk cell - mediated elimination of tumor cells induced the subsequent development of tumor - specific t - cell responses to the parental tumor cells [ 11 , 13 ] as a bridge between innate and adaptive immune responses . in addition to their endogenous protective role in tumor models , nk cells are also mediators of the antitumor effects of several recombinant cytokines , such as il-2 , il-12 , il-18 , and il-21 . so far , these studies in vitro and in animal models show the role of nk cells in cancer immunosurveillance . however , in the last few years , novel studies have revealed the phenotypic status and functionality of nk cells in tumor site and also in peripheral blood ( pb ) of cancer patients . first studies about tumor immune infiltration have shown that the presence of nk cells represents a positive prognostic marker in different carcinomas [ 1921 ] . it has been suggested that despite of low nk cell numbers in tumors , due to their inefficient homing into malignant tissues , this situation may be overcome by cytokine - mediated activation in immunotherapeutical regimens . in this respect , all this evidence about nk importance in tumor immune control enhances the appeal of nk cell - based immunotherapeutic approaches . for instance , the beneficial role of nk cells in controlling human malignancies stems from clinical studies of leukemia patients who received alloreactive nk cells in the course of allogeneic hematopoietic stem cell transplantation [ 25 , 26 ] . in the present work we will revise key aspects of nk cells role from molecular and cellular characteristics to therapeutic applications in cancer patients . a developing tumor influences and is influenced by its stroma , initiates angiogenesis , and interacts with both the adaptive and innate immune systems . the clinicopathological significance of the tumor - infiltrating lymphocytes ( tils ) in various human cancers has been an issue of great interest . cd8 cytotoxic t cells ( ctl ) and nk cells are the most likely effectors for an effective antitumor immunity . several studies have shown that the infiltration of lymphocytes significantly correlates with a prolonged survival time of patients , at least in certain types of cancer [ 3234 ] . typically , nk cells are not found in large numbers in advanced human neoplasms , indicating that they do not normally home efficiently to malignant tissues . moreover , the percentage of intrahepatic nk ( cd56 ) cells was also decreased in patients with metastases compared to those without , being almost twice lower than cd8 and cd4 . in human nonsmall cell lung cancers ( nsclc ) nk cells percentage within cd45 mononuclear cells ( mcs ) isolated from lung tumors was consistently lower than in mc from the pb counterpart and was comparable to that in mc from peritumoral lung tissue . with regard to their localization , nk cells were found in tumor stroma , whereas they were not in direct contact with cancer cells . however , novel studies of tumor - associated nk cells demonstrated a striking phenotype , supporting the notion that tumor - induced alterations of activating nk cell receptor expression may hamper immune surveillance and promote tumor progression . in this sense , several authors studied the balance between inhibitory and activating receptors of nk cells that infiltrate tumors , observing in some cases downregulation of activating receptors and in others , overexpression of inhibitory ones . in a study which analyzed the receptor repertoire and functional integrity of nk cells in peritoneal effusions from patients with ovarian carcinoma , tumor - associated nk cells expressed reduced levels of the activating dnam-1 , 2b4 , and cd16 receptors and were hyporesponsive to hla class i - deficient k562 cells and to coactivation via dnam-1 and 2b4 , relative to autologous peripheral blood nk cells . moreover , tumor - associated nk cells were also refractory to cd16 receptor stimulation , resulting in diminished ab - dependent cellular cytotoxicity ( adcc ) against autologous tumor cells . in another study , tongue cancer intraepithelial nk cells expressed nkg2a , an inhibitory receptor that recognizes hla - e , more frequently than those in the stroma or in lichen planus . collectively , the intraepithelial cd8 t cells and nk cells were phenotypically inactivated , whereas stromal counterparts were phenotypically just as active as those in lichen planus , suggesting the suppressed state of the intraepithelial nk cells . carrega and coworkers described both relevant molecule expression and function of nk cells infiltrating nsclc in comparison with autologous nk cells isolated from either peritumoral normal lung tissues or pb . remarkably , the cytolytic potential of nk cells isolated from cancer tissues was lower than that of nk cells from pb or normal lung tissue , whereas no difference was observed regarding their capability of producing cytokines . bidirectional signaling following cd137-cd137l interaction induced the release of the immunomodulatory cytokines il-10 and tnf by aml cells and directly diminished granule mobilization , cytotoxicity , and ifn- production by human nk cells , demonstrating an immune evasion of aml cells by impairing nk - cell tumor surveillance . not only did nk infiltrating tumor cells show a different phenotype or reduced cytotoxicity , but in some kind of cancers this was also observed in nk cells from pb . in a study in metastatic melanoma ( mm ) patients , nk cells presented decreased activity and ifn- production and also had a redistribution of nk cell subsets . these results are in agreement with another study in mm where a significantly lower percentage of nk cells expressed cd16 , nkp30 , and nkp46 activating receptors as compared to healthy donors . these observations imply that down - regulated nk litic receptor expression in mm patients may affect nk cells ability to recognize and eliminate tumor cells . likewise , the frequency of nk cells expressing the activating receptors nkp30 , nkp44 , nkp46 , nkg2d , and nkg2c was significantly decreased in aml patients compared to the nk cells of normal controls . although the molecular mechanisms responsible for the reduced receptor expression in pb nk cells remains elusive , elevated serum levels of soluble nk cell receptor ligands shed by tumor cells have been associated with downregulation of the nk cell receptors and might contribute to the decreased levels of nk cell activity . in this section , we cited works which showed some impaired nk cells characteristics in cancer patients which determine their inability to eliminate tumor cells ( depicted in figure 1 ) . on the other hand , it is a fact that nk cells are potentially active against tumor cells and it is feasible to manipulate them to restore and/or improve their antitumoral activity . the ability of some mabs to disrupt signalling pathways involved in the maintenance of the malignant phenotype has received widespread attention . in addition , abs exhibit various immunomodulatory properties and , by directly activating or inhibiting molecules of the immune system , they can promote the induction of antitumor immune responses . abs are linked to immune effector functions by the fc fragment , which is capable of initiating adcc when binding to fc receptors , specially fcriii ( cd16 ) on nk cells , which initiates a sequence of cellular events culminating in the release of cytotoxic granzyme - containing granules and inf- secretion [ 4547 ] . several studies have established the importance of fc - fcr interactions for the antitumor effects of certain mabs in murine models and clinical trials . a seminal paper showed that the antitumor activities of the anti - her-2 mab , trastuzumab , and the anti - cd20 mab , rituximab , were lower in fcr - deficient mice than in wild - type mice . the role of fcr in the antitumor response has been further supported by the finding that polymorphisms in the gene encoding fcriii , which lead to higher binding of ab to fcriii , are associated with high response rates to rituximab in patients with follicular non - hodgkin 's lymphoma . adcc enhancement through fc domain modification has shown promise in the development of next generation mabs . in previous reports , we and others proved that nk cells produce cetuximab - mediated adcc of metastatic crc ( mcrc ) and that this activity is not affected by the mutational status of the downstream molecule k - ras [ 51 , 52 ] . this effect is expected because nk cells recognize the surface - bound abs and are able to kill tumor cells independently of the egfr pathway activation . nevertheless , it remains to be answered why in mcrc patients with k - ras mutated status cetuximab - mediated adcc does not induce clinical remissions . as we discussed , one of the possibilities is the low proportion of nk cells infiltrating crc tumors and the low functional capacity of these cells observed in cancer patients [ 54 , 55 ] . therefore , if it were possible to enhance the activity of nk cells , the efficacy of treatment with cetuximab could be increased . the anti - vegf - a mab , bevacizumab , is also active in mcrc , regardless of the k - ras mutation status ; nevertheless , it needs to be administered in combination with a cytotoxic agent . because panitumumab , an igg2a anti - egfr mab , does not effectively interact with fc receptors on the nk cell surface , it was unable to initiate adcc and , as expected , lenalidomide had no effect because its activity is reliant on the augmentation of nk cell signaling downstream of fcr . as explained previously , many studies demonstrated the impairment of nk cell activity in cancer patients . watanabe and coworkers evaluated whether il-21 could improve the impairment of adcc in patients with escc as il-21 was reported to have the ability to activate nk cells . as in the previous example , nk cell response to cetuximab - coated tumor cells could be enhanced by the administration of nk cell stimulatory cytokines il-2 , il-12 , or il-21 and resulted in higher ifn- production than was observed with either agent alone . costimulated nk cells also secreted elevated levels of chemokines ( il-8 , macrophage inflammatory protein-1a , and rantes ) that could direct the migration of naive and activated t cells . in other experimental study , it was analyzed the correlation between egfr expression in lung cancer cell lines and the adcc activity of cetuximab as well as the influence of il-2 and chemotherapy on adcc activity . in addition , nk cell - mediated adcc was enhanced by il-2 and such cells were also less susceptible to immunosuppression by chemotherapy than in lung cancer patients . moreover , nk cells purified from patients with advanced disease indicated less herceptin - mediated adcc in comparison with that from healthy donors , whereas monocytes purified from the patients showed an almost equal amount of herceptin - mediated adcc in comparison with that from healthy individuals , indicating that nk cell dysfunction contributed to the impaired herceptin - mediated adcc in gastric cancer patients . furthermore , the nk - cell dysfunction on herceptin - mediated adcc correlated with the downregulation of cd16 expression in the patients , and il-2 ex vivo treatment of nk cells could restore the impairment of herceptin - mediated adcc , concomitant to the normalization of the expression of cd16 molecules . furthermore , il-15 could replace il-2 in most of its immunologic activities , stimulating nk cells ability to produce ifn- , paralleling the upregulation of activating receptors . these results show that cetuximab - mediated nk cell activity can be significantly enhanced in the presence of nk cell stimulatory cytokines . adoptive cell transfer ( act ) therapy can be considered as a strategy aimed at replacing , repairing , or enhancing the biological function of the immune system by means of autologous or allogeneic cells . act therapy may include ( i ) removal or enrichment of various cell populations ; ( ii ) expansion of hematopoietic cell subsets ; ( iii ) activation of lymphocytes for immunotherapy ; ( iv ) genetic modification of lymphoid cells , when these cells are intended to engraft transiently in the recipient and/or be used in the treatment of cancer . as we will discuss , the use of lymphocytes and/or nk cells as a strategic weapon in preventing or curing the neoplastic relapse after surgery / chemotherapy has been applied to the treatment of hematological malignancies and solid tumors . nevertheless , problems with deriving sufficient numbers of these cells from patients , downregulation of mhc class i ligands and costimulatory ligands on tumor cells , combined with the lack of persistence of transferred cells have precluded broad utilization of these cells for the treatment of cancer patients . the transfer of other immune subsets , such as nk cells which do not require prior sensitization to respond to target cells and can potently induce a cytolytic response , represents a good alternate or auxiliary cell type for immunotherapy . one of the main hurdles involved in developing adoptive transfer of immune cells has been to define good manufacturing procedures ( gmps ) compliant methods to isolate defined subsets with the number of cells required that guarantee the safety of the injection to patients . in this respect , clinical - grade production of nk cells has proven efficient , and large - scale expansion method has been possible for human nk cells , using a cytokine - based culture system for ex vivo expansion of nk cells from hematopoietic progenitor cells from umbilical cord blood . nevertheless , improved technologies for nk cells expansion lately have been tested . even when the first works were based on transplantation of autologous ex vivo expanded cells , and in addition to what we earlier discussed on cancer patients nk cells dysfunctions , it is now understood that the failure of autologous nk therapy is partially due to the downregulation of nk cell killing that occurs with recognition of self - class i mhc on tumor cells [ 7173 ] , making allogeneic cell transfer more attractive . during allogeneic hematopoietic stem cell transplantation , nk cells have been implicated in the suppression of graft versus host disease ( gvhd ) , the promotion of bone marrow engraftment , and mediation of a graft versus leukemia ( gvl ) effect . in the setting of allogeneic act in leukemias , the results of nk cells activity depend on the directionality of lysis [ 75 , 76 ] . when the nk cells are donor - derived and the recipient cells lack expression of the cognate kir ligand , the donor nk cell lysis of recipient target cells can result in gvl and/or gvhd , depending on the tissue origin of the nk cell target . however , if the target cell is of donor origin , and the nk effector cell is of recipient origin , nk cell lysis can result in graft rejection . it has been estimated that nk cell alloreactivity can be expected to occur in about 50% of unrelated donor transplants with velardi and coworkers announced a new era in the exploitation of nk cells for cancer immunotherapy with a pioneering study of hematopoietic stem cell transplantation that stratified patients according to kir - ligand mismatch between donor cells and recipient leukemic cells . this reveals that alloreactive nk cells , unrestrained by inhibitory signals from the recipient hla ligands , protects against disease relapse . this kir - ligand mismatch phenomenon has attracted researchers to study it , and similar observations have since been made in other trials of leukemia immunotherapy [ 7981 ] . these findings might be crucial to elucidating the mechanism of the decreased risk of relapse on the basis of hla molecule haplotype ( figure 2 ) . the effectiveness of adoptive transfer of expanded nk cells for the treatment of relapsed leukemia has been demonstrated , and ongoing efforts are designed to evaluate this approach in the treatment of solid tumor malignancies as well [ 8385 ] . despite this therapy has been proven safe for patients and feasible , more effective strategies to augment in vivo nk cell persistence and expansion are needed to test the clinical benefit of nk cells against solid tumors , as literature supports the notion that there is a cell dose response in act . to provide a greater number of nk cells with a differentiated activated phenotype for adoptive immunotherapy , some authors described cytokine - based methods useful for clinical - scale nk cell production . furthermore , they observed a modification in nk - cell receptor expression pattern with upregulation of the activating receptor nkg2d , but also of the inhibiting receptors kir2dl1 and kir2dl2 . safety and feasibility of administering ex vivo expanded nk and nk - like t - effector cells as donor lymphocytes infusion to five cancer patients after stem cell transplantation was evaluated . not only do all these data support a role of nk cell during act therapy , but results also link nk cell recovery with a gvl rather than with a gvhd effect , as recipient 's nk cells have been shown to be the first of lymphoid lineage cells to reconstitute following allogeneic transplantation , and adequate recovery of nk cell number in the early posttransplant period has been associated with improved relapse - free outcome . in recent years there have been significant advances in the discovery of the molecular mechanisms that govern the reactivity of nk cells against tumors . in this paper we have reviewed how the expression and function of both inhibitory and activating nk receptors are involved in the molecular regulation of innate immunity in malignant settings . nk cells are also potential tools for cancer therapy , both due to cytotoxic ability enhanced by antibodies and the possibility of ex vivo expansion and adoptive transfer .

according to current recommendations , a clinical diagnosis of copd is primarily based on the detection of airflow obstruction in spirometric measures.1,2 however , the spirometric criteria defining airflow obstruction in copd are still subject to lively debate.3,4 the global initiative for chronic obstructive lung disease recommends the use of a fixed ratio ( fr ) of 0.7 for postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) to confirm the diagnosis.1 this approach has been criticized as arbitrary and argued to particularly overestimate prevalences in advanced age.5 alternatively , the use of the fifth percentile of the predicted value ( lower limit of normal , lln ) for fev1/fvc has been proposed by respiratory societies , eg , the american thoracic society and european respiratory society.6,7 it has been shown that the two copd definitions lead to deviating prevalences.8 in addition , data and subjects in advanced age beyond 75 years are often underrepresented in the studied population - based samples . besides airflow obstruction , copd is accompanied by a variety of systemic impairments.9 comorbidities of copd include cardiovascular disease , diabetes , depression , and cancer,10 and it has been reported that copd may accelerate the progression of some of these comorbidities.9 moreover , copd is characterized by low - grade systemic inflammation.11 different biomarkers such as c - reactive protein ( crp)12 and interleukin ( il)-613 have been associated with a poor prognosis in copd participants . celli et al14 showed that using a whole panel of inflammatory biomarkers ( eg , crp , il-6 , il-8 , and fibrinogen ) improved the clinical prediction of mortality in copd participants . still , in how far circulating biomarker levels are affected by spirometric criteria is less investigated . the aim of this investigation was 1 ) to quantify the impact of disease definition by different spirometric criteria ( fr vs lln ) on copd prevalence in a population - based sample and 2 ) to evaluate the association of fr vs lln criteria with comorbidity prevalences and selected inflammatory biomarkers . within a population - based cohort from the augsburg region ( kora , cooperative health research in the augsburg region , germany),15 two follow - up studies , kora - f416 and kora - age,17 were launched from the monica / kora surveys s1s4 . the kora - f4 and kora - age studies were approved by the ethics committee of the bavarian medical association , and written informed consent was obtained from all participants . details of the kora platform and surveys have been described earlier.15,18 briefly , spirometry was performed in 1,321 subjects aged 4163 years in kora - f4 ( 20062008 ) and in 935 subjects aged 6590 years in kora - age ( 2009 ) . measurement conditions for spirometry and the examiners were the same in both studies . standing height and weight were measured with subjects wearing only light clothes and no shoes . details on spirometric measurements have been reported earlier.16 in short , standard spirometry without bronchodilation was performed in line with the american thoracic society / european respiratory society recommendations in an upright sitting position , while subjects were wearing nose clips using a pneumotachograph - type spirometer ( masterscope pc ; carefusion , hchberg , germany ) . the participants performed at least three spirometric maneuvers in order to obtain a minimum of two acceptable and reproducible values . subjects were classified as having copd if fev1/fvc was < 0.7 for the fr criterion and if fev1/fvc was < lln for the lln criterion . reference values of the global lungs initiative for fvc , fev1 , and fev1/fvc were used.19 the severity of copd was graded according to the stages of disease based on fev1 results into categories as defined by the global initiative for chronic obstructive lung disease.1 self - reported information on myocardial infarction ( mi ) , stroke , cancer , diabetes , inflammatory joint disease / rheumatic disease , gastrointestinal disease ( gid ) , and copd / chronic bronchitis diagnosis was collected in standardized interviews , which were partly performed via telephone and self - administered questionnaires within the comprehensive kora assessments.20 information on current medication was assessed by asking all study participants to bring original packaging of their medications used during the last 7 days before the study examination . hypertension was defined as blood pressure values 140/90 mmhg measured during the visit and/or intake of antihypertensive medication , given that the participant was aware of having hypertension as assessed in the standardized interview or questionnaire . diabetes mellitus was defined as having a self - reported physician diagnosis of diabetes and/or intake of antidiabetic medication . the presence of an anxiety disorder was defined using the generalized anxiety disorder scale-721 in the standardized interview or questionnaire with a score of 10 indicating the presence of an anxiety disorder . depression was assessed using the depression module of the brief patient health questionnaire - d ( phq - d ) in kora - f4 ( age 4163 years ) , whereas in kora - age ( age 6590 years ) it was assessed using the geriatric depression scale-15 ( gds-15 ) during the standardized interview , where participants with a score of 10 were classified as being depressive.22 smoking habits and symptoms of chronic bronchitis , defined as cough and sputum on most days during 3 or more months per year , were assessed within the standardized interview or questionnaire . furthermore , information on the presence of a physician diagnosis of copd or chronic bronchitis was assessed during the standardized interview . information on medication against obstructive respiratory disease comprised sympathomimetics , anticholinergics , xanthines , leukotriene receptor antagonists , and corticosteroids . serum and ethylenediaminetetraacetic acid ( edta ) plasma samples were obtained from all study participants and were stored at 80c until analysis . levels of high - sensitivity crp were measured in serum ( kora - age ) or edta plasma samples ( kora - f4 ) using cardio phase hscrp ( siemens , eschborn , germany ) . basic study results are presented according to three age groups ( < 65 years/65 years and < 75 years/75 years ) and spirometric results on the basis of fev1/fvc categories . as the distributions of the biomarker levels were skewed , results were log10 transformed before further analysis . for the outcome variables of comorbidities , self - reported physician diagnosis of copd / chronic bronchitis , use of lung medication , chronic bronchitis symptoms , and ever - smoking logistic regression models were generated including sex and age as factors . the influence of the following dichotomous variables was then tested by separately including them into these basic models : 1 ) cases vs healthy subjects by the fr criterion , 2 ) cases vs healthy subjects by the lln criterion , and 3 ) cases by the fr criterion only vs cases by the lln criterion . all lln - defined copd cases were included in the respective fr - defined group except for one subject in the age group of 4044 years who was excluded from these analyses . analyses were performed using the commercially available software sas 9.3 ( sas institute inc . , cary , nc , usa ) and statgraphics centurion xvii ( statpoint technologies inc . , warrenton , va , usa ) ; additional plots were done using sigmaplot 12.0 ( systat software inc . within a population - based cohort from the augsburg region ( kora , cooperative health research in the augsburg region , germany),15 two follow - up studies , kora - f416 and kora - age,17 were launched from the monica / kora surveys s1s4 . the kora - f4 and kora - age studies were approved by the ethics committee of the bavarian medical association , and written informed consent was obtained from all participants . details of the kora platform and surveys have been described earlier.15,18 briefly , spirometry was performed in 1,321 subjects aged 4163 years in kora - f4 ( 20062008 ) and in 935 subjects aged 6590 years in kora - age ( 2009 ) . measurement conditions for spirometry and the examiners were the same in both studies . standing height and weight were measured with subjects wearing only light clothes and no shoes . details on spirometric measurements have been reported earlier.16 in short , standard spirometry without bronchodilation was performed in line with the american thoracic society / european respiratory society recommendations in an upright sitting position , while subjects were wearing nose clips using a pneumotachograph - type spirometer ( masterscope pc ; carefusion , hchberg , germany ) . the participants performed at least three spirometric maneuvers in order to obtain a minimum of two acceptable and reproducible values . subjects were classified as having copd if fev1/fvc was < 0.7 for the fr criterion and if fev1/fvc was < lln for the lln criterion . reference values of the global lungs initiative for fvc , fev1 , and fev1/fvc were used.19 the severity of copd was graded according to the stages of disease based on fev1 results into categories as defined by the global initiative for chronic obstructive lung disease.1 self - reported information on myocardial infarction ( mi ) , stroke , cancer , diabetes , inflammatory joint disease / rheumatic disease , gastrointestinal disease ( gid ) , and copd / chronic bronchitis diagnosis was collected in standardized interviews , which were partly performed via telephone and self - administered questionnaires within the comprehensive kora assessments.20 information on current medication was assessed by asking all study participants to bring original packaging of their medications used during the last 7 days before the study examination . hypertension was defined as blood pressure values 140/90 mmhg measured during the visit and/or intake of antihypertensive medication , given that the participant was aware of having hypertension as assessed in the standardized interview or questionnaire . diabetes mellitus was defined as having a self - reported physician diagnosis of diabetes and/or intake of antidiabetic medication . the presence of an anxiety disorder was defined using the generalized anxiety disorder scale-721 in the standardized interview or questionnaire with a score of 10 indicating the presence of an anxiety disorder . depression was assessed using the depression module of the brief patient health questionnaire - d ( phq - d ) in kora - f4 ( age 4163 years ) , whereas in kora - age ( age 6590 years ) it was assessed using the geriatric depression scale-15 ( gds-15 ) during the standardized interview , where participants with a score of 10 were classified as being depressive.22 smoking habits and symptoms of chronic bronchitis , defined as cough and sputum on most days during 3 or more months per year , were assessed within the standardized interview or questionnaire . furthermore , information on the presence of a physician diagnosis of copd or chronic bronchitis was assessed during the standardized interview . information on medication against obstructive respiratory disease comprised sympathomimetics , anticholinergics , xanthines , leukotriene receptor antagonists , and corticosteroids . serum and ethylenediaminetetraacetic acid ( edta ) plasma samples were obtained from all study participants and were stored at 80c until analysis . levels of high - sensitivity crp were measured in serum ( kora - age ) or edta plasma samples ( kora - f4 ) using cardio phase hscrp ( siemens , eschborn , germany ) . basic study results are presented according to three age groups ( < 65 years/65 years and < 75 years/75 years ) and spirometric results on the basis of fev1/fvc categories . as the distributions of the biomarker levels were skewed , results were log10 transformed before further analysis . for the outcome variables of comorbidities , self - reported physician diagnosis of copd / chronic bronchitis , use of lung medication , chronic bronchitis symptoms , and ever - smoking logistic regression models were generated including sex and age as factors . the influence of the following dichotomous variables was then tested by separately including them into these basic models : 1 ) cases vs healthy subjects by the fr criterion , 2 ) cases vs healthy subjects by the lln criterion , and 3 ) cases by the fr criterion only vs cases by the lln criterion . all lln - defined copd cases were included in the respective fr - defined group except for one subject in the age group of 4044 years who was excluded from these analyses . for the outcome variables , log10(crp ) and log10(il-6 ) general linear models were generated including sex and age as predictors . analyses were performed using the commercially available software sas 9.3 ( sas institute inc . , cary , nc , usa ) and statgraphics centurion xvii ( statpoint technologies inc . , warrenton , va , usa ) in total , 2,256 individuals were included : 1,321 from kora - f4 ( 618 men and 703 women ) and 935 from kora - age ( 474 men and 461 women ) . table 1 summarizes descriptive data of our study participants from both cohorts separately and combined . all study participants from kora - f4 were younger than 65 years ( 4163 years ) , and all participants from kora - age were 65 years and older ( 6590 years ) . the prevalence of copd defined by spirometry without bronchodilation according to the fr criterion increased with age from ~10% for the age group below 65 years to 21% for the age group between 65 years and 75 years , and finally 26% for individuals in the age group of 75 years and older ( table 2 ) , but was predominantly confined to mild and moderate stages of copd . severe and very severe copd grades were rare in our population - based study ( nine cases in total ) and did not differ between fr and lln criteria for all age groups . the prevalence of copd on the basis of the lln criterion remained stable below 10% up to an age of 90 years . the drifting apart with increasing age between both copd definitions , depending on the diagnostic criteria used ( fr vs lln ) , became most prominent at age beyond 65 years ( figure 1 ) . as shown in table 3 , the presence of a self - reported physician diagnosis of copd / chronic bronchitis in cases of spirometrically defined copd was generally rather low ranging between 14% and 38% . however , it was predominantly higher for lln - defined copd than for the fr criterion as was true for the current use of lung medication and the presence of chronic bronchitis symptoms . in the logistic regression model , this difference between the spirometric criteria was observed for all three aspects ( table 4 ) . regarding ever smoking , differences of cases vs no copd by both criteria were most prominent in the youngest age group ( table 3 ) , while no differences between the two copd criteria were observed . table 5 shows the odds ratio estimates for the influence of copd according to fr and lln criteria on disease prevalence from the logistic regression models ; primary prevalences of comorbidities are given in figure s1a c . by trend , differences between subjects with and without copd were most prominent in males for the lln criterion for mi , cancer , stroke , and anxiety in the middle and oldest age group and for gid in the youngest age group . although a similar trend was observed for gid in females in the youngest age group , other comorbidities did not show consistent tendencies in women . on the other hand , as shown in table 5 , the prevalence of obesity was consistently lower in copd cases , which was independent of the criterion . beyond this observation , still , the occurrence of two or more comorbidities was lower in subjects with vs without copd defined by the lln criterion ( table 5 ) . the estimates for the effect of copd on levels of crp and il-6 are presented in table 6 ; median crp and il-6 levels are shown in figure s2a and b. levels of both bio - markers slightly increased with age in all participants without copd , but consistent differences between fr- and lln - defined copd were not detectable . still , both crp and il-6 tended to be higher in cases by both criteria ( table 6 ) . in epidemiology , the definition of copd disease status is frequently based on either spirometric results , often without bronchodilation , or questionnaire data of physician diagnosis . in this population - based observational study , the impact of disease definition by different spirometric criteria ( fr vs lln ) on copd prevalence was evident and increased with age ( figure 1 ) . the tendency of copd being possibly over - diagnosed by the fr criterion becomes more pronounced in advanced age , most prominently beyond 65 years in our sample . however , neither the prevalence of common comorbidities nor levels of the inflammatory biomarkers crp and il-6 were affected by the selection of the copd criterion . our results have to be considered in light of the fact that in our epidemiological cohort we predominantly observed participants with mild - to - moderate airflow obstruction based on spirometry without bronchodilation . the prevalence of respiratory obstruction we observed for both spirometric criteria compared well with recent population - based analyses that were performed in a similar fashion . for example , in a large canadian sample , an overall obstruction prevalence of 17% by the fr criterion has been reported for the age range of 4093 years,23 turkeshi et al24 found an fr - based prevalence of 27% in a belgian sample of very old adults aged 80 years or older . however , the prevalence of ~7% we found for lln was slightly lower than the range of 9%11% reported in these studies . unexpectedly , study participants with spirometrically defined copd generally did not suffer from more comorbidities than participants of the same age without copd irrespective of the applied spirometric criterion . although a tendency toward higher prevalences of mi , cancer , stroke , and anxiety was observed in participants with lln - defined copd compared with participants without lln - defined copd , there was no distinct difference after taking account of the sex and age in logistic regression models . on the contrary , we found a lower prevalence of obesity in copd subjects for both criteria , which might be explained by a higher fraction of smokers among these subjects.25 none of the other comorbidities evaluated were more prevalent among copd participants , which is in contrast to findings from other studies.2628 the lack of a difference may partly be explained by the fact that our study was conducted in the general population with predominantly mild cases as opposed to a clinical setting with more severe stages of copd . the prevalences we found in spirometrically defined copd for mi , stroke , anxiety , and diabetes compared well with findings described by divo et al29 in a recent analysis of copd patients based on the bode registry , when considering comparable age groups . however , our prevalences for hypertension were markedly higher , while those for depression were lower , which might be attributed to differences in the assessment of those comorbidities . furthermore , the copd cases as defined in our study were predominantly mild and mostly not yet confirmed by a physician , as indicated by the rather poor overlap with physician diagnoses , ( table 3 ) suggesting early undiagnosed cases to a large degree . moreover , the increased fraction of ever smokers especially in the youngest age group points to the fact that this factor is of importance predominantly in copd at comparably young age . it might be speculated that possible concomitant systemic impairments in these cases are in a comparably early stage and thus are not or not yet diagnosed by a physician . alternatively , a limitation in airway function may precede the affections of other organs that are later described as comorbidities in copd . the exploration of a possible sequence in this regard , however , would require a longitudinal evaluation , which was beyond the scope of the current cross - sectional study . in recent years , systemic inflammation has come into the focus of copd research , and increased serum levels of il-6 and crp in copd participants have been reported.30 although we found a tendency toward elevated values for both markers in copd participants , the overall effect was comparatively small . the lack of findings , particularly in the aged , may , in our case , be partly explained by harvesting , where subjects in poor health did not survive or did not participate in the study as they were unable to get into the study center . a recent study by silva et al31 did not find markedly elevated crp in nonsmoking copd patients compared to a control group indicating the possible impact of active smoking on this parameter . however , the tendency toward higher il-6 levels in lln - defined copd cases in older age for males suggests that this criterion may be diagnostically more specific in identifying participants with ( early stage ) systemic inflammation than the fr and needs confirmation in longitudinal settings . consequently , these findings indicate that the application of the lln criterion in epidemiological or clinical settings will result in an identification of subjects with more concomitant typical characteristics of copd , eg , chronic bronchitis symptoms , and a higher probability of a physician diagnosis of copd compared to fr ( table 4 ) . however , since no consistent differences in comorbidity prevalences or biomarker levels were observed , the preference of one criterion over the other in this regard could not be derived from our study . beyond the fev1/fvc ratio , the use of more advanced lung function measurements such as body plethysmography32 could provide a refined characterization of patient subgroups with a higher number or specific patterns of comorbidities . the limitation of the current study might be the fact that the definition of copd cases here , as a rather common epidemiological approach , was solely based on spirometric results as opposed to a usually more comprehensive approach in clinical practice including anamnesis , symptoms , risk factors , and individual clinical history . however , the restriction to different interpretations of a spirometric measure allowed us to specifically focus on the impact of this particular component of the diagnostic sequence in predominantly early , often undiagnosed cases of airway obstruction according to our research question . a clear strength of our study is the inclusion of participants from the general population up to an advanced age of 90 years , an age group that is often underrepresented in copd studies . in a population - based sample of adults in middle and advanced age , we found that the prevalence of spirometrically defined copd was higher when using the fr criterion as opposed to lln and this difference increased with increasing age . however , for the predominantly mild cases observed in our cohort , there was no conclusive impact of the different spirometric criteria on the prevalence of comorbidities and on common inflammatory biomarker levels . ( a c ) prevalence of comorbidities in subjects without copd ( light grey ) or with copd ( dark grey ) by fr ( fev1/fvc < 0.7 ; solid color ) and lln ( fev1/fvc < lower limit of normal ; hatched ) criterion ( percentages ) . abbreviations : fr , fixed ratio ; lln , lower limit of normal ; fev1 , forced expiratory volume in 1 second ; fvc , forced vital capacity ; y , years . ( a b ) levels of crp and il-6 in subjects without copd ( light grey ) and copd cases ( dark grey ) based on fr ( fev1/fvc < 0.7 ; solid color ) or lln ( fev1/fvc < lower limit of normal ; hatched ) criterion ( number of subjects per group are given in brackets ) . abbreviations : fr , fixed ratio ; lln , lower limit of normal ; fev1 , forced expiratory volume in 1 second ; fvc , forced vital capacity ; y , years .

backgroundthere is an ongoing debate about the appropriate spirometric criterion for airway obstruction to detect copd . furthermore , the association of different criteria with comorbidity prevalence and inflammatory biomarkers in advanced age is unclear.materials and methodsspirometry was performed in a population - based study ( n=2,256 ) covering an age range of 4190 years . copd was spirometrically determined either by a fixed ratio ( fr ) of < 0.7 for forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) or by fev1/fvc below the lower limit of normal ( lln ) . comorbidity prevalences and circulating biomarker levels ( c - reactive protein [ crp ] , interleukin [ il]-6 ) were compared between subjects with or without copd by the two criteria using logistic and multiple regression models , adjusting for sex and age.resultsthe prevalence of spirometrically defined copd by fr increased with age from 10% in subjects aged < 65 years to 26% in subjects aged 75 years . for lln - defined copd , it remained below 10% for all age groups . overall , copd diagnosis was not associated with specific comorbidities , except for a lower prevalence of obesity in both fr- and lln - defined cases . both crp and il-6 tended to be higher in cases by both criteria.conclusionin a population - based cohort of adults up to the age of 90 years , the prevalence of spirometrically defined copd was higher for the fr criterion than for the lln criterion . this difference increased with age . neither prevalences of common comorbidities nor levels of the biomarkers , crp or il-6 , were conclusively associated with the selection of the copd criterion . results have to be considered in light of the predominantly mild cases of airway obstruction in the examined study population .

Background Materials and methods Study participants Spirometry Comorbidities and biomarkers Statistical analysis Results Discussion Conclusion Supplementary materials

according to current recommendations , a clinical diagnosis of copd is primarily based on the detection of airflow obstruction in spirometric measures.1,2 however , the spirometric criteria defining airflow obstruction in copd are still subject to lively debate.3,4 the global initiative for chronic obstructive lung disease recommends the use of a fixed ratio ( fr ) of 0.7 for postbronchodilator forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) to confirm the diagnosis.1 this approach has been criticized as arbitrary and argued to particularly overestimate prevalences in advanced age.5 alternatively , the use of the fifth percentile of the predicted value ( lower limit of normal , lln ) for fev1/fvc has been proposed by respiratory societies , eg , the american thoracic society and european respiratory society.6,7 it has been shown that the two copd definitions lead to deviating prevalences.8 in addition , data and subjects in advanced age beyond 75 years are often underrepresented in the studied population - based samples . besides airflow obstruction , copd is accompanied by a variety of systemic impairments.9 comorbidities of copd include cardiovascular disease , diabetes , depression , and cancer,10 and it has been reported that copd may accelerate the progression of some of these comorbidities.9 moreover , copd is characterized by low - grade systemic inflammation.11 different biomarkers such as c - reactive protein ( crp)12 and interleukin ( il)-613 have been associated with a poor prognosis in copd participants . celli et al14 showed that using a whole panel of inflammatory biomarkers ( eg , crp , il-6 , il-8 , and fibrinogen ) improved the clinical prediction of mortality in copd participants . still , in how far circulating biomarker levels are affected by spirometric criteria is less investigated . the aim of this investigation was 1 ) to quantify the impact of disease definition by different spirometric criteria ( fr vs lln ) on copd prevalence in a population - based sample and 2 ) to evaluate the association of fr vs lln criteria with comorbidity prevalences and selected inflammatory biomarkers . within a population - based cohort from the augsburg region ( kora , cooperative health research in the augsburg region , germany),15 two follow - up studies , kora - f416 and kora - age,17 were launched from the monica / kora surveys s1s4 . the kora - f4 and kora - age studies were approved by the ethics committee of the bavarian medical association , and written informed consent was obtained from all participants . details of the kora platform and surveys have been described earlier.15,18 briefly , spirometry was performed in 1,321 subjects aged 4163 years in kora - f4 ( 20062008 ) and in 935 subjects aged 6590 years in kora - age ( 2009 ) . details on spirometric measurements have been reported earlier.16 in short , standard spirometry without bronchodilation was performed in line with the american thoracic society / european respiratory society recommendations in an upright sitting position , while subjects were wearing nose clips using a pneumotachograph - type spirometer ( masterscope pc ; carefusion , hchberg , germany ) . subjects were classified as having copd if fev1/fvc was < 0.7 for the fr criterion and if fev1/fvc was < lln for the lln criterion . reference values of the global lungs initiative for fvc , fev1 , and fev1/fvc were used.19 the severity of copd was graded according to the stages of disease based on fev1 results into categories as defined by the global initiative for chronic obstructive lung disease.1 self - reported information on myocardial infarction ( mi ) , stroke , cancer , diabetes , inflammatory joint disease / rheumatic disease , gastrointestinal disease ( gid ) , and copd / chronic bronchitis diagnosis was collected in standardized interviews , which were partly performed via telephone and self - administered questionnaires within the comprehensive kora assessments.20 information on current medication was assessed by asking all study participants to bring original packaging of their medications used during the last 7 days before the study examination . the presence of an anxiety disorder was defined using the generalized anxiety disorder scale-721 in the standardized interview or questionnaire with a score of 10 indicating the presence of an anxiety disorder . depression was assessed using the depression module of the brief patient health questionnaire - d ( phq - d ) in kora - f4 ( age 4163 years ) , whereas in kora - age ( age 6590 years ) it was assessed using the geriatric depression scale-15 ( gds-15 ) during the standardized interview , where participants with a score of 10 were classified as being depressive.22 smoking habits and symptoms of chronic bronchitis , defined as cough and sputum on most days during 3 or more months per year , were assessed within the standardized interview or questionnaire . levels of high - sensitivity crp were measured in serum ( kora - age ) or edta plasma samples ( kora - f4 ) using cardio phase hscrp ( siemens , eschborn , germany ) . basic study results are presented according to three age groups ( < 65 years/65 years and < 75 years/75 years ) and spirometric results on the basis of fev1/fvc categories . as the distributions of the biomarker levels were skewed , results were log10 transformed before further analysis . for the outcome variables of comorbidities , self - reported physician diagnosis of copd / chronic bronchitis , use of lung medication , chronic bronchitis symptoms , and ever - smoking logistic regression models were generated including sex and age as factors . the influence of the following dichotomous variables was then tested by separately including them into these basic models : 1 ) cases vs healthy subjects by the fr criterion , 2 ) cases vs healthy subjects by the lln criterion , and 3 ) cases by the fr criterion only vs cases by the lln criterion . all lln - defined copd cases were included in the respective fr - defined group except for one subject in the age group of 4044 years who was excluded from these analyses . within a population - based cohort from the augsburg region ( kora , cooperative health research in the augsburg region , germany),15 two follow - up studies , kora - f416 and kora - age,17 were launched from the monica / kora surveys s1s4 . the kora - f4 and kora - age studies were approved by the ethics committee of the bavarian medical association , and written informed consent was obtained from all participants . details of the kora platform and surveys have been described earlier.15,18 briefly , spirometry was performed in 1,321 subjects aged 4163 years in kora - f4 ( 20062008 ) and in 935 subjects aged 6590 years in kora - age ( 2009 ) . details on spirometric measurements have been reported earlier.16 in short , standard spirometry without bronchodilation was performed in line with the american thoracic society / european respiratory society recommendations in an upright sitting position , while subjects were wearing nose clips using a pneumotachograph - type spirometer ( masterscope pc ; carefusion , hchberg , germany ) . subjects were classified as having copd if fev1/fvc was < 0.7 for the fr criterion and if fev1/fvc was < lln for the lln criterion . reference values of the global lungs initiative for fvc , fev1 , and fev1/fvc were used.19 the severity of copd was graded according to the stages of disease based on fev1 results into categories as defined by the global initiative for chronic obstructive lung disease.1 self - reported information on myocardial infarction ( mi ) , stroke , cancer , diabetes , inflammatory joint disease / rheumatic disease , gastrointestinal disease ( gid ) , and copd / chronic bronchitis diagnosis was collected in standardized interviews , which were partly performed via telephone and self - administered questionnaires within the comprehensive kora assessments.20 information on current medication was assessed by asking all study participants to bring original packaging of their medications used during the last 7 days before the study examination . depression was assessed using the depression module of the brief patient health questionnaire - d ( phq - d ) in kora - f4 ( age 4163 years ) , whereas in kora - age ( age 6590 years ) it was assessed using the geriatric depression scale-15 ( gds-15 ) during the standardized interview , where participants with a score of 10 were classified as being depressive.22 smoking habits and symptoms of chronic bronchitis , defined as cough and sputum on most days during 3 or more months per year , were assessed within the standardized interview or questionnaire . levels of high - sensitivity crp were measured in serum ( kora - age ) or edta plasma samples ( kora - f4 ) using cardio phase hscrp ( siemens , eschborn , germany ) . basic study results are presented according to three age groups ( < 65 years/65 years and < 75 years/75 years ) and spirometric results on the basis of fev1/fvc categories . as the distributions of the biomarker levels were skewed , results were log10 transformed before further analysis . for the outcome variables of comorbidities , self - reported physician diagnosis of copd / chronic bronchitis , use of lung medication , chronic bronchitis symptoms , and ever - smoking logistic regression models were generated including sex and age as factors . the influence of the following dichotomous variables was then tested by separately including them into these basic models : 1 ) cases vs healthy subjects by the fr criterion , 2 ) cases vs healthy subjects by the lln criterion , and 3 ) cases by the fr criterion only vs cases by the lln criterion . all lln - defined copd cases were included in the respective fr - defined group except for one subject in the age group of 4044 years who was excluded from these analyses . for the outcome variables , log10(crp ) and log10(il-6 ) general linear models were generated including sex and age as predictors . the prevalence of copd defined by spirometry without bronchodilation according to the fr criterion increased with age from ~10% for the age group below 65 years to 21% for the age group between 65 years and 75 years , and finally 26% for individuals in the age group of 75 years and older ( table 2 ) , but was predominantly confined to mild and moderate stages of copd . severe and very severe copd grades were rare in our population - based study ( nine cases in total ) and did not differ between fr and lln criteria for all age groups . the prevalence of copd on the basis of the lln criterion remained stable below 10% up to an age of 90 years . the drifting apart with increasing age between both copd definitions , depending on the diagnostic criteria used ( fr vs lln ) , became most prominent at age beyond 65 years ( figure 1 ) . as shown in table 3 , the presence of a self - reported physician diagnosis of copd / chronic bronchitis in cases of spirometrically defined copd was generally rather low ranging between 14% and 38% . however , it was predominantly higher for lln - defined copd than for the fr criterion as was true for the current use of lung medication and the presence of chronic bronchitis symptoms . in the logistic regression model , this difference between the spirometric criteria was observed for all three aspects ( table 4 ) . regarding ever smoking , differences of cases vs no copd by both criteria were most prominent in the youngest age group ( table 3 ) , while no differences between the two copd criteria were observed . table 5 shows the odds ratio estimates for the influence of copd according to fr and lln criteria on disease prevalence from the logistic regression models ; primary prevalences of comorbidities are given in figure s1a c . by trend , differences between subjects with and without copd were most prominent in males for the lln criterion for mi , cancer , stroke , and anxiety in the middle and oldest age group and for gid in the youngest age group . on the other hand , as shown in table 5 , the prevalence of obesity was consistently lower in copd cases , which was independent of the criterion . beyond this observation , still , the occurrence of two or more comorbidities was lower in subjects with vs without copd defined by the lln criterion ( table 5 ) . the estimates for the effect of copd on levels of crp and il-6 are presented in table 6 ; median crp and il-6 levels are shown in figure s2a and b. levels of both bio - markers slightly increased with age in all participants without copd , but consistent differences between fr- and lln - defined copd were not detectable . still , both crp and il-6 tended to be higher in cases by both criteria ( table 6 ) . in this population - based observational study , the impact of disease definition by different spirometric criteria ( fr vs lln ) on copd prevalence was evident and increased with age ( figure 1 ) . the tendency of copd being possibly over - diagnosed by the fr criterion becomes more pronounced in advanced age , most prominently beyond 65 years in our sample . however , neither the prevalence of common comorbidities nor levels of the inflammatory biomarkers crp and il-6 were affected by the selection of the copd criterion . our results have to be considered in light of the fact that in our epidemiological cohort we predominantly observed participants with mild - to - moderate airflow obstruction based on spirometry without bronchodilation . the prevalence of respiratory obstruction we observed for both spirometric criteria compared well with recent population - based analyses that were performed in a similar fashion . for example , in a large canadian sample , an overall obstruction prevalence of 17% by the fr criterion has been reported for the age range of 4093 years,23 turkeshi et al24 found an fr - based prevalence of 27% in a belgian sample of very old adults aged 80 years or older . however , the prevalence of ~7% we found for lln was slightly lower than the range of 9%11% reported in these studies . unexpectedly , study participants with spirometrically defined copd generally did not suffer from more comorbidities than participants of the same age without copd irrespective of the applied spirometric criterion . although a tendency toward higher prevalences of mi , cancer , stroke , and anxiety was observed in participants with lln - defined copd compared with participants without lln - defined copd , there was no distinct difference after taking account of the sex and age in logistic regression models . on the contrary , we found a lower prevalence of obesity in copd subjects for both criteria , which might be explained by a higher fraction of smokers among these subjects.25 none of the other comorbidities evaluated were more prevalent among copd participants , which is in contrast to findings from other studies.2628 the lack of a difference may partly be explained by the fact that our study was conducted in the general population with predominantly mild cases as opposed to a clinical setting with more severe stages of copd . the prevalences we found in spirometrically defined copd for mi , stroke , anxiety , and diabetes compared well with findings described by divo et al29 in a recent analysis of copd patients based on the bode registry , when considering comparable age groups . however , our prevalences for hypertension were markedly higher , while those for depression were lower , which might be attributed to differences in the assessment of those comorbidities . furthermore , the copd cases as defined in our study were predominantly mild and mostly not yet confirmed by a physician , as indicated by the rather poor overlap with physician diagnoses , ( table 3 ) suggesting early undiagnosed cases to a large degree . moreover , the increased fraction of ever smokers especially in the youngest age group points to the fact that this factor is of importance predominantly in copd at comparably young age . it might be speculated that possible concomitant systemic impairments in these cases are in a comparably early stage and thus are not or not yet diagnosed by a physician . in recent years , systemic inflammation has come into the focus of copd research , and increased serum levels of il-6 and crp in copd participants have been reported.30 although we found a tendency toward elevated values for both markers in copd participants , the overall effect was comparatively small . the lack of findings , particularly in the aged , may , in our case , be partly explained by harvesting , where subjects in poor health did not survive or did not participate in the study as they were unable to get into the study center . however , the tendency toward higher il-6 levels in lln - defined copd cases in older age for males suggests that this criterion may be diagnostically more specific in identifying participants with ( early stage ) systemic inflammation than the fr and needs confirmation in longitudinal settings . consequently , these findings indicate that the application of the lln criterion in epidemiological or clinical settings will result in an identification of subjects with more concomitant typical characteristics of copd , eg , chronic bronchitis symptoms , and a higher probability of a physician diagnosis of copd compared to fr ( table 4 ) . however , since no consistent differences in comorbidity prevalences or biomarker levels were observed , the preference of one criterion over the other in this regard could not be derived from our study . however , the restriction to different interpretations of a spirometric measure allowed us to specifically focus on the impact of this particular component of the diagnostic sequence in predominantly early , often undiagnosed cases of airway obstruction according to our research question . a clear strength of our study is the inclusion of participants from the general population up to an advanced age of 90 years , an age group that is often underrepresented in copd studies . in a population - based sample of adults in middle and advanced age , we found that the prevalence of spirometrically defined copd was higher when using the fr criterion as opposed to lln and this difference increased with increasing age . however , for the predominantly mild cases observed in our cohort , there was no conclusive impact of the different spirometric criteria on the prevalence of comorbidities and on common inflammatory biomarker levels . ( a c ) prevalence of comorbidities in subjects without copd ( light grey ) or with copd ( dark grey ) by fr ( fev1/fvc < 0.7 ; solid color ) and lln ( fev1/fvc < lower limit of normal ; hatched ) criterion ( percentages ) . abbreviations : fr , fixed ratio ; lln , lower limit of normal ; fev1 , forced expiratory volume in 1 second ; fvc , forced vital capacity ; y , years . ( a b ) levels of crp and il-6 in subjects without copd ( light grey ) and copd cases ( dark grey ) based on fr ( fev1/fvc < 0.7 ; solid color ) or lln ( fev1/fvc < lower limit of normal ; hatched ) criterion ( number of subjects per group are given in brackets ) . abbreviations : fr , fixed ratio ; lln , lower limit of normal ; fev1 , forced expiratory volume in 1 second ; fvc , forced vital capacity ; y , years .

hybrid systems featuring -conjugated molecular building blocks and exfoliated graphite hold enormous promises for various applications due to the possibility to modulate the electronic nature of the graphene flakes . since its seminal realization , graphene and its production methods have been at the forefront of multidisciplinary studies aiming at its application in , for example , next generation organic electronic devices . in general , graphene is prepared by either physical or chemical processes , whereby its quantity and quality decisively impacts the development and performance of prospective applications . the liquid phase exfoliation of graphite represents a particularly appealing strategy as it opens up new avenues to construct unprecedented hybrid systems . even though this approach still poses myriads of challenges , hybrid materials consisting of functionalized polycyclic aromatic scaffolds and ( nano)graphene came to the forefront in recent years . overall , the degree of individualization is controlled by the nature of the molecular exfoliant the use of planar and rigid scaffolds including porphyrins , phthalocyanines , or perylene-3,4,9,10-tetracarboxylic dianhydrides amongst others turned out to be particularly effective for graphite exfoliation . depending on the electron - deficient or -rich nature of the exfoliant , the liquid phase delamination of graphite represents a versatile methodology to produce p- or n - type doped ( nano)graphene hybrids , respectively . the more so , as the careful chemical modification of the molecular exfoliant enables precise control over the degree of doping . through this approach graphene flakes with tailored electronic properties are in reach and the scope of potential applications of graphene in nanoelectronics seems sheer endless . to the best of our knowledge , apart from the previous report by stoddart et al . , who employed 2,9-diazaperopyrenium di - cation a to directly exfoliate graphite in aqueous media ( fig . 1 ) , positively charged polycyclic aromatic exfoliants have never been utilized . as a matter of fact , it has been documented that the comparatively small size of 2,7-diazapyrenium dication b is insufficient for an intercalation between graphite layers and , in turn , exfoliation of single graphene flakes from graphite . a careful consideration of structural prerequisites such as rigidity and planarity of an extended -conjugation paired with inherent redox activity prompted us to design and realize n , n-dialkylated 3,10-diazapicenium dication c. as such , dication c combines the features of an exfoliant , a stabilizer , and a p - dopant . the parent 3,10-diazapicene scaffold 1 , a member of the hitherto rather underrepresented family of hetero[n]-phenacenes , was accessible through microwave - assisted photocyclization of the corresponding diazastilbenes . to enhance the solubility of 1 and to introduce electron - accepting properties , n - dodecyl alkyl chains were introduced at the pyridyl nitrogens ( for synthetic details , see esi ) . in analogy to the reaction conditions optimized for the dialkylation of related 4,4-bipyridines , so - called viologens , reaction of 1 with 1-bromododecane at 130 c in n , n - dimethylformamide ( dmf ) under microwave irradiation furnished the n , n-didodecyl decorated 3,10-diazapicenium dibromide salt 2 in 89% yield ( scheme 1 ) . to study the impact of the counterion on the photophysical and electrochemical properties , anion exchange reactions were carried out . for that purpose , addition of ammonium hexafluorophosphate to a solution of dibromide 2 in an etoh / h2o mixture resulted in nearly quantitative precipitation of 3,10-diazapicenium bis(hexafluorophosphate ) 3 . compounds 2 and 3 were isolated as air stable , yellow powders , which show sufficient solubility in common organic solvents . to gain insights into the characteristics of 3,10-diazapicene 1 and its dialkylated salts 2 and 3 , absorption and fluorescence studies were performed in etoh ( fig . strong absorptions in the uv region ( 240340 nm for 1 , 250350 nm for 2 and 3 ) followed by weak absorptions centered in the visible region ( 340390 nm for 1 , 370440 nm for 2 and 3 ) were observed due to s0 s2 and s0 s1 transitions , respectively . the vibrational fine structure with a spacing in the range from 1350 to 1430 cm stems from several in - plane c h bending and ring breathing modes as previously reported for picene and smaller rigid viologens . upon dialkylation of 1 , the spectroscopic features are bathochromically shifted by about 42 nm for 2 and 3 and are independent on the nature of the counterion . energies of the fundamental s0 s1 transitions were estimated as 3.18 , 2.79 , and 2.77 ev for 1 , 2 and 3 , respectively , ( table 1 ) . fluorescence quantum yields measured in etoh with coumarin 47 as reference ( ref = 0.73 ) . half - wave potentials recorded by cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 ( scan rate 200 mv s , referenced vs. fc / fc ) . for example , the fluorescence of 3,10-diazapicene 1 maximizes at 386 , 408 , 432 , and 459 nm . in contrast , the fluorescence of 2 and 3 is far less resolved with a maximum centered at 442 nm and a shoulder at 464 nm . all compounds 13 feature similar fluorescence quantum yields with 0.210.22 and stokes shifts of 910 cm . the excitation spectra of 13 closely resemble their absorption spectra and , thus , confirm their monomeric nature . these results clearly show that the spectroscopic features of n , n-dialkylated 3,10-diazapicenium dications are not impacted by the counterion pointing to the presence of solvent separated ion pairs in solution . the fluorescence spectra are reasonable mirror images of the absorption spectra including vibrational fine structure indicative of a rigid aromatic fluorophore . to determine the fluorescence lifetimes , time - correlated - single - photon - counting ( tcspc ) measurements were performed in etoh with an excitation wavelength of 403 nm and the resulting decays were fit by a monoexponential fitting function . we find that the bromide salt 2 and the hexafluorophosphate salt 3 display comparable fluorescence lifetimes of 11.4 and 10.7 ns ( table 1 ) . the characterization of diazapicenium bromide 2 was complemented by femtosecond transient absorption spectroscopy to gather a deeper insight into the excited state features and their dynamics . the deconvoluted transient absorption spectra for 2 in etoh upon excitation at 387 nm taken from a global analysis are shown in fig . , the singlet excited state features were found in the visible as well as in the near infrared region with maxima at 455 , 480 , 630 , 920 , and 1035 nm . all of them are formed instantaneously upon excitation and feature a lifetime of 9.7 1.0 ns . the corresponding intersystem crossing produces a triplet excited state , whose lifetime is 280 30 ns , and whose signature is a 615 nm maximum . to evaluate the electron accepting strength of 2 and 3 , their electrochemical properties were examined by means of cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 as a supporting electrolyte at a scan rate of 200 mv s. glassy carbon served as a working electrode , a platinum wire as counter electrode , and a ag / agcl as pseudo - reference electrode . overall , the redox properties , which are independent on the nature of the counterions , give rise to two irreversible one - electron reductions at 1.10 v vs. fc / fc ( e lumo = 3.7 ev ) and 1.51 v vs. fc / fc for 2 and 3 ( table 1 , see esi ) . both reductions to afford the corresponding radical cation and subsequently neutral quinoidal form of the n , n-dialkylated 3,10-diazapicenium salts are comparable to those reported for n , n-dialkylated 3,8-diazaphenanthrenium viologens and related compounds ( fig . 4 ) . notably , the first reduction of 2 and 3 is within the range of electron - deficient aromatics as previously used to fabricate non - covalently p - type doped graphene hybrid materials . based on these results , spectroelectrochemical studies were performed for 2 in dry dmf containing 0.1 m nbu4npf6 as supporting electrolyte . as working electrode , a platinum mesh was used , while the counter electrode was a platinum plate and a silver wire was used as reference electrode . in agreement with the findings from cyclic voltammetry , the differential absorption spectrum recorded at 0.70 v vs. ag wire ( ca . 1.10 v vs. fc / fc ) reveals characteristic changes in the absorption , which were ascribed to the one - electron reduction of 2 to the corresponding radical cation ( fig . 4 ) . in agreement with other related viologens , absorptions at 350400 nm as well as at 450500 nm increase upon reduction and new absorptions evolve between 500650 nm with a maximum centered at 555 nm . with the key features of the n , n-dialkylated 3,10-diazapicendiium salt 2 and 3 at hand , we focused on the preparation of the hybrid materials with graphene by adapting a previously reported procedure . initially , a metastable graphene dispersion ( eg ) was generated in etoh by ultrasonication for at least 1 h at room temperature and subsequent centrifugation of graphene flakes . this was followed by a stepwise addition of eg to a 3 10 m solution of 2 in etoh affording the hybrid system eg/2 which was analyzed by steady state absorption spectroscopy , ( for details , see esi ) . our procedure led to exfoliated graphene flakes , which are stabilized by the diazapicenium cations through interactions . the inherent positive charge of 2 thereby aids in the dispersion of graphene flakes via charge repulsion and in that way minimizes their agglomeration in solution . thus , as the graphene concentration is increased the absorption features of 2 diminish , indicating strong interaction with the basal plane of graphene ( fig . notably , the peak at about 270 nm , which steadily increases upon adding various aliquots of the graphene dispersion is due to a van hove singularity in the density of states of graphene . in turn , the fluorescence of 2 is quenched with gradually increasing concentration of eg in the dispersion , which is in agreement with previous reports of closely related positively charged exfoliants by stoddart et al . a comprehensive tool to disclose ground state interactions between graphene and 2 is based on raman spectroscopy . the sample preparation involved drop casting of eg/2 onto a si / sio2 wafer followed by subsequent drying . to validate our results with eg/2 , similar experiments were performed with a reference graphene sample , that is , eg . eg was prepared in analogy to eg/2 without , however , the addition of 2 . statistical analyses of the coated wafer were carried out by measuring mappings consisting of 1000 individual spectra recorded upon an excitation wavelength of 532 nm . from 2d / g - ratios on the order of 0.65 as well as a full width at half maximum ( fwhm ) of the 2d - band of about 70 cm we conclude that the majority of the spectra resemble turbostratic graphene flakes . restacking of the individual graphene flakes during the drying process is a likely rationale for this finding . to shed light onto doping effects present in the hybrid material , we focused our attention on the position of the individual raman bands ( fig . . a careful comparison of the histograms regarding the g- and 2d - band positions of eg/2 and reference eg discloses shifts to higher wavenumbers in eg/2 . such a p - doping is in sound agreement with the electrochemical measurements , in which the low reduction potentials of 2 were documented . in other words , the diazapicenium salt not only acts as a stabilizing agent for exfoliated graphene flakes exploiting the charge repulsion between the cationic scaffolds , but also results in p - doping of the graphene flakes owing to strong electronic coupling in the resulting hybrid material . to further study the nature of the obtained graphene flakes , atomic force microscopy ( afm ) and transmission electron microscopy ( tem ) were employed . in typical tem images of eg/2 , drop casted from etoh onto a lacey carbon coated copper grid , few layer graphene with lateral sizes ranging from a few microns as maximum size down to flakes sizes in the nm regime was noted ( fig . often , the graphene flakes were folded , rolled up , and intertwined . from the height profiles taken from a characteristic afm image of eg/2 , which was also used for the raman measurements , we infer that next to solvent residues and/or some contaminants thin graphene flakes with average heights of 1.92.5 nm are present . in combination with the raman results , these findings demonstrate that the hybrid material consists of turbostratic graphene flakes with a thickness of 5 to 8 layers when probing solid state samples . it is , however , reasonable to assume that the hybrid material consists in the liquid phase of fewer layers due to the repulsive forces introduced from 2 . next to steady state measurements , also transient absorption studies were carried out to probe eg/2 . in the near - infrared region of the spectrum , the usual graphene related bleaching is discernable , for which a lifetime of 0.4 ps was determined . in the visible region of the spectrum , a new transient maximum at 595 nm is discernible for eg/2 ( see esi ) . this finding is in sharp contrast to the singlet and triplet excited state features noted for just 2 in etoh ( see fig . global analysis of eg/2 afforded a single evolution associated spectrum with a rather short lifetime of 26 3 ps . tentatively , we assign this species to that of the charge separated state . as derived from raman experiments , strong interactions between eg and 2 can be regarded as the inception to a doping of eg in the ground state . as such , it is reasonable to expect that a partial shift of charge density in the ground state leads to rapid charge separation kinetics in the excited state . moreover , the comparison of the deconvoluted transient absorption spectrum with the spectroelectrochemical results for 2 confirms that this newly formed transient relates to the one - electron reduced form of 2 ( fig . 8) . in comparison to the absorption spectrum of the electrochemically generated radical cation of 2 a possible rationale for this shift is based on the additional stabilization of the radical cation resulting from electronic coupling with the basal plane of the exfoliated graphene flakes in eg/2 . conclusively , the excitation of eg/2 leads to an electron transfer event from graphene to 2 resulting in a hybrid system consisting of p - doped graphene flakes and radical cations of 2 . the parent 3,10-diazapicene scaffold 1 , a member of the hitherto rather underrepresented family of hetero[n]-phenacenes , was accessible through microwave - assisted photocyclization of the corresponding diazastilbenes . to enhance the solubility of 1 and to introduce electron - accepting properties , n - dodecyl alkyl chains were introduced at the pyridyl nitrogens ( for synthetic details , see esi ) . in analogy to the reaction conditions optimized for the dialkylation of related 4,4-bipyridines , so - called viologens , reaction of 1 with 1-bromododecane at 130 c in n , n - dimethylformamide ( dmf ) under microwave irradiation furnished the n , n-didodecyl decorated 3,10-diazapicenium dibromide salt 2 in 89% yield ( scheme 1 ) . to study the impact of the counterion on the photophysical and electrochemical properties , anion exchange reactions were carried out . for that purpose , addition of ammonium hexafluorophosphate to a solution of dibromide 2 in an etoh / h2o mixture resulted in nearly quantitative precipitation of 3,10-diazapicenium bis(hexafluorophosphate ) 3 . compounds 2 and 3 were isolated as air stable , yellow powders , which show sufficient solubility in common organic solvents . to gain insights into the characteristics of 3,10-diazapicene 1 and its dialkylated salts 2 and 3 , absorption and fluorescence studies were performed in etoh ( fig . strong absorptions in the uv region ( 240340 nm for 1 , 250350 nm for 2 and 3 ) followed by weak absorptions centered in the visible region ( 340390 nm for 1 , 370440 nm for 2 and 3 ) were observed due to s0 s2 and s0 s1 transitions , respectively . the vibrational fine structure with a spacing in the range from 1350 to 1430 cm stems from several in - plane c h bending and ring breathing modes as previously reported for picene and smaller rigid viologens . upon dialkylation of 1 , the spectroscopic features are bathochromically shifted by about 42 nm for 2 and 3 and are independent on the nature of the counterion . energies of the fundamental s0 s1 transitions were estimated as 3.18 , 2.79 , and 2.77 ev for 1 , 2 and 3 , respectively , ( table 1 ) . fluorescence quantum yields measured in etoh with coumarin 47 as reference ( ref = 0.73 ) . half - wave potentials recorded by cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 ( scan rate 200 mv s , referenced vs. fc / fc ) . for example , the fluorescence of 3,10-diazapicene 1 maximizes at 386 , 408 , 432 , and 459 nm . in contrast , the fluorescence of 2 and 3 is far less resolved with a maximum centered at 442 nm and a shoulder at 464 nm . all compounds 13 feature similar fluorescence quantum yields with 0.210.22 and stokes shifts of 910 cm . the excitation spectra of 13 closely resemble their absorption spectra and , thus , confirm their monomeric nature . these results clearly show that the spectroscopic features of n , n-dialkylated 3,10-diazapicenium dications are not impacted by the counterion pointing to the presence of solvent separated ion pairs in solution . the fluorescence spectra are reasonable mirror images of the absorption spectra including vibrational fine structure indicative of a rigid aromatic fluorophore . to determine the fluorescence lifetimes , time - correlated - single - photon - counting ( tcspc ) measurements were performed in etoh with an excitation wavelength of 403 nm and the resulting decays were fit by a monoexponential fitting function . we find that the bromide salt 2 and the hexafluorophosphate salt 3 display comparable fluorescence lifetimes of 11.4 and 10.7 ns ( table 1 ) . the characterization of diazapicenium bromide 2 was complemented by femtosecond transient absorption spectroscopy to gather a deeper insight into the excited state features and their dynamics . the deconvoluted transient absorption spectra for 2 in etoh upon excitation at 387 nm taken from a global analysis are shown in fig . , the singlet excited state features were found in the visible as well as in the near infrared region with maxima at 455 , 480 , 630 , 920 , and 1035 nm . all of them are formed instantaneously upon excitation and feature a lifetime of 9.7 1.0 ns . the corresponding intersystem crossing produces a triplet excited state , whose lifetime is 280 30 ns , and whose signature is a 615 nm maximum . to evaluate the electron accepting strength of 2 and 3 , their electrochemical properties were examined by means of cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 as a supporting electrolyte at a scan rate of 200 mv s. glassy carbon served as a working electrode , a platinum wire as counter electrode , and a ag / agcl as pseudo - reference electrode . overall , the redox properties , which are independent on the nature of the counterions , give rise to two irreversible one - electron reductions at 1.10 v vs. fc / fc ( e lumo = 3.7 ev ) and 1.51 v vs. fc / fc for 2 and 3 ( table 1 , see esi ) . both reductions to afford the corresponding radical cation and subsequently neutral quinoidal form of the n , n-dialkylated 3,10-diazapicenium salts are comparable to those reported for n , n-dialkylated 3,8-diazaphenanthrenium viologens and related compounds ( fig . 4 ) . notably , the first reduction of 2 and 3 is within the range of electron - deficient aromatics as previously used to fabricate non - covalently p - type doped graphene hybrid materials . based on these results , spectroelectrochemical studies were performed for 2 in dry dmf containing 0.1 m nbu4npf6 as supporting electrolyte . as working electrode , a platinum mesh was used , while the counter electrode was a platinum plate and a silver wire was used as reference electrode . in agreement with the findings from cyclic voltammetry , the differential absorption spectrum recorded at 0.70 v vs. ag wire ( ca . 1.10 v vs. fc / fc ) reveals characteristic changes in the absorption , which were ascribed to the one - electron reduction of 2 to the corresponding radical cation ( fig . 4 ) . in agreement with other related viologens , absorptions at 350400 nm as well as at 450500 nm increase upon reduction and with the key features of the n , n-dialkylated 3,10-diazapicendiium salt 2 and 3 at hand , we focused on the preparation of the hybrid materials with graphene by adapting a previously reported procedure . initially , a metastable graphene dispersion ( eg ) was generated in etoh by ultrasonication for at least 1 h at room temperature and subsequent centrifugation of graphene flakes . this was followed by a stepwise addition of eg to a 3 10 m solution of 2 in etoh affording the hybrid system eg/2 which was analyzed by steady state absorption spectroscopy , ( for details , see esi ) . our procedure led to exfoliated graphene flakes , which are stabilized by the diazapicenium cations through interactions . the inherent positive charge of 2 thereby aids in the dispersion of graphene flakes via charge repulsion and in that way minimizes their agglomeration in solution . thus , as the graphene concentration is increased the absorption features of 2 diminish , indicating strong interaction with the basal plane of graphene ( fig . 5 ) . notably , the peak at about 270 nm , which steadily increases upon adding various aliquots of the graphene dispersion is due to a van hove singularity in the density of states of graphene . in turn , the fluorescence of 2 is quenched with gradually increasing concentration of eg in the dispersion , which is in agreement with previous reports of closely related positively charged exfoliants by stoddart et al . a comprehensive tool to disclose ground state interactions between graphene and 2 is based on raman spectroscopy . the sample preparation involved drop casting of eg/2 onto a si / sio2 wafer followed by subsequent drying . to validate our results with eg/2 , similar experiments were performed with a reference graphene sample , that is , eg . eg was prepared in analogy to eg/2 without , however , the addition of 2 . statistical analyses of the coated wafer were carried out by measuring mappings consisting of 1000 individual spectra recorded upon an excitation wavelength of 532 nm . from 2d / g - ratios on the order of 0.65 as well as a full width at half maximum ( fwhm ) of the 2d - band of about 70 cm we conclude that the majority of the spectra resemble turbostratic graphene flakes . restacking of the individual graphene flakes during the drying process is a likely rationale for this finding . to shed light onto doping effects present in the hybrid material , we focused our attention on the position of the individual raman bands ( fig . . a careful comparison of the histograms regarding the g- and 2d - band positions of eg/2 and reference eg discloses shifts to higher wavenumbers in eg/2 . such a p - doping is in sound agreement with the electrochemical measurements , in which the low reduction potentials of 2 were documented . in other words , the diazapicenium salt not only acts as a stabilizing agent for exfoliated graphene flakes exploiting the charge repulsion between the cationic scaffolds , but also results in p - doping of the graphene flakes owing to strong electronic coupling in the resulting hybrid material . to further study the nature of the obtained graphene flakes , atomic force microscopy ( afm ) and transmission electron microscopy ( tem ) were employed . in typical tem images of eg/2 , drop casted from etoh onto a lacey carbon coated copper grid , few layer graphene with lateral sizes ranging from a few microns as maximum size down to flakes sizes in the nm regime was noted ( fig . 7 ) . often , the graphene flakes were folded , rolled up , and intertwined . from the height profiles taken from a characteristic afm image of eg/2 , which was also used for the raman measurements , we infer that next to solvent residues and/or some contaminants thin graphene flakes with average heights of 1.92.5 nm are present . in combination with the raman results , these findings demonstrate that the hybrid material consists of turbostratic graphene flakes with a thickness of 5 to 8 layers when probing solid state samples . it is , however , reasonable to assume that the hybrid material consists in the liquid phase of fewer layers due to the repulsive forces introduced from 2 . next to steady state measurements , also transient absorption studies were carried out to probe eg/2 . in the near - infrared region of the spectrum , the usual graphene related bleaching is discernable , for which a lifetime of 0.4 ps was determined . in the visible region of the spectrum , a new transient maximum at 595 nm is discernible for eg/2 ( see esi ) . this finding is in sharp contrast to the singlet and triplet excited state features noted for just 2 in etoh ( see fig . 4 ) . global analysis of eg/2 afforded a single evolution associated spectrum with a rather short lifetime of 26 3 ps . as derived from raman experiments , strong interactions between eg and 2 can be regarded as the inception to a doping of eg in the ground state . as such , it is reasonable to expect that a partial shift of charge density in the ground state leads to rapid charge separation kinetics in the excited state . moreover , the comparison of the deconvoluted transient absorption spectrum with the spectroelectrochemical results for 2 confirms that this newly formed transient relates to the one - electron reduced form of 2 ( fig . 8) . in comparison to the absorption spectrum of the electrochemically generated radical cation of 2 a possible rationale for this shift is based on the additional stabilization of the radical cation resulting from electronic coupling with the basal plane of the exfoliated graphene flakes in eg/2 . conclusively , the excitation of eg/2 leads to an electron transfer event from graphene to 2 resulting in a hybrid system consisting of p - doped graphene flakes and radical cations of 2 . two novel n , n-didodecyl 3,10-diazapicenium salts with bromide and hexafluorophosphate counterions were synthesized as an exfoliant , a stabilizer , and a p - dopant for liquid phase exfoliated graphene . when studied alone , it was demonstrated that the two different counterions of the n , n-didodecyl 3,10-diazapicenium salts have only negligible influence on the photophysical and electrochemical properties : both n , n-didodecyl 3,10-diazapicenium salts exhibited comparable absorption , fluorescence , and reduction , which were predominantly dictated by the polycyclic -conjugated scaffold . complementary spectroelectrochemical investigations with the 3,10-diazapicenium dibromide revealed that their reductions were accompanied by characteristic changes in the absorption features . the bromide salt was successfully applied to exfoliate graphite and to stabilize the graphene flakes . the resulting hybrids were characterized by an arsenal of steady state and time - resolved spectroscopic as well as microscopic techniques . as such , they corroborate that the ground state of these hybrids is dominated by a shift of charge density from the basal plane of graphene to the positively charged 3,10-diazapicenium : result of this shift is a p - doped graphene . for the excited state , we see an efficient charge transfer from graphene to the positively charged 3,10-diazapicenium . here , the result is the one - electron reduction of 2 and a hole delocalized on the graphene flakes . unambiguously , the presence of multilayered turbostratic graphene flakes with modified electronic properties was demonstrated and , hence , the newly synthesized n , n-dialkylated 3,10-diazapicenium salts open up highly promising new avenues towards the fabrication of hybrid ( nano)graphene materials with electronic properties tailored at will .

we report on the preparation of a hybrid system consisting of exfoliated graphene and n , n-didodecyl 3,10-diazapicenium salts by solution based methods . the obtained hybrids were characterized by steady state and time - resolved spectroscopic as well as microscopic techniques to corroborate the p - doped character .

Introduction Results and discussion Synthesis and photophysical characterization of Exfoliation and characterization of graphene/3,10-diazapicenium hybrid systems Conclusions

since its seminal realization , graphene and its production methods have been at the forefront of multidisciplinary studies aiming at its application in , for example , next generation organic electronic devices . in general , graphene is prepared by either physical or chemical processes , whereby its quantity and quality decisively impacts the development and performance of prospective applications . even though this approach still poses myriads of challenges , hybrid materials consisting of functionalized polycyclic aromatic scaffolds and ( nano)graphene came to the forefront in recent years . depending on the electron - deficient or -rich nature of the exfoliant , the liquid phase delamination of graphite represents a versatile methodology to produce p- or n - type doped ( nano)graphene hybrids , respectively . to the best of our knowledge , apart from the previous report by stoddart et al . , who employed 2,9-diazaperopyrenium di - cation a to directly exfoliate graphite in aqueous media ( fig . 1 ) , positively charged polycyclic aromatic exfoliants have never been utilized . a careful consideration of structural prerequisites such as rigidity and planarity of an extended -conjugation paired with inherent redox activity prompted us to design and realize n , n-dialkylated 3,10-diazapicenium dication c. as such , dication c combines the features of an exfoliant , a stabilizer , and a p - dopant . the parent 3,10-diazapicene scaffold 1 , a member of the hitherto rather underrepresented family of hetero[n]-phenacenes , was accessible through microwave - assisted photocyclization of the corresponding diazastilbenes . in analogy to the reaction conditions optimized for the dialkylation of related 4,4-bipyridines , so - called viologens , reaction of 1 with 1-bromododecane at 130 c in n , n - dimethylformamide ( dmf ) under microwave irradiation furnished the n , n-didodecyl decorated 3,10-diazapicenium dibromide salt 2 in 89% yield ( scheme 1 ) . to study the impact of the counterion on the photophysical and electrochemical properties , anion exchange reactions were carried out . the vibrational fine structure with a spacing in the range from 1350 to 1430 cm stems from several in - plane c h bending and ring breathing modes as previously reported for picene and smaller rigid viologens . upon dialkylation of 1 , the spectroscopic features are bathochromically shifted by about 42 nm for 2 and 3 and are independent on the nature of the counterion . half - wave potentials recorded by cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 ( scan rate 200 mv s , referenced vs. fc / fc ) . for example , the fluorescence of 3,10-diazapicene 1 maximizes at 386 , 408 , 432 , and 459 nm . all compounds 13 feature similar fluorescence quantum yields with 0.210.22 and stokes shifts of 910 cm . these results clearly show that the spectroscopic features of n , n-dialkylated 3,10-diazapicenium dications are not impacted by the counterion pointing to the presence of solvent separated ion pairs in solution . the fluorescence spectra are reasonable mirror images of the absorption spectra including vibrational fine structure indicative of a rigid aromatic fluorophore . to determine the fluorescence lifetimes , time - correlated - single - photon - counting ( tcspc ) measurements were performed in etoh with an excitation wavelength of 403 nm and the resulting decays were fit by a monoexponential fitting function . the characterization of diazapicenium bromide 2 was complemented by femtosecond transient absorption spectroscopy to gather a deeper insight into the excited state features and their dynamics . , the singlet excited state features were found in the visible as well as in the near infrared region with maxima at 455 , 480 , 630 , 920 , and 1035 nm . the corresponding intersystem crossing produces a triplet excited state , whose lifetime is 280 30 ns , and whose signature is a 615 nm maximum . to evaluate the electron accepting strength of 2 and 3 , their electrochemical properties were examined by means of cyclic voltammetry in ch2cl2 with 0.1 m nbu4npf6 as a supporting electrolyte at a scan rate of 200 mv s. glassy carbon served as a working electrode , a platinum wire as counter electrode , and a ag / agcl as pseudo - reference electrode . overall , the redox properties , which are independent on the nature of the counterions , give rise to two irreversible one - electron reductions at 1.10 v vs. fc / fc ( e lumo = 3.7 ev ) and 1.51 v vs. fc / fc for 2 and 3 ( table 1 , see esi ) . both reductions to afford the corresponding radical cation and subsequently neutral quinoidal form of the n , n-dialkylated 3,10-diazapicenium salts are comparable to those reported for n , n-dialkylated 3,8-diazaphenanthrenium viologens and related compounds ( fig . notably , the first reduction of 2 and 3 is within the range of electron - deficient aromatics as previously used to fabricate non - covalently p - type doped graphene hybrid materials . based on these results , spectroelectrochemical studies were performed for 2 in dry dmf containing 0.1 m nbu4npf6 as supporting electrolyte . 1.10 v vs. fc / fc ) reveals characteristic changes in the absorption , which were ascribed to the one - electron reduction of 2 to the corresponding radical cation ( fig . 4 ) . in agreement with other related viologens , absorptions at 350400 nm as well as at 450500 nm increase upon reduction and new absorptions evolve between 500650 nm with a maximum centered at 555 nm . with the key features of the n , n-dialkylated 3,10-diazapicendiium salt 2 and 3 at hand , we focused on the preparation of the hybrid materials with graphene by adapting a previously reported procedure . initially , a metastable graphene dispersion ( eg ) was generated in etoh by ultrasonication for at least 1 h at room temperature and subsequent centrifugation of graphene flakes . this was followed by a stepwise addition of eg to a 3 10 m solution of 2 in etoh affording the hybrid system eg/2 which was analyzed by steady state absorption spectroscopy , ( for details , see esi ) . our procedure led to exfoliated graphene flakes , which are stabilized by the diazapicenium cations through interactions . the inherent positive charge of 2 thereby aids in the dispersion of graphene flakes via charge repulsion and in that way minimizes their agglomeration in solution . thus , as the graphene concentration is increased the absorption features of 2 diminish , indicating strong interaction with the basal plane of graphene ( fig . notably , the peak at about 270 nm , which steadily increases upon adding various aliquots of the graphene dispersion is due to a van hove singularity in the density of states of graphene . a comprehensive tool to disclose ground state interactions between graphene and 2 is based on raman spectroscopy . the sample preparation involved drop casting of eg/2 onto a si / sio2 wafer followed by subsequent drying . to validate our results with eg/2 , similar experiments were performed with a reference graphene sample , that is , eg . statistical analyses of the coated wafer were carried out by measuring mappings consisting of 1000 individual spectra recorded upon an excitation wavelength of 532 nm . from 2d / g - ratios on the order of 0.65 as well as a full width at half maximum ( fwhm ) of the 2d - band of about 70 cm we conclude that the majority of the spectra resemble turbostratic graphene flakes . to shed light onto doping effects present in the hybrid material , we focused our attention on the position of the individual raman bands ( fig . . a careful comparison of the histograms regarding the g- and 2d - band positions of eg/2 and reference eg discloses shifts to higher wavenumbers in eg/2 . such a p - doping is in sound agreement with the electrochemical measurements , in which the low reduction potentials of 2 were documented . in other words , the diazapicenium salt not only acts as a stabilizing agent for exfoliated graphene flakes exploiting the charge repulsion between the cationic scaffolds , but also results in p - doping of the graphene flakes owing to strong electronic coupling in the resulting hybrid material . to further study the nature of the obtained graphene flakes , atomic force microscopy ( afm ) and transmission electron microscopy ( tem ) were employed . often , the graphene flakes were folded , rolled up , and intertwined . next to steady state measurements , also transient absorption studies were carried out to probe eg/2 . in the visible region of the spectrum , a new transient maximum at 595 nm is discernible for eg/2 ( see esi ) . global analysis of eg/2 afforded a single evolution associated spectrum with a rather short lifetime of 26 3 ps . tentatively , we assign this species to that of the charge separated state . moreover , the comparison of the deconvoluted transient absorption spectrum with the spectroelectrochemical results for 2 confirms that this newly formed transient relates to the one - electron reduced form of 2 ( fig . in comparison to the absorption spectrum of the electrochemically generated radical cation of 2 a possible rationale for this shift is based on the additional stabilization of the radical cation resulting from electronic coupling with the basal plane of the exfoliated graphene flakes in eg/2 . conclusively , the excitation of eg/2 leads to an electron transfer event from graphene to 2 resulting in a hybrid system consisting of p - doped graphene flakes and radical cations of 2 . in analogy to the reaction conditions optimized for the dialkylation of related 4,4-bipyridines , so - called viologens , reaction of 1 with 1-bromododecane at 130 c in n , n - dimethylformamide ( dmf ) under microwave irradiation furnished the n , n-didodecyl decorated 3,10-diazapicenium dibromide salt 2 in 89% yield ( scheme 1 ) . to study the impact of the counterion on the photophysical and electrochemical properties , anion exchange reactions were carried out . for that purpose , addition of ammonium hexafluorophosphate to a solution of dibromide 2 in an etoh / h2o mixture resulted in nearly quantitative precipitation of 3,10-diazapicenium bis(hexafluorophosphate ) 3 . compounds 2 and 3 were isolated as air stable , yellow powders , which show sufficient solubility in common organic solvents . to gain insights into the characteristics of 3,10-diazapicene 1 and its dialkylated salts 2 and 3 , absorption and fluorescence studies were performed in etoh ( fig . strong absorptions in the uv region ( 240340 nm for 1 , 250350 nm for 2 and 3 ) followed by weak absorptions centered in the visible region ( 340390 nm for 1 , 370440 nm for 2 and 3 ) were observed due to s0 s2 and s0 s1 transitions , respectively . the vibrational fine structure with a spacing in the range from 1350 to 1430 cm stems from several in - plane c h bending and ring breathing modes as previously reported for picene and smaller rigid viologens . upon dialkylation of 1 , the spectroscopic features are bathochromically shifted by about 42 nm for 2 and 3 and are independent on the nature of the counterion . these results clearly show that the spectroscopic features of n , n-dialkylated 3,10-diazapicenium dications are not impacted by the counterion pointing to the presence of solvent separated ion pairs in solution . the fluorescence spectra are reasonable mirror images of the absorption spectra including vibrational fine structure indicative of a rigid aromatic fluorophore . to determine the fluorescence lifetimes , time - correlated - single - photon - counting ( tcspc ) measurements were performed in etoh with an excitation wavelength of 403 nm and the resulting decays were fit by a monoexponential fitting function . , the singlet excited state features were found in the visible as well as in the near infrared region with maxima at 455 , 480 , 630 , 920 , and 1035 nm . overall , the redox properties , which are independent on the nature of the counterions , give rise to two irreversible one - electron reductions at 1.10 v vs. fc / fc ( e lumo = 3.7 ev ) and 1.51 v vs. fc / fc for 2 and 3 ( table 1 , see esi ) . both reductions to afford the corresponding radical cation and subsequently neutral quinoidal form of the n , n-dialkylated 3,10-diazapicenium salts are comparable to those reported for n , n-dialkylated 3,8-diazaphenanthrenium viologens and related compounds ( fig . notably , the first reduction of 2 and 3 is within the range of electron - deficient aromatics as previously used to fabricate non - covalently p - type doped graphene hybrid materials . based on these results , spectroelectrochemical studies were performed for 2 in dry dmf containing 0.1 m nbu4npf6 as supporting electrolyte . as working electrode , a platinum mesh was used , while the counter electrode was a platinum plate and a silver wire was used as reference electrode . 4 ) . in agreement with other related viologens , absorptions at 350400 nm as well as at 450500 nm increase upon reduction and with the key features of the n , n-dialkylated 3,10-diazapicendiium salt 2 and 3 at hand , we focused on the preparation of the hybrid materials with graphene by adapting a previously reported procedure . initially , a metastable graphene dispersion ( eg ) was generated in etoh by ultrasonication for at least 1 h at room temperature and subsequent centrifugation of graphene flakes . this was followed by a stepwise addition of eg to a 3 10 m solution of 2 in etoh affording the hybrid system eg/2 which was analyzed by steady state absorption spectroscopy , ( for details , see esi ) . our procedure led to exfoliated graphene flakes , which are stabilized by the diazapicenium cations through interactions . 5 ) . notably , the peak at about 270 nm , which steadily increases upon adding various aliquots of the graphene dispersion is due to a van hove singularity in the density of states of graphene . a comprehensive tool to disclose ground state interactions between graphene and 2 is based on raman spectroscopy . statistical analyses of the coated wafer were carried out by measuring mappings consisting of 1000 individual spectra recorded upon an excitation wavelength of 532 nm . from 2d / g - ratios on the order of 0.65 as well as a full width at half maximum ( fwhm ) of the 2d - band of about 70 cm we conclude that the majority of the spectra resemble turbostratic graphene flakes . to shed light onto doping effects present in the hybrid material , we focused our attention on the position of the individual raman bands ( fig . . a careful comparison of the histograms regarding the g- and 2d - band positions of eg/2 and reference eg discloses shifts to higher wavenumbers in eg/2 . such a p - doping is in sound agreement with the electrochemical measurements , in which the low reduction potentials of 2 were documented . in other words , the diazapicenium salt not only acts as a stabilizing agent for exfoliated graphene flakes exploiting the charge repulsion between the cationic scaffolds , but also results in p - doping of the graphene flakes owing to strong electronic coupling in the resulting hybrid material . to further study the nature of the obtained graphene flakes , atomic force microscopy ( afm ) and transmission electron microscopy ( tem ) were employed . 7 ) . often , the graphene flakes were folded , rolled up , and intertwined . from the height profiles taken from a characteristic afm image of eg/2 , which was also used for the raman measurements , we infer that next to solvent residues and/or some contaminants thin graphene flakes with average heights of 1.92.5 nm are present . it is , however , reasonable to assume that the hybrid material consists in the liquid phase of fewer layers due to the repulsive forces introduced from 2 . next to steady state measurements , also transient absorption studies were carried out to probe eg/2 . in the near - infrared region of the spectrum , the usual graphene related bleaching is discernable , for which a lifetime of 0.4 ps was determined . as derived from raman experiments , strong interactions between eg and 2 can be regarded as the inception to a doping of eg in the ground state . in comparison to the absorption spectrum of the electrochemically generated radical cation of 2 a possible rationale for this shift is based on the additional stabilization of the radical cation resulting from electronic coupling with the basal plane of the exfoliated graphene flakes in eg/2 . conclusively , the excitation of eg/2 leads to an electron transfer event from graphene to 2 resulting in a hybrid system consisting of p - doped graphene flakes and radical cations of 2 . two novel n , n-didodecyl 3,10-diazapicenium salts with bromide and hexafluorophosphate counterions were synthesized as an exfoliant , a stabilizer , and a p - dopant for liquid phase exfoliated graphene . when studied alone , it was demonstrated that the two different counterions of the n , n-didodecyl 3,10-diazapicenium salts have only negligible influence on the photophysical and electrochemical properties : both n , n-didodecyl 3,10-diazapicenium salts exhibited comparable absorption , fluorescence , and reduction , which were predominantly dictated by the polycyclic -conjugated scaffold . the bromide salt was successfully applied to exfoliate graphite and to stabilize the graphene flakes . the resulting hybrids were characterized by an arsenal of steady state and time - resolved spectroscopic as well as microscopic techniques . as such , they corroborate that the ground state of these hybrids is dominated by a shift of charge density from the basal plane of graphene to the positively charged 3,10-diazapicenium : result of this shift is a p - doped graphene . here , the result is the one - electron reduction of 2 and a hole delocalized on the graphene flakes . unambiguously , the presence of multilayered turbostratic graphene flakes with modified electronic properties was demonstrated and , hence , the newly synthesized n , n-dialkylated 3,10-diazapicenium salts open up highly promising new avenues towards the fabrication of hybrid ( nano)graphene materials with electronic properties tailored at will .

severe trauma can cause a profound imbalance in immune function and predispose the injured host to opportunistic infections . in this respect , nosocomial infections are a common clinical problem in trauma intensive care units in injured patients and are associated with increased morbidity , length of hospital stay , and mortality . a number of studies have demonstrated that splenocyte dysfunction often observed as t - lymphocyte dysfunction and t - helper 1 ( th1 ) depression are associated with increased susceptibility to severe infection and/or multiple organ dysfunction ( mods ) after trauma [ 24 ] . myeloid - derived suppressor cells ( mdscs ) , a heterogeneous cd11bgr-1 cell population consisting of immature myeloid cells and myeloid progenitor cells that can suppress t - cell responses by a variety of mechanisms [ 5 , 6 ] , have recently been associated with the profound immunosuppression seen in sepsis and trauma . ochoa and colleagues [ 7 , 8 ] found that a marked increase of cd11bgr-1 mdscs is associated with suppressed t - cell functions via the production of arginase-1 in the mouse spleen at 24 h after surgical trauma . however , under septic conditions , moldawer and colleagues found that a different response where cd11bgr-1 mdscs expanded in the spleen , in the bone marrow , and in the peripheral lymph nodes by 3 days after sepsis and several approaches to prevent the expansion of these cells deteriorated the immunosuppression and worsened outcomes [ 9 , 10 ] . the relatively different roles of mdscs in sepsis and trauma thus warrant a characterization of cd11bgr-1 mdscs in different lymphoid compartments in traumatized mice over time . high mobility group box nuclear protein 1 ( hmgb1 ) is a nuclear dna binding protein that has been shown to be a key late mediator in sepsis and an early trigger of sterile inflammation in severe trauma when released from cells [ 12 , 13 ] . although hmgb1 released as the result of tissue injury or stress is involved in many aspects of inflammation , it is unknown whether hmgb1 mediates trauma - associated immune suppression . hmgb1 has multiple functions in the regulation of immunity and inflammation , and studies have shown that hmgb1 has variable effects on t - cell responses depending on dose , redox status , and disease setting [ 14 , 15 ] . furthermore hmgb1 has been demonstrated to lead to mdsc expansion after surgical trauma in the setting of cancer ; however , hmgb1 has been shown to bind to and enhance the cytokine - induced effect of several inflammatory mediators , such as il-1 , il-6 , and tnf- , which themselves have been shown to drive the expansion of mdsc [ 20 , 21 ] . in this study , we evaluated the role of hmgb1 in trauma - associated immune suppression through examination of the contribution of hmgb1 to mdsc expansion and splenocyte responses after peripheral tissue trauma . systemic neutralization of hmgb1 was achieved with administration of anti - hmgb1 monoclonal antibody , 2g7 after injury . using a murine trauma model , the pseudofracture ( pf ) model , mice used in the experimental protocols were housed in accordance with university of pittsburgh ( pittsburgh , pa ) and national institutes of health ( nih , bethesda , md ) animal care guidelines in specific pathogen - free conditions . a total of 175 male c57bl/6 mice were used , aged 812 weeks , weighing 2030 g , and were obtained from charles rivers laboratories international ( wilmington , mass ) . the animals were maintained in the university of pittsburgh animal research center with a 12 h light - dark cycle and free access to standard laboratory food and water . all animals were acclimatized for 7 days prior to being used and fasted for 12 h prior to experimental manipulation . mice were injected s.c . with a total volume of 200 l of solution containing 40 g of a mouse monoclonal anti - hmgb1 antibody 2g7 ( igg2b , noncommercial antibody , gift from dr . k. tracey ) right after and at 24 h after the pseudofracture ( pf ) trauma model . pf is a model of peripheral tissue trauma , which incorporates all the elements of this injury type including the soft tissue and fracture components [ 22 , 23 ] . as described previously [ 22 , 23 ] , donor mice were euthanized with overdoses of isoflurane ( abbott laboratories , chicago , ill ) , and the long bones of the lower extremities were harvested sterilely . to obtain standardized concentrations , two femurs and two tibias were removed from collected tissues and crushed using a sterile mortar and pestle in the biological safety hood . the crushed bone fragments were resuspended with 2 ml phosphate - buffer solution ( pbs ) , crushed again , and kept in a sterile tube on ice . , lake forest , il , for ovation pharmaceuticals , deerfield , il ) and inhaled isoflurane , and both thighs were squeezed with a hemostat for 30 seconds to induce a soft tissue injury . then , 0.15 ml of bone components suspension prepared above was injected using an 18 g needle in the area of the soft tissue injury of each thigh . buprenorphine ( 0.10 mg / kg , bedford laboratories , bedford , oh ) to minimize discomfort . anaesthetized mice that received no experimental manipulation were used as uninjured controls . at different time points ( 1 , 6 , 24 , 48 , and 72 h ) after pf , mice were sacrificed with overdoses of isoflurane ( hospira , lake forest , il , usa ) , cardiac puncture was directed and performed to collect whole blood , and spleens and bone marrow were collected aseptically for further analysis . splenocytes were isolated and the red blood cells were lysed by exposure for 5 min to 2 ml of red blood cell lysis buffer ( sigma , st . after washing the cells twice with complete rpmi 1640 , isolated splenocytes were diluted to appropriate concentrations , detailed as follows , in complete rpmi 1640 medium ( lonza , walkersville , md , usa ) , which contained 10% fetal bovine serum ( fbs , sigma ) , 2 mm glutamine ( gibco laboratories , grand island , ny ) , 1% penicillin - streptomycin ( gibco laboratories ) , and 0.05 mm 2-mercaptoethanol ( 2-me , sigma ) . a 100 l aliquot of whole blood and bone marrow was lysed with 2 ml of lysing buffer . whole blood , splenocyte , and bone marrow suspension were then blocked with 1 g / ml fc blocker ( bd pharmingen , mississauga , on , canada ) and stained with appropriately diluted antibodies directly conjugated with fitc or pe according to the standard procedure and followed by fixation in 1% paraformaldehyde . antibodies used for staining were the following : fitc - labeled anti - mouse cd11b and pe - labeled gr1 ( bd pharmingen ) . fluorescence was measured using guava easycyte 8 flow cytometer ( millipore , hayward , ca ) , and data analysis was performed using expresspro 8.1 software ( millipore ) . the ability of the splenocyte cultures to produce cytokines in response to a mitogenic challenge was assessed by incubating total splenocyte cultures ( 5 10 cells / ml ) for 48 h ( at 37c , 5% co2 , and 90% humidity ) in the presence of 0.25 g / ml concanavalin a ( con a , perkin elmer life science , boston , ma , usa ) or 0.1 g / ml anti - cd3 mab ( clone 145 - 2c11 , bd pharmingen , san diego ) . after that period , the supernatants were harvested , aliquoted , and stored at 20c until being assayed for cytokines . a second portion of the splenocyte suspension was placed in a 96-well u - bottomed plate ( becton dickinson , franklin lakers , nj , usa ) in aliquots of 100 l . the cells ' ability to proliferate in response to mitogenic stimulation with 0 ( negative control ) , 0.25 g / ml con a , or 0.1 g / ml anti - cd3 mab was determined by incubation for 48 h at 37c in a 5% co2 atmosphere with 90% humidity . the cultures were pulsed with [ h ] thymidine ( 0.5 ci / well ) for the last 18 to 24 h of incubation , cells were harvested , and radioactivity of [ h ] thymidine incorporation was counted using a scintillation counter ( perkin elmer life science , boston , ma ) . [ h ] thymidine uptake was expressed as the mean counts per minute ( c.p.m . ) of sextuplicate wells . plasma interleukin il-6 levels were used as a means of evaluating systemic inflammation and were quantified with commercial elisa kits ( r&d systems inc . il-2 levels in supernatants were used in analysis of t helper lymphocyte subclasses th1 cytokines and il-10 as a th2 cytokine . , toronto , canada ) . to assess hepatic function and cellular injury following pf , plasma levels of alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) were measured using the dri - chem 7000 chemistry analyzer ( heska co. , loveland , co , usa , slides from fujifilm corp . , 0.2 l plasma in a 20 l system was separated by sds - page and transferred onto a nitrocellulose membrane . lipopolysaccharides - stimulated macrophages were used as positive controls . the membrane was blocked for 1 h in pbs - tween ( 0.1% ) with 5% milk , followed by immunostaining with optimized dilutions of a polyclonal rabbit anti - mouse hmgb1 antibody ( 1 : 1,000 ) in 1% milk in pbs - tween overnight at 4c . the membrane was then incubated in pbs - tween + 1% milk containing hrp - conjugated goat anti - rabbit igg ( pierce ) for 1 h at room temperature . chemiluminescent signal was developed using supersignal west pico reagents ( pierce ) and imaged on x - ray film . analysis of area values complementary to western blot results was performed using imagej software ( us national institutes of health , bethesda , ma , usa ) [ 24 , 25 ] . comparisons between groups were performed using one - way analysis of variances ( anova ) and a post hoc tukey test , and differences were considered significant at p < 0.05 . the individual studies described in the results section are representative of at least three independent studies . to examine the changes of immunoinflammatory response across time after acute peripheral tissue trauma , we examined circulating cytokine mediators , t - cell proliferation , and in vitro th1/th2 cytokines production at time intervals of 1 , 6 , 24 , 48 , and 72 h after pf . pf is a model of peripheral tissue trauma , which incorporates all the elements of this injury type including the soft tissue and fracture components [ 22 , 23 ] . we had previously identified the early inflammation in pf mice , which showed a similar reproducible response to that found with the bilateral femur fracture model [ 26 , 27 ] . here we further found that the pf - induced early inflammatory response , which was assessed using systemic il-6 levels ( supplementary figure 1a available online at ) , was upregulated early with its peak at 1 h and recovered to normal levels by 24 h after trauma . as expected , hepatic injury , assessed by circulating ast and alt levels , was elevated by 6 h and recovered to normal levels by 48 h ( supplementary figures 1b and 1c ) . the time course of splenocyte proliferation in response to stimulation with con a in cells isolated from pf mice is shown in supplementary figure 2a . splenocyte proliferation was depressed by 48 h after injury and recovered to normal levels by 72 h , when compared with responses of cells from uninjured controls . next we assessed the th1/th2 cytokines released by the splenocytes from pf mice at 48 h after trauma . the in vitro release of cytokines by t - lymphocytes is shown in supplementary figure 2b . the production of th1 ( ifn- and il-2 ) cytokines by splenocytes was significantly lower in pf mice than in controls , while the production of th2 ( il-10 ) cytokines was significantly higher in pf mice . the t - cell proliferative responses and th1/2 shift induced by anti - cd3 were similar to those induced by con a in these groups ( data not shown ) . thus , these results suggest that peripheral tissue trauma elicits an early inflammatory response and a late attenuated t - lymphocyte response . recently , the accumulation of mdscs in the spleen has been reported to play a key role in the immunosuppression after physical injury . therefore , we determined whether peripheral tissue trauma has any effect on the expansion of cd11bgr-1 mdscs in bone marrow , blood , or spleen across 3 days after injury . as can be seen in figure 1 , the increased percentage of cd11bgr-1 mdsc in bone marrow ( 73% versus 43% ) , blood ( 26% versus 8% ) , and spleen ( 9% versus 4% ) is significant by 24 h after injury . the greatest increases were measured at 48 h with a 3-fold increase seen in the spleen . all the changes in mdsc percentages in different lymphocyte compartments returned to baseline by 72 h , the last time point assessed . circulating hmgb1 levels were estimated using western blot analysis and further quantified by hmgb1 elisa . the time course study showed that plasma hmgb1 level increased at 1 h , peaked at 24 h , and remained partially elevated at 48 h after injury ( figure 2 , supplementary table 1 , supplementary table 2 ) . this finding is consistent with others that showed elevated circulating hmgb1 levels after blunt trauma [ 28 , 29 ] . it was reasoned that this hmgb1 elevation might interfere with the immune function after injury . using a neutralizing anti - hmgb1 monoclonal antibody , 2g7 , we next examined the role of elevated hmgb1 on the deleterious effects of trauma on immune system . the increased plasma il-6 levels at 6 h , which return to baseline by 48 h after pf , were seen in both the control mab- and the anti - hmgb1 mab - injected mice ( figures 3(a)3(c ) ) . however , the attenuated t - cell proliferation and th1 cytokine responses observed in injured mice that received control mab remained intact in anti - hmgb1 mab - injected mice ( figures 3(d)3(f ) ) . thus the impaired t - cell function appears to be associated with the elevated hmgb1 after peripheral tissue trauma . since mdsc can be dissimilarly affected in different lymphocyte compartments , we next examined these possible differences in anti - hmgb1 neutralizing antibody injected mice . figures 4(a ) and 4(b ) show the expansion of cd11bgr-1 mdscs in bone marrow , as well as their mobilization in blood and accumulation in spleen by 48 h after pf in the control mab - injected mice . these cd11bgr-1 mdsc responses to trauma were blocked in the anti - hmgb1 mab - injected mice . collectively , our finding that anti - hmgb1 antibody ameliorates the impaired t - cell response and the expansion of mdsc in spleen strongly suggests that trauma - induced immunosuppression requires hmgb1 . functional alterations have been a consistent immunological finding after severe trauma in patients and in animal models [ 3032 ] . postinjury aberrations in t - cell function are most consistently found in th1 cytokine production [ 3133 ] . in addition , the accumulation and induction of mdscs in the spleen have been suggested recently as a contributor to the immunosuppression that occurs after injury [ 7 , 8 ] . the main finding of this study is that an hmgb1 neutralizing antibody ameliorates the attenuated t - cell response and increases in cd11bgr-1 myeloid cells in the spleen two days after peripheral tissue trauma in mice . in this study , a pf model was used to examine trauma - induced immune imbalance over time . as a model of peripheral tissue trauma , pf incorporates the soft tissue and fracture components of this injury type [ 22 , 23 ] . we have previously shown that this model induces an early systemic inflammatory response of a similar magnitude to that seen with bilateral femur fracture in mice [ 22 , 23 , 26 , 27 ] . however , unlike the bilateral femur fracture model , the pf model does not involve breaking the native bones . thus , the mice can be recovered , allowing for late posttraumatic survival studies . here , we show that pf results in a depression in immune functions such as cell proliferation and cytokine production in splenocytes , similar to that seen in other trauma models [ 34 , 35 ] . therefore , we conclude that this pf model is useful for assessing the immune changes following peripheral tissue trauma . this study and our previous findings show a significant decrease in the mitogen - induced proliferation in mice spleen at 48 hours after pf compared with uninjured controls . in addition , we also see the expected suppression of th1 cytokine production and increases in representative th2 cytokines at 48 h released by activated splenocytes isolated from injured animals , consistent with previous reports [ 31 , 33 ] . however , this is in contrast to the findings of chaudry et al . who showed splenic and peritoneal macrophages , as well as t - cell functions depressed in male animals early , within 30 min after trauma - hemorrhage . this discrepancy may be due to a difference in the severity of the trauma models ; the work by chaudry et al . focused more on a model of systemic ischemia and reperfusion , whereas our model focuses on peripheral tissue injury . myeloid - derived suppressor cells have gained significant attention in recent years for their role in acute inflammatory processes such as those that occur during sepsis and trauma [ 9 , 17 , 37 ] . we therefore asked whether peripheral tissue trauma was associated with expansion of cd11bgr-1 cells in the bone marrow , as well as their mobilization to the circulating blood and accumulation in the spleen at various time points across 3 days after injury . we found a significant increase in numbers of cd11bgr-1 mdscs in bone marrow , in blood , and in spleen at 24 h after trauma , while by 48 h only the increase in spleen remained . this study identifies a significant accumulation of cells with mdsc markers ( cd11bgr-1 myeloid cells ) early after trauma , yet this study does not directly show their immunosuppressive capacity . however , the findings are in agreement with a previous study , where trauma - induced cd11bgr-1 mdscs , which show functional immunosuppressive capacity , also exhibit significant early accumulation , importantly highlighting the timeline differences from other disease models . for example , sepsis expands this population in days while tumor implantation does so in weeks [ 9 , 10 ] . accumulating evidence has shown that mdscs regulate immune responses in infections , acute and chronic inflammation , trauma , and surgical sepsis , although initial observations and most of the current information regarding the role of mdscs have come from studies about cancer . in many mouse tumor models , as many as 2040% of nucleated splenocytes are represented by cd11bgr-1 mdscs ( in contrast to the 35% seen in normal mice in the present study ) . it has been demonstrated that the expansion of mdscs is influenced by several factors that are produced mainly by tumor cells or by activated t - cells . however , in contrast to the prolonged expansion shown in cancer and chronic infection [ 9 , 17 ] , the expansion of mdscs in the setting of acute trauma is transient . this transient cd11bgr-1 myeloid population may possibly mediate the suppressive functions that are characteristic of mdscs . however , because the acute conditions are short - lived , the suppressive functions of this transient population may have a minimal impact on the overall immune response . therefore , these cells may function as important gatekeepers that prevent pathologic immune - mediated damage . indeed , cd11bgr-1 mdscs have been demonstrated to infiltrate the spleen and suppress t - cell function and increase susceptibility to listeria monocytogenes infection in a trauma model [ 7 , 38 ] . furthermore , some evidence suggests that mdscs can induce expansion of regulatory t - cells , another suppressive regulator of the immune system . interestingly , li et al . have shown that an mdsc expansion after surgical trauma in the setting of cancer promoted peritoneal metastasis . collectively , it is reasoned that this transient expansion of mdscs is involved in the immune imbalance to trauma . hmgb1 levels are known to be elevated early following trauma in humans [ 28 , 29 ] and remain elevated . prompted by the pattern of elevated hmgb1 , we hypothesized that treatment with a neutralizing anti - hmgb1 monoclonal antibody would impact the immunosuppression at late posttraumatic term . when measurements were made at 48 h after injury , treatment with two doses of s.c . anti - hmgb1 antibody provided significant protection against the development of trauma - induced immunosuppression including the depressed t - cell response and accumulation of mdscs in spleen . the precise mechanisms for protective effects of anti - hmgb1 mab on trauma - induced immune dysfunction in the spleen remain to be established . here we showed that early inflammation manifested by circulatory il-6 levels was unresolved while late immune suppression was ameliorated in the hmgb1 mab treated mice after trauma . in the context of the present study , it is worth pointing out that the mechanistic relationship between the early hyperinflammatory and the posttraumatic immunosuppression is not known . these two components comprise the current paradigm of the host response to injury , which has been widely accepted over the last 2 decades . this paradigm is directly based on the clinical manifestations of trauma and is supported by an immense body of literature , which has catalogued the complex response to injury that disrupts the immune system homeostasis [ 4044 ] . current changes to this paradigm suggest an altered timeline , due to recent findings by xiao et al . and others which have reported that hyperinflammatory and hypoinflammatory responses were concurrent following blunt trauma in humans . however a definitive understanding of the mechanistic correlation of these two components remains to be determined . our previous work has highlighted the distinct hyperinflammatory and immunosuppressive responses to trauma ; we identified that even though toll - like receptor ( tlr ) 4 contributed to both responses , tlr9 contributed only to immunosuppressed t - lymphocyte responses after peripheral tissue injury . it is of note that hmgb1 is one of several known ligands for both of these receptors . therein , these findings suggest that hyperinflammatory response and immunosuppression are not always mechanistically linked and also indicate that hmgb1 may use some alternate mechanisms following tissue trauma alone that lead to the immunosuppression . hmgb1 is also known to mediate different functions dependent on the redox status of the protein and its binding targets . several inflammatory mediators , such as il-1 , il-6 , tnf- , ifn- , and proinflammatory s100 proteins , have been demonstrated to be able to drive the expansion of mdsc [ 20 , 21 ] . additionally , there is abundant evidence to support a molecular chaperon property of hmgb1 through enhancement of presentation of bound mediators to their cellular receptors . this notion is supported by many studies , showing that hmgb1 binds il-1 , il-6 , tnf- , or ifn- for an enhanced cytokine - induced effect [ 5056 ] . thus , hmgb1 may drive the expansion of cd11bgr-1 mdscs through binding with these mediators in a lymphoid compartment after trauma . indeed , this deduction is supported by our results showing that treatment with anti - hmgb1 antibody ameliorated the expansion of cd11bgr-1 mdscs and the accumulation in spleen , in the late posttraumatic term . it is possible , of course , that hmgb1 is not directly involved in the expansion of mdscs , but rather responsible for the release of mediators , which in turn drive the expansion following trauma . although the molecular mechanisms responsible for hmgb1 in the expansion of cd11bgr-1 mdscs and in the cd11bgr-1 mdscs - mediation of t - cell suppression remain to be determined , our data suggest that expansion of cd11bgr-1 myeloid cells in mice after peripheral tissue injury requires hmgb1 . although this study demonstrates new insights regarding the role of tissue - derived hmgb1 in the response of t - cells and the expansion of cd11bgr-1 mdscs to trauma , it has limitations . for example , we may not be measuring the whole of the immune compartment through our current methodology . for example , t - cell numbers were not measured in the thymus or lymph nodes . ezernitchi et al . have reported that t - cell dysfunction is induced in the spleen , peripheral blood , and bone marrow , but not in lymph nodes , and correlates with elevated levels of mdsc . in summary , we showed for the first time that treatment with a neutralizing anti - hmgb1 monoclonal antibody can ameliorate attenuated t - cell response and accumulation of cd11bgr-1 mdsc in spleen in a clinically relevant model of peripheral tissue trauma . it is likely that expansion of mdscs involves elevated hmgb1 levels and contributes to the immunosuppression after trauma . our findings suggest that anti - hmgb1 antibodies strategies warrant further evaluation as a therapeutic to reduce infection and mods after trauma .

although tissue - derived high mobility group box 1 ( hmgb1 ) is involved in many aspects of inflammation and tissue injury after trauma , its role in trauma - induced immune suppression remains elusive . using an established mouse model of peripheral tissue trauma , which includes soft tissue and fracture components , we report here that treatment with anti - hmgb1 monoclonal antibody ameliorated the trauma - induced attenuated t - cell responses and accumulation of cd11b+gr-1 + myeloid - derived suppressor cells in the spleens seen two days after injury . our data suggest that hmgb1 released after tissue trauma contributes to signaling pathways that lead to attenuation of t - lymphocyte responses and enhancement of myeloid - derived suppressor cell expansion .

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

in this respect , nosocomial infections are a common clinical problem in trauma intensive care units in injured patients and are associated with increased morbidity , length of hospital stay , and mortality . a number of studies have demonstrated that splenocyte dysfunction often observed as t - lymphocyte dysfunction and t - helper 1 ( th1 ) depression are associated with increased susceptibility to severe infection and/or multiple organ dysfunction ( mods ) after trauma [ 24 ] . myeloid - derived suppressor cells ( mdscs ) , a heterogeneous cd11bgr-1 cell population consisting of immature myeloid cells and myeloid progenitor cells that can suppress t - cell responses by a variety of mechanisms [ 5 , 6 ] , have recently been associated with the profound immunosuppression seen in sepsis and trauma . ochoa and colleagues [ 7 , 8 ] found that a marked increase of cd11bgr-1 mdscs is associated with suppressed t - cell functions via the production of arginase-1 in the mouse spleen at 24 h after surgical trauma . however , under septic conditions , moldawer and colleagues found that a different response where cd11bgr-1 mdscs expanded in the spleen , in the bone marrow , and in the peripheral lymph nodes by 3 days after sepsis and several approaches to prevent the expansion of these cells deteriorated the immunosuppression and worsened outcomes [ 9 , 10 ] . high mobility group box nuclear protein 1 ( hmgb1 ) is a nuclear dna binding protein that has been shown to be a key late mediator in sepsis and an early trigger of sterile inflammation in severe trauma when released from cells [ 12 , 13 ] . although hmgb1 released as the result of tissue injury or stress is involved in many aspects of inflammation , it is unknown whether hmgb1 mediates trauma - associated immune suppression . hmgb1 has multiple functions in the regulation of immunity and inflammation , and studies have shown that hmgb1 has variable effects on t - cell responses depending on dose , redox status , and disease setting [ 14 , 15 ] . furthermore hmgb1 has been demonstrated to lead to mdsc expansion after surgical trauma in the setting of cancer ; however , hmgb1 has been shown to bind to and enhance the cytokine - induced effect of several inflammatory mediators , such as il-1 , il-6 , and tnf- , which themselves have been shown to drive the expansion of mdsc [ 20 , 21 ] . in this study , we evaluated the role of hmgb1 in trauma - associated immune suppression through examination of the contribution of hmgb1 to mdsc expansion and splenocyte responses after peripheral tissue trauma . systemic neutralization of hmgb1 was achieved with administration of anti - hmgb1 monoclonal antibody , 2g7 after injury . using a murine trauma model , the pseudofracture ( pf ) model , mice used in the experimental protocols were housed in accordance with university of pittsburgh ( pittsburgh , pa ) and national institutes of health ( nih , bethesda , md ) animal care guidelines in specific pathogen - free conditions . the animals were maintained in the university of pittsburgh animal research center with a 12 h light - dark cycle and free access to standard laboratory food and water . with a total volume of 200 l of solution containing 40 g of a mouse monoclonal anti - hmgb1 antibody 2g7 ( igg2b , noncommercial antibody , gift from dr . pf is a model of peripheral tissue trauma , which incorporates all the elements of this injury type including the soft tissue and fracture components [ 22 , 23 ] . to obtain standardized concentrations , two femurs and two tibias were removed from collected tissues and crushed using a sterile mortar and pestle in the biological safety hood . , lake forest , il , for ovation pharmaceuticals , deerfield , il ) and inhaled isoflurane , and both thighs were squeezed with a hemostat for 30 seconds to induce a soft tissue injury . then , 0.15 ml of bone components suspension prepared above was injected using an 18 g needle in the area of the soft tissue injury of each thigh . after washing the cells twice with complete rpmi 1640 , isolated splenocytes were diluted to appropriate concentrations , detailed as follows , in complete rpmi 1640 medium ( lonza , walkersville , md , usa ) , which contained 10% fetal bovine serum ( fbs , sigma ) , 2 mm glutamine ( gibco laboratories , grand island , ny ) , 1% penicillin - streptomycin ( gibco laboratories ) , and 0.05 mm 2-mercaptoethanol ( 2-me , sigma ) . antibodies used for staining were the following : fitc - labeled anti - mouse cd11b and pe - labeled gr1 ( bd pharmingen ) . the ability of the splenocyte cultures to produce cytokines in response to a mitogenic challenge was assessed by incubating total splenocyte cultures ( 5 10 cells / ml ) for 48 h ( at 37c , 5% co2 , and 90% humidity ) in the presence of 0.25 g / ml concanavalin a ( con a , perkin elmer life science , boston , ma , usa ) or 0.1 g / ml anti - cd3 mab ( clone 145 - 2c11 , bd pharmingen , san diego ) . the cells ' ability to proliferate in response to mitogenic stimulation with 0 ( negative control ) , 0.25 g / ml con a , or 0.1 g / ml anti - cd3 mab was determined by incubation for 48 h at 37c in a 5% co2 atmosphere with 90% humidity . plasma interleukin il-6 levels were used as a means of evaluating systemic inflammation and were quantified with commercial elisa kits ( r&d systems inc . il-2 levels in supernatants were used in analysis of t helper lymphocyte subclasses th1 cytokines and il-10 as a th2 cytokine . the membrane was blocked for 1 h in pbs - tween ( 0.1% ) with 5% milk , followed by immunostaining with optimized dilutions of a polyclonal rabbit anti - mouse hmgb1 antibody ( 1 : 1,000 ) in 1% milk in pbs - tween overnight at 4c . the membrane was then incubated in pbs - tween + 1% milk containing hrp - conjugated goat anti - rabbit igg ( pierce ) for 1 h at room temperature . the individual studies described in the results section are representative of at least three independent studies . to examine the changes of immunoinflammatory response across time after acute peripheral tissue trauma , we examined circulating cytokine mediators , t - cell proliferation , and in vitro th1/th2 cytokines production at time intervals of 1 , 6 , 24 , 48 , and 72 h after pf . pf is a model of peripheral tissue trauma , which incorporates all the elements of this injury type including the soft tissue and fracture components [ 22 , 23 ] . we had previously identified the early inflammation in pf mice , which showed a similar reproducible response to that found with the bilateral femur fracture model [ 26 , 27 ] . here we further found that the pf - induced early inflammatory response , which was assessed using systemic il-6 levels ( supplementary figure 1a available online at ) , was upregulated early with its peak at 1 h and recovered to normal levels by 24 h after trauma . splenocyte proliferation was depressed by 48 h after injury and recovered to normal levels by 72 h , when compared with responses of cells from uninjured controls . next we assessed the th1/th2 cytokines released by the splenocytes from pf mice at 48 h after trauma . the in vitro release of cytokines by t - lymphocytes is shown in supplementary figure 2b . the t - cell proliferative responses and th1/2 shift induced by anti - cd3 were similar to those induced by con a in these groups ( data not shown ) . thus , these results suggest that peripheral tissue trauma elicits an early inflammatory response and a late attenuated t - lymphocyte response . recently , the accumulation of mdscs in the spleen has been reported to play a key role in the immunosuppression after physical injury . therefore , we determined whether peripheral tissue trauma has any effect on the expansion of cd11bgr-1 mdscs in bone marrow , blood , or spleen across 3 days after injury . as can be seen in figure 1 , the increased percentage of cd11bgr-1 mdsc in bone marrow ( 73% versus 43% ) , blood ( 26% versus 8% ) , and spleen ( 9% versus 4% ) is significant by 24 h after injury . the greatest increases were measured at 48 h with a 3-fold increase seen in the spleen . the time course study showed that plasma hmgb1 level increased at 1 h , peaked at 24 h , and remained partially elevated at 48 h after injury ( figure 2 , supplementary table 1 , supplementary table 2 ) . it was reasoned that this hmgb1 elevation might interfere with the immune function after injury . using a neutralizing anti - hmgb1 monoclonal antibody , 2g7 , we next examined the role of elevated hmgb1 on the deleterious effects of trauma on immune system . the increased plasma il-6 levels at 6 h , which return to baseline by 48 h after pf , were seen in both the control mab- and the anti - hmgb1 mab - injected mice ( figures 3(a)3(c ) ) . however , the attenuated t - cell proliferation and th1 cytokine responses observed in injured mice that received control mab remained intact in anti - hmgb1 mab - injected mice ( figures 3(d)3(f ) ) . thus the impaired t - cell function appears to be associated with the elevated hmgb1 after peripheral tissue trauma . since mdsc can be dissimilarly affected in different lymphocyte compartments , we next examined these possible differences in anti - hmgb1 neutralizing antibody injected mice . figures 4(a ) and 4(b ) show the expansion of cd11bgr-1 mdscs in bone marrow , as well as their mobilization in blood and accumulation in spleen by 48 h after pf in the control mab - injected mice . these cd11bgr-1 mdsc responses to trauma were blocked in the anti - hmgb1 mab - injected mice . collectively , our finding that anti - hmgb1 antibody ameliorates the impaired t - cell response and the expansion of mdsc in spleen strongly suggests that trauma - induced immunosuppression requires hmgb1 . postinjury aberrations in t - cell function are most consistently found in th1 cytokine production [ 3133 ] . in addition , the accumulation and induction of mdscs in the spleen have been suggested recently as a contributor to the immunosuppression that occurs after injury [ 7 , 8 ] . the main finding of this study is that an hmgb1 neutralizing antibody ameliorates the attenuated t - cell response and increases in cd11bgr-1 myeloid cells in the spleen two days after peripheral tissue trauma in mice . in this study , a pf model was used to examine trauma - induced immune imbalance over time . as a model of peripheral tissue trauma , pf incorporates the soft tissue and fracture components of this injury type [ 22 , 23 ] . here , we show that pf results in a depression in immune functions such as cell proliferation and cytokine production in splenocytes , similar to that seen in other trauma models [ 34 , 35 ] . therefore , we conclude that this pf model is useful for assessing the immune changes following peripheral tissue trauma . this study and our previous findings show a significant decrease in the mitogen - induced proliferation in mice spleen at 48 hours after pf compared with uninjured controls . in addition , we also see the expected suppression of th1 cytokine production and increases in representative th2 cytokines at 48 h released by activated splenocytes isolated from injured animals , consistent with previous reports [ 31 , 33 ] . who showed splenic and peritoneal macrophages , as well as t - cell functions depressed in male animals early , within 30 min after trauma - hemorrhage . this discrepancy may be due to a difference in the severity of the trauma models ; the work by chaudry et al . focused more on a model of systemic ischemia and reperfusion , whereas our model focuses on peripheral tissue injury . myeloid - derived suppressor cells have gained significant attention in recent years for their role in acute inflammatory processes such as those that occur during sepsis and trauma [ 9 , 17 , 37 ] . we therefore asked whether peripheral tissue trauma was associated with expansion of cd11bgr-1 cells in the bone marrow , as well as their mobilization to the circulating blood and accumulation in the spleen at various time points across 3 days after injury . we found a significant increase in numbers of cd11bgr-1 mdscs in bone marrow , in blood , and in spleen at 24 h after trauma , while by 48 h only the increase in spleen remained . this study identifies a significant accumulation of cells with mdsc markers ( cd11bgr-1 myeloid cells ) early after trauma , yet this study does not directly show their immunosuppressive capacity . however , the findings are in agreement with a previous study , where trauma - induced cd11bgr-1 mdscs , which show functional immunosuppressive capacity , also exhibit significant early accumulation , importantly highlighting the timeline differences from other disease models . accumulating evidence has shown that mdscs regulate immune responses in infections , acute and chronic inflammation , trauma , and surgical sepsis , although initial observations and most of the current information regarding the role of mdscs have come from studies about cancer . in many mouse tumor models , as many as 2040% of nucleated splenocytes are represented by cd11bgr-1 mdscs ( in contrast to the 35% seen in normal mice in the present study ) . it has been demonstrated that the expansion of mdscs is influenced by several factors that are produced mainly by tumor cells or by activated t - cells . however , in contrast to the prolonged expansion shown in cancer and chronic infection [ 9 , 17 ] , the expansion of mdscs in the setting of acute trauma is transient . indeed , cd11bgr-1 mdscs have been demonstrated to infiltrate the spleen and suppress t - cell function and increase susceptibility to listeria monocytogenes infection in a trauma model [ 7 , 38 ] . furthermore , some evidence suggests that mdscs can induce expansion of regulatory t - cells , another suppressive regulator of the immune system . have shown that an mdsc expansion after surgical trauma in the setting of cancer promoted peritoneal metastasis . collectively , it is reasoned that this transient expansion of mdscs is involved in the immune imbalance to trauma . prompted by the pattern of elevated hmgb1 , we hypothesized that treatment with a neutralizing anti - hmgb1 monoclonal antibody would impact the immunosuppression at late posttraumatic term . when measurements were made at 48 h after injury , treatment with two doses of s.c . anti - hmgb1 antibody provided significant protection against the development of trauma - induced immunosuppression including the depressed t - cell response and accumulation of mdscs in spleen . the precise mechanisms for protective effects of anti - hmgb1 mab on trauma - induced immune dysfunction in the spleen remain to be established . here we showed that early inflammation manifested by circulatory il-6 levels was unresolved while late immune suppression was ameliorated in the hmgb1 mab treated mice after trauma . in the context of the present study , it is worth pointing out that the mechanistic relationship between the early hyperinflammatory and the posttraumatic immunosuppression is not known . these two components comprise the current paradigm of the host response to injury , which has been widely accepted over the last 2 decades . our previous work has highlighted the distinct hyperinflammatory and immunosuppressive responses to trauma ; we identified that even though toll - like receptor ( tlr ) 4 contributed to both responses , tlr9 contributed only to immunosuppressed t - lymphocyte responses after peripheral tissue injury . it is of note that hmgb1 is one of several known ligands for both of these receptors . therein , these findings suggest that hyperinflammatory response and immunosuppression are not always mechanistically linked and also indicate that hmgb1 may use some alternate mechanisms following tissue trauma alone that lead to the immunosuppression . additionally , there is abundant evidence to support a molecular chaperon property of hmgb1 through enhancement of presentation of bound mediators to their cellular receptors . this notion is supported by many studies , showing that hmgb1 binds il-1 , il-6 , tnf- , or ifn- for an enhanced cytokine - induced effect [ 5056 ] . thus , hmgb1 may drive the expansion of cd11bgr-1 mdscs through binding with these mediators in a lymphoid compartment after trauma . indeed , this deduction is supported by our results showing that treatment with anti - hmgb1 antibody ameliorated the expansion of cd11bgr-1 mdscs and the accumulation in spleen , in the late posttraumatic term . it is possible , of course , that hmgb1 is not directly involved in the expansion of mdscs , but rather responsible for the release of mediators , which in turn drive the expansion following trauma . although the molecular mechanisms responsible for hmgb1 in the expansion of cd11bgr-1 mdscs and in the cd11bgr-1 mdscs - mediation of t - cell suppression remain to be determined , our data suggest that expansion of cd11bgr-1 myeloid cells in mice after peripheral tissue injury requires hmgb1 . although this study demonstrates new insights regarding the role of tissue - derived hmgb1 in the response of t - cells and the expansion of cd11bgr-1 mdscs to trauma , it has limitations . for example , we may not be measuring the whole of the immune compartment through our current methodology . for example , t - cell numbers were not measured in the thymus or lymph nodes . have reported that t - cell dysfunction is induced in the spleen , peripheral blood , and bone marrow , but not in lymph nodes , and correlates with elevated levels of mdsc . in summary , we showed for the first time that treatment with a neutralizing anti - hmgb1 monoclonal antibody can ameliorate attenuated t - cell response and accumulation of cd11bgr-1 mdsc in spleen in a clinically relevant model of peripheral tissue trauma . it is likely that expansion of mdscs involves elevated hmgb1 levels and contributes to the immunosuppression after trauma . our findings suggest that anti - hmgb1 antibodies strategies warrant further evaluation as a therapeutic to reduce infection and mods after trauma .

the wafacs is a randomized , double - blind , placebo - controlled trial evaluating the effects of a combination pill of folic acid ( 2.5 mg / day ) , vitamin b6 ( 50 mg / day ) , and vitamin b12 ( 1 mg / day ) in the secondary prevention of important vascular events among high - risk women with either a history of cvd or at least three cardiovascular risk factors . briefly , the wafacs began in 1998 , when the folic acid , vitamin b6 , and vitamin b12 component was added to the women 's antioxidant cardiovascular study ( wacs ) , an ongoing 222 factorial trial of three antioxidant vitamins ( vitamins c and e and -carotene ) , which expanded it to a four - group factorial trial . details of the overall trial design and the main results from the wafacs and wacs have been reported previously ( 2426 ) . the study was sponsored by the national heart , lung , and blood institute of the national institutes of health . the study vitamins and matching placebo were provided by basf corporation ( mount olive , nj ) . the trial was approved by the institutional review board of brigham and women 's hospital ( boston , ma ) , and all patients provided written informed consent . an external independent data and safety - monitoring board monitored the safety of the participants and the overall quality and scientific integrity of the study . in the wacs parent trial , 8,171 female health professionals were randomized into the trial from june 1995 through october 1996 to receive vitamin c ( 500 mg / day ) , vitamin e ( 600 iu every other day ) , and -carotene ( 50 mg every other day ) versus respective matching placebos . women were eligible for wacs if they were at least 40 years old , postmenopausal or had no intention of becoming pregnant , and had a self - reported history of cvd ( including myocardial infarction , stroke , coronary revascularization , or angina ) or had at least three traditional cardiac risk factors ( 26 ) . in april 1998 , 5,442 of these women provided consent and were additionally randomized in a factorial design to receive a combination pill containing 2.5 mg folic acid , 50 mg vitamin b6 , and 1 mg vitamin b12 ( active treatment ) or a matching placebo daily . all study investigators , staff , and participants were unaware of the participants ' treatment assignments . from the 5,442 women randomized in the wafacs trial , we excluded those with diabetes at baseline ( n = 1,190 ) for the present analyses , leaving 4,252 nondiabetic women at baseline ( fig . flow diagram illustrating diabetes outcomes in the randomly assigned treatment of folic acid and vitamins b6 and b12 of the wafacs . a total of 1,190 participants who had a diagnosis of diabetes at baseline were excluded in the analysis . following randomization and annually thereafter , participants were mailed monthly calendar packs containing active agents or placebos , along with questionnaires on adherence , use of nonstudy supplements , and occurrence of major illnesses or adverse events . a semiquantitative food - frequency questionnaire at baseline was used to assess dietary nutrient intake ( 27 ) . written permission for medical records was sought from participants who reported cardiovascular end points or from the next of kin in case of death . an end points committee of physicians who were blinded to randomized treatment assignment adjudicated all primary and secondary cardiovascular outcome events . study medications and end point ascertainment were continued in a blinded fashion until the scheduled end of the trial , 31 july 2005 , for a follow - up duration of 7.3 years . at the scheduled end of the trial , if assessed in terms of person - time , mortality and morbidity information was complete for 98.9 and 98.0% , respectively , of person - years of follow - up . adherence was assessed through self - report on annual study questionnaires and was defined as taking at least two - thirds of the study pills . average adherence over the course of follow - up was 83% for active and placebo agents , with no significant difference between active and placebo groups . use of open - label folic acid supplements , vitamin b6 , or vitamin b12 supplements containing more than the recommended daily allowance for at least 4 days per month ranged from 2 to 11% in the active group to 2 to 13% in the placebo group over the course of the study . women in the wafacs provided a baseline blood sample in 1996 , prior to the initiation of background dietary folic acid fortification in the u.s . food supply in 1998 . randomly selected from participants who were adherent with study medications , 300 ( 150 in the active treatment and 150 in the placebo group ) provided a blood sample at the end of randomized treatment . as reported previously ( 25 ) , median folate and homocysteine levels were similar between the active treatment group and the placebo group at baseline . at the end of study follow - up , the median folate level increased significantly in both groups ; however , the relative increase in folate level was greater in the active treatment group . despite significant increases in folate levels among the placebo group , there was no apparent reduction in homocysteine levels at the end of the study compared with participants measured at the beginning of the study in the placebo group ( p = 0.99 ) . in comparison , homocysteine levels were significantly reduced in the active treatment group ; the geometric mean homocysteine level was decreased by 18.5% ( 95% ci 12.524.1 ; p < 0.001 ) in the active group over that observed in the placebo group for a difference of 2.27 mol / l ( 1.542.96 ) from the placebo geometric mean homocysteine level of 12.28 diabetes status was evaluated at baseline , and all the participants were also asked annually whether and when they had been diagnosed with diabetes after randomization . women who reported a diagnosis of diabetes during the follow - up were mailed supplementary questionnaires to confirm their self - reported diagnoses . the supplementary diabetes questionnaire was specifically designed to collect further detailed information on diabetes symptoms , screening test , and hypoglycemic medication . based on the american diabetes association diagnostic criteria ( 28 ) , actual glucose levels at fasting or oral glucose tolerance testing , diabetic symptoms , and/or hypoglycemic medication were combined together to confirm the self - reported incident cases of diabetes in a blinded fashion . the screening rate of having blood glucose testing among our study population was relatively high ( 8590% ) . the observed high agreement between annual follow - up questionnaire and supplementary questionnaire ( positive predictive value = 96% ) suggests that self - reported diabetes possesses excellent predictive ability for true diabetes status in this cohort of u.s . female health professionals , who are likely to report accurate diagnostic information ( 29 ) . thus , we believe that self - reported type 2 diabetes is valid in our study population . primary analyses were performed on an intention - to - treat basis , including all randomized women after excluding those with self - reported diabetes at baseline . baseline characteristics were compared by randomized groups using two - sample t tests for continuous variables and statistics for categorical variables . kaplan - meier survival curves were used to estimate the overall cumulative incidence over time for the active vitamin group and the placebo group . we used cox proportional hazards models to calculate the estimates of hazard ratio expressed as relative risks ( rrs ) and 95% ci for randomized treatment versus placebo , after adjustment for age and other randomized treatments ( vitamin e , vitamin c , and -carotene ) . to test the proportionality assumption , we included an interaction term for treatment with the logarithm of time in the cox models . the tests showed that the proportional hazard assumption was not violated for any of the models . to examine the effect of actual as opposed to assigned folic acid / b vitamin treatment , we carried out a sensitivity analysis according to compliance . women were censored if and when they stopped taking at least two - thirds of their study pills or were missing compliance information . subgroup analyses were conducted to examine the effect of active treatment on risk of type 2 diabetes according to prespecified risk factors for type 2 diabetes at baseline , including age - groups ( 4554 , 5564 , and 65 years ) , bmi ( kg / m ) , smoking status ( current , past , or never ) , alcohol use ( never / rarely or one or more drinks per month ) , family history of diabetes ( yes or no ) , physical activity ( estimated energy expenditure from leisure activities of < 1,000 or 1,000 kcal per week ) , menopausal status and hormone therapy ( uncertain menopausal status , premenopausal , or postmenopausal including current , past , or never users of hormone therapy ) , history of hypertension ( yes or no ) , history of hyperlipidemia ( yes or no ) , and baseline dietary intakes of folate , vitamin b6 , or vitamin b12 ( tertiles for each ) . we assessed effect modification using interaction terms between subgroup indicators and randomized assignment , testing for trend when subgroup categories were ordinal . all analyses were conducted with sas version 9 ( sas institute , cary , nc ) , and a two - sided test with a significance level of = 0.05 ( p < 0.05 ) was used . following randomization and annually thereafter , participants were mailed monthly calendar packs containing active agents or placebos , along with questionnaires on adherence , use of nonstudy supplements , and occurrence of major illnesses or adverse events . a semiquantitative food - frequency questionnaire at baseline was used to assess dietary nutrient intake ( 27 ) . written permission for medical records was sought from participants who reported cardiovascular end points or from the next of kin in case of death . an end points committee of physicians who were blinded to randomized treatment assignment adjudicated all primary and secondary cardiovascular outcome events . study medications and end point ascertainment were continued in a blinded fashion until the scheduled end of the trial , 31 july 2005 , for a follow - up duration of 7.3 years . at the scheduled end of the trial , if assessed in terms of person - time , mortality and morbidity information was complete for 98.9 and 98.0% , respectively , of person - years of follow - up . adherence was assessed through self - report on annual study questionnaires and was defined as taking at least two - thirds of the study pills . average adherence over the course of follow - up was 83% for active and placebo agents , with no significant difference between active and placebo groups . use of open - label folic acid supplements , vitamin b6 , or vitamin b12 supplements containing more than the recommended daily allowance for at least 4 days per month ranged from 2 to 11% in the active group to 2 to 13% in the placebo group over the course of the study . women in the wafacs provided a baseline blood sample in 1996 , prior to the initiation of background dietary folic acid fortification in the u.s . food supply in 1998 . randomly selected from participants who were adherent with study medications , 300 ( 150 in the active treatment and 150 in the placebo group ) provided a blood sample at the end of randomized treatment . as reported previously ( 25 ) , median folate and homocysteine levels were similar between the active treatment group and the placebo group at baseline . at the end of study follow - up , the median folate level increased significantly in both groups ; however , the relative increase in folate level was greater in the active treatment group . despite significant increases in folate levels among the placebo group , there was no apparent reduction in homocysteine levels at the end of the study compared with participants measured at the beginning of the study in the placebo group ( p = 0.99 ) . in comparison , homocysteine levels were significantly reduced in the active treatment group ; the geometric mean homocysteine level was decreased by 18.5% ( 95% ci 12.524.1 ; p < 0.001 ) in the active group over that observed in the placebo group for a difference of 2.27 mol / l ( 1.542.96 ) from the placebo geometric mean homocysteine level of 12.28 diabetes status was evaluated at baseline , and all the participants were also asked annually whether and when they had been diagnosed with diabetes after randomization . women who reported a diagnosis of diabetes during the follow - up were mailed supplementary questionnaires to confirm their self - reported diagnoses . the supplementary diabetes questionnaire was specifically designed to collect further detailed information on diabetes symptoms , screening test , and hypoglycemic medication . based on the american diabetes association diagnostic criteria ( 28 ) , actual glucose levels at fasting or oral glucose tolerance testing , diabetic symptoms , and/or hypoglycemic medication were combined together to confirm the self - reported incident cases of diabetes in a blinded fashion . the screening rate of having blood glucose testing among our study population was relatively high ( 8590% ) . the observed high agreement between annual follow - up questionnaire and supplementary questionnaire ( positive predictive value = 96% ) suggests that self - reported diabetes possesses excellent predictive ability for true diabetes status in this cohort of u.s . female health professionals , who are likely to report accurate diagnostic information ( 29 ) . thus , we believe that self - reported type 2 diabetes is valid in our study population . primary analyses were performed on an intention - to - treat basis , including all randomized women after excluding those with self - reported diabetes at baseline . baseline characteristics were compared by randomized groups using two - sample t tests for continuous variables and statistics for categorical variables . kaplan - meier survival curves were used to estimate the overall cumulative incidence over time for the active vitamin group and the placebo group . we used cox proportional hazards models to calculate the estimates of hazard ratio expressed as relative risks ( rrs ) and 95% ci for randomized treatment versus placebo , after adjustment for age and other randomized treatments ( vitamin e , vitamin c , and -carotene ) . to test the proportionality assumption , we included an interaction term for treatment with the logarithm of time in the cox models . the tests showed that the proportional hazard assumption was not violated for any of the models . to examine the effect of actual as opposed to assigned folic acid / b vitamin treatment , we carried out a sensitivity analysis according to compliance . women were censored if and when they stopped taking at least two - thirds of their study pills or were missing compliance information . subgroup analyses were conducted to examine the effect of active treatment on risk of type 2 diabetes according to prespecified risk factors for type 2 diabetes at baseline , including age - groups ( 4554 , 5564 , and 65 years ) , bmi ( kg / m ) , smoking status ( current , past , or never ) , alcohol use ( never / rarely or one or more drinks per month ) , family history of diabetes ( yes or no ) , physical activity ( estimated energy expenditure from leisure activities of < 1,000 or 1,000 kcal per week ) , menopausal status and hormone therapy ( uncertain menopausal status , premenopausal , or postmenopausal including current , past , or never users of hormone therapy ) , history of hypertension ( yes or no ) , history of hyperlipidemia ( yes or no ) , and baseline dietary intakes of folate , vitamin b6 , or vitamin b12 ( tertiles for each ) . we assessed effect modification using interaction terms between subgroup indicators and randomized assignment , testing for trend when subgroup categories were ordinal . all analyses were conducted with sas version 9 ( sas institute , cary , nc ) , and a two - sided test with a significance level of = 0.05 ( p < 0.05 ) was used . during a median follow - up period of 7.3 years , 504 women were diagnosed with type 2 diabetes among 4,252 nondiabetic participants who underwent randomization in the wafacs . there were no statistically significant differences in baseline characteristics between the folic acid / b vitamin active treatment group and its corresponding placebo group ( table 1 ) . baseline characteristics of nondiabetic women according to randomized groups in the wafacs data are means sd unless otherwise indicated . overall , there was no significant effect of folic acid / b vitamins on the development of type 2 diabetes compared with the placebo group ( table 2 and fig . 2 ) . in total , there were 245 incident cases ( 11.5% ) in the active treatment group and 259 ( 12.2% ) in the placebo group ( 172.5/10,000 person - years vs. 184.8/10,000 person - years ) , corresponding to an overall rr of 0.94 ( 95% ci 0.791.11 ; p = 0.46 ) after controlling for age and antioxidant treatment assignments ( table 2 ) . figure 2 shows the cumulative incidence of type 2 diabetes events among women in the treatment and placebo groups by year of follow - up . there appeared to be a trend toward a modest reduction in risk of type 2 diabetes for the treatment group versus placebo group over the follow - up period , but the log - rank test for the overall difference was not statistically significant ( p for log - rank test = 0.44 ) ( fig . 2 ) . rrs of self - reported type 2 diabetes by randomized folic acid and b vitamins intervention in the wafacs * adjusted for age and other randomized assignments ( vitamins c and e and -carotene ) . additionally adjusted for baseline variables , including bmi , smoking status , postmenopausal hormone use , multivitamin use , alcohol intake , coffee intake , physical activity , and family history of diabetes . cumulative incidence of self - reported type 2 diabetes by randomized treatment assignment ( active treatment versus placebo ) in the wafacs . when we subdivided the period of risk into years 1 and 2 combined and year 3 onward combined , we did not observe a statistically significant effect in any time period ( table 2 ) . in sensitivity analyses to minimize potential bias due to the inclusion of undiagnosed diabetes at baseline , folic acid / b vitamin treatment was not significantly associated with risk of type 2 diabetes when excluding those cases that occurred in the first 2 years ( rr 0.98 [ 95% ci 0.801.21 ] ; p = 0.87 ) . in a separate analysis where women were censored at the time they stopped taking at least two - thirds of their study pills or started to use outside folic acid / b vitamin supplements on 4 days per month , the results were unchanged after further adjustment for diabetes risk factors including bmi , physical activity , family history of diabetes , smoking , postmenopausal hormone use , multivitamin use , alcohol intake , and coffee consumption . to determine whether certain subgroups of women were at particularly high or low risk for type 2 diabetes with folic acid / b vitamin treatment , we conducted multiple subgroup analyses stratified by several prespecified diabetes risk factors ( fig . 3 ) . overall , there was no evidence that any of these factors modified the treatment effect on the risk of type 2 diabetes . similarly , neither baseline dietary folate intake nor intake of vitamin b6 or vitamin b12 modified the treatment effect . there was a significant reduction in risk of type 2 diabetes among women who had a family history of diabetes ( rr 0.77 [ 95% ci 0.600.99 ] ; p = 0.04 ) but not among those who had no such family history ( 1.09 [ 0.851.41 ] ; p = 0.49 ) , and the interaction was marginally significant ( p = 0.06 ) . however , these results from such subgroup analyses should be treated with caution , since they could be explained by chance alone due to multiple comparisons or imbalance of diabetes risk factors at baseline in small subgroups . finally , we did a separate analysis to assess whether the randomly assigned treatment with vitamin e , vitamin c , or -carotene may have modified the results ( table 3 ) . in total , there were 245 incident cases ( 11.5% ) in the active treatment group and 259 ( 12.2% ) in the placebo group ( 172.5/10,000 person - years vs. 184.8/10,000 person - years ) , corresponding to an overall rr of 0.94 ( 95% ci 0.791.11 ; p = 0.46 ) after controlling for age and antioxidant treatment assignments ( table 2 ) . figure 2 shows the cumulative incidence of type 2 diabetes events among women in the treatment and placebo groups by year of follow - up . there appeared to be a trend toward a modest reduction in risk of type 2 diabetes for the treatment group versus placebo group over the follow - up period , but the log - rank test for the overall difference was not statistically significant ( p for log - rank test = 0.44 ) ( fig . 2 ) . rrs of self - reported type 2 diabetes by randomized intervention ( active treatment versus placebo ) within subgroups in the wafacs . rrs of self - reported type 2 diabetes by homocysteine - lowering intervention according to other randomized treatment assignment groups in the wafacs * adjusted for age and two randomized assignments other than stratified groups . in this large , randomized , double - blind , placebo - controlled trial with 7.3 years of treatment among 4,252 women at high risk for cvd , we found no significant effect of homocysteine - lowering treatment by a combination pill of folic acid and vitamins b6 and b12 on risk of type 2 diabetes . there remained no evidence for a treatment effect in a sensitivity analysis restricted to women compliant with their study pills over the follow - up period . our study provides the first randomized trial data regarding the long - term effect of folic acid / b vitamin supplementation on the risk of type 2 diabetes , although our findings remain to be corroborated by future research . it has been hypothesized that b vitamins may help reduce the risk of type 2 diabetes by ameliorating metabolic abnormalities , such as oxidative damage , inflammation , and endothelial dysfunction , which characterize all phases of insulin resistance and pancreatic -cell function and are implicated in the development and progression of type 2 diabetes . due to their relative safety and low cost , b vitamin supplements have been targeted as potential therapeutic agents in previous randomized controlled trials for prevention of vascular diseases in high - risk populations . in contrast , direct evidence from randomized trials of b vitamin supplementation for type 2 diabetes has been very limited . some ( 2023 ) but not all ( 30 ) secondary prevention trials have suggested that folic acid supplementation may be effective in the improvement of oxidative stress ( 23 ) and endothelial dysfunction ( 2022 ) in patients with type 2 diabetes . to our knowledge , no large clinical trials have specifically examined homocysteine - lowering interventions on the primary prevention of type 2 diabetes . in the present study , we provide evidence that folic acid and b vitamins have a neutral effect on the risk of type 2 diabetes among nondiabetic women ; this is consistent with the absence of benefit from this intervention in lowering risk of cardiovascular events ( 31 ) . given the efficacy of the intervention in reducing homocysteine levels , our trial casts doubt on the etiologic role of hyperhomocysteinemia in the development of type 2 diabetes . a major concern has been raised about the limited effect of folic acid treatment in a folate - fortified population . to assess the changes in homocysteine levels in response to background folate fortification in the u.s . population and to the randomized treatment with folic acid and b vitamins in our trial , albert et al . ( 25 ) have conducted an analysis of baseline and follow - up folate and homocysteine levels in a randomly chosen subpopulation ( 150 in the active group and 150 in the placebo group ) of wafacs participants . the prefortification prevalence of hyperhomocysteinemia ( 15.0 mol / l ) in our population ( 27.7% ) was larger than the average prevalence estimates of increased homocysteine levels ( 13.0 mol / l ) for men and women ( 25% ) in the national health and nutrition examination survey ( 32 ) and in the framingham population ( 19% ) ( 33 ) . despite significant elevation in plasma folate levels due to mandatory folate fortification , homocysteine levels changed relatively little over a 7-year period . in contrast , our folic acid / b vitamin intervention lowered homocysteine levels by 18.5% ( 2.27 this reduction in homocysteine levels , however , was not associated with protection against the development of type 2 diabetes in this randomized trial . it is unknown whether a greater magnitude of homocysteine - lowering would have conferred protection against diabetes . genetic variations in enzymes involved in homocysteine metabolism may modulate the effect of treatment on risk of type 2 diabetes via their effects on circulating homocysteine levels ( 34 ) . interindividual genetic variability in our study population is an unlikely explanation for our null findings , however , because genetic factors should have been comparable in the active treatment and placebo groups by the randomization procedure . there may be subgroups of individuals with b vitamin deficiencies or genetic variants in the pathway of homocysteine metabolism that could particularly benefit from homocysteine - lowering therapy . future well - designed and large - scale genetic studies within randomized trial settings are warranted to test the hypothesis . of interest , our prespecified subgroup analyses showed a trend toward decreased risk associated with folic acid / vitamins b6 and b12 among women with a family history of diabetes , but these findings may have been due to chance and need to be confirmed in future investigations . some limitations of our trial also deserve consideration . first , declining compliance over time in the trial may have diluted the findings . in sensitivity analyses restricted to women compliant with their study pills , second , the use of a combination pill did not allow us to investigate the effects of individual components or potential interactions among them in relation to type 2 diabetes . third , misclassification in the self - reported diagnosis of type 2 diabetes is another concern . since the proportions of undiagnosed cases are likely to be similar in the treatment and placebo groups due to effective randomization and double - blinding strategies , such a misclassification is more likely to be nondifferential but could have attenuated the results . fourth , we did not measure homocysteine and folate levels in all participants to assess a modifying effect of baseline levels ; however , we found no evidence of benefits of b vitamin therapy among individuals with different levels of dietary folate , vitamin b6 , and vitamin b12 intakes at baseline . fifth , confounding by extraneous risk factors can not be completely excluded , although the baseline characteristics were well balanced in the treatment and placebo group , as expected in a large - scale trial with effective randomization . finally , our results based on women at high risk for cvd may not be generalizable to men or to the general population . in conclusion , in this study within a large , randomized , placebo - controlled trial among over 5,400 women at high risk for cvd , we observed no apparent benefit or harm of folic acid and vitamins b6 and b12 supplementation on the risk of type 2 diabetes over 7 years of treatment . our findings do not support recommending b vitamin supplements for diabetes prevention , although additional evidence from future large trials in populations with moderate - to - severe hyperhomocysteinemia or in regions without grain fortification will be needed .

objectivehomocysteinemia may play an etiologic role in the pathogenesis of type 2 diabetes by promoting oxidative stress , systemic inflammation , and endothelial dysfunction . we investigated whether homocysteine - lowering treatment by b vitamin supplementation prevents the risk of type 2 diabetes.research design and methodsthe women 's antioxidant and folic acid cardiovascular study ( wafacs ) , a randomized , double - blind , placebo - controlled trial of 5,442 female health professionals aged 40 years with a history of cardiovascular disease ( cvd ) or three or more cvd risk factors , included 4,252 women free of diabetes at baseline . participants were randomly assigned to either an active treatment group ( daily intake of a combination pill of 2.5 mg folic acid , 50 mg vitamin b6 , and 1 mg vitamin b12 ) or to the placebo group.resultsduring a median follow - up of 7.3 years , 504 women had an incident diagnosis of type 2 diabetes . overall , there was no significant difference between the active treatment group and the placebo group in diabetes risk ( relative risk 0.94 [ 95% ci 0.791.11 ] ; p = 0.46 ) , despite significant lowering of homocysteine levels . also , there was no evidence for effect modifications by baseline intakes of dietary folate , vitamin b6 , and vitamin b12 . in a sensitivity analysis , the null result remained for women compliant with their study pills ( 0.92 [ 0.761.10 ] ; p = 0.36).conclusionslowering homocysteine levels by daily supplementation with folic acid and vitamins b6 and b12 did not reduce the risk of developing type 2 diabetes among women at high risk for cvd .

RESEARCH DESIGN AND METHODS Follow-up procedures. Ascertainment of incident type 2 diabetes. Statistical analysis. RESULTS DISCUSSION

the wafacs is a randomized , double - blind , placebo - controlled trial evaluating the effects of a combination pill of folic acid ( 2.5 mg / day ) , vitamin b6 ( 50 mg / day ) , and vitamin b12 ( 1 mg / day ) in the secondary prevention of important vascular events among high - risk women with either a history of cvd or at least three cardiovascular risk factors . briefly , the wafacs began in 1998 , when the folic acid , vitamin b6 , and vitamin b12 component was added to the women 's antioxidant cardiovascular study ( wacs ) , an ongoing 222 factorial trial of three antioxidant vitamins ( vitamins c and e and -carotene ) , which expanded it to a four - group factorial trial . in the wacs parent trial , 8,171 female health professionals were randomized into the trial from june 1995 through october 1996 to receive vitamin c ( 500 mg / day ) , vitamin e ( 600 iu every other day ) , and -carotene ( 50 mg every other day ) versus respective matching placebos . women were eligible for wacs if they were at least 40 years old , postmenopausal or had no intention of becoming pregnant , and had a self - reported history of cvd ( including myocardial infarction , stroke , coronary revascularization , or angina ) or had at least three traditional cardiac risk factors ( 26 ) . in april 1998 , 5,442 of these women provided consent and were additionally randomized in a factorial design to receive a combination pill containing 2.5 mg folic acid , 50 mg vitamin b6 , and 1 mg vitamin b12 ( active treatment ) or a matching placebo daily . from the 5,442 women randomized in the wafacs trial , we excluded those with diabetes at baseline ( n = 1,190 ) for the present analyses , leaving 4,252 nondiabetic women at baseline ( fig . flow diagram illustrating diabetes outcomes in the randomly assigned treatment of folic acid and vitamins b6 and b12 of the wafacs . a total of 1,190 participants who had a diagnosis of diabetes at baseline were excluded in the analysis . study medications and end point ascertainment were continued in a blinded fashion until the scheduled end of the trial , 31 july 2005 , for a follow - up duration of 7.3 years . average adherence over the course of follow - up was 83% for active and placebo agents , with no significant difference between active and placebo groups . use of open - label folic acid supplements , vitamin b6 , or vitamin b12 supplements containing more than the recommended daily allowance for at least 4 days per month ranged from 2 to 11% in the active group to 2 to 13% in the placebo group over the course of the study . women in the wafacs provided a baseline blood sample in 1996 , prior to the initiation of background dietary folic acid fortification in the u.s . randomly selected from participants who were adherent with study medications , 300 ( 150 in the active treatment and 150 in the placebo group ) provided a blood sample at the end of randomized treatment . as reported previously ( 25 ) , median folate and homocysteine levels were similar between the active treatment group and the placebo group at baseline . at the end of study follow - up , the median folate level increased significantly in both groups ; however , the relative increase in folate level was greater in the active treatment group . despite significant increases in folate levels among the placebo group , there was no apparent reduction in homocysteine levels at the end of the study compared with participants measured at the beginning of the study in the placebo group ( p = 0.99 ) . in comparison , homocysteine levels were significantly reduced in the active treatment group ; the geometric mean homocysteine level was decreased by 18.5% ( 95% ci 12.524.1 ; p < 0.001 ) in the active group over that observed in the placebo group for a difference of 2.27 mol / l ( 1.542.96 ) from the placebo geometric mean homocysteine level of 12.28 diabetes status was evaluated at baseline , and all the participants were also asked annually whether and when they had been diagnosed with diabetes after randomization . women who reported a diagnosis of diabetes during the follow - up were mailed supplementary questionnaires to confirm their self - reported diagnoses . based on the american diabetes association diagnostic criteria ( 28 ) , actual glucose levels at fasting or oral glucose tolerance testing , diabetic symptoms , and/or hypoglycemic medication were combined together to confirm the self - reported incident cases of diabetes in a blinded fashion . kaplan - meier survival curves were used to estimate the overall cumulative incidence over time for the active vitamin group and the placebo group . we used cox proportional hazards models to calculate the estimates of hazard ratio expressed as relative risks ( rrs ) and 95% ci for randomized treatment versus placebo , after adjustment for age and other randomized treatments ( vitamin e , vitamin c , and -carotene ) . to examine the effect of actual as opposed to assigned folic acid / b vitamin treatment , we carried out a sensitivity analysis according to compliance . subgroup analyses were conducted to examine the effect of active treatment on risk of type 2 diabetes according to prespecified risk factors for type 2 diabetes at baseline , including age - groups ( 4554 , 5564 , and 65 years ) , bmi ( kg / m ) , smoking status ( current , past , or never ) , alcohol use ( never / rarely or one or more drinks per month ) , family history of diabetes ( yes or no ) , physical activity ( estimated energy expenditure from leisure activities of < 1,000 or 1,000 kcal per week ) , menopausal status and hormone therapy ( uncertain menopausal status , premenopausal , or postmenopausal including current , past , or never users of hormone therapy ) , history of hypertension ( yes or no ) , history of hyperlipidemia ( yes or no ) , and baseline dietary intakes of folate , vitamin b6 , or vitamin b12 ( tertiles for each ) . all analyses were conducted with sas version 9 ( sas institute , cary , nc ) , and a two - sided test with a significance level of = 0.05 ( p < 0.05 ) was used . study medications and end point ascertainment were continued in a blinded fashion until the scheduled end of the trial , 31 july 2005 , for a follow - up duration of 7.3 years . average adherence over the course of follow - up was 83% for active and placebo agents , with no significant difference between active and placebo groups . use of open - label folic acid supplements , vitamin b6 , or vitamin b12 supplements containing more than the recommended daily allowance for at least 4 days per month ranged from 2 to 11% in the active group to 2 to 13% in the placebo group over the course of the study . women in the wafacs provided a baseline blood sample in 1996 , prior to the initiation of background dietary folic acid fortification in the u.s . randomly selected from participants who were adherent with study medications , 300 ( 150 in the active treatment and 150 in the placebo group ) provided a blood sample at the end of randomized treatment . as reported previously ( 25 ) , median folate and homocysteine levels were similar between the active treatment group and the placebo group at baseline . at the end of study follow - up , the median folate level increased significantly in both groups ; however , the relative increase in folate level was greater in the active treatment group . despite significant increases in folate levels among the placebo group , there was no apparent reduction in homocysteine levels at the end of the study compared with participants measured at the beginning of the study in the placebo group ( p = 0.99 ) . in comparison , homocysteine levels were significantly reduced in the active treatment group ; the geometric mean homocysteine level was decreased by 18.5% ( 95% ci 12.524.1 ; p < 0.001 ) in the active group over that observed in the placebo group for a difference of 2.27 mol / l ( 1.542.96 ) from the placebo geometric mean homocysteine level of 12.28 diabetes status was evaluated at baseline , and all the participants were also asked annually whether and when they had been diagnosed with diabetes after randomization . women who reported a diagnosis of diabetes during the follow - up were mailed supplementary questionnaires to confirm their self - reported diagnoses . based on the american diabetes association diagnostic criteria ( 28 ) , actual glucose levels at fasting or oral glucose tolerance testing , diabetic symptoms , and/or hypoglycemic medication were combined together to confirm the self - reported incident cases of diabetes in a blinded fashion . kaplan - meier survival curves were used to estimate the overall cumulative incidence over time for the active vitamin group and the placebo group . to examine the effect of actual as opposed to assigned folic acid / b vitamin treatment , we carried out a sensitivity analysis according to compliance . subgroup analyses were conducted to examine the effect of active treatment on risk of type 2 diabetes according to prespecified risk factors for type 2 diabetes at baseline , including age - groups ( 4554 , 5564 , and 65 years ) , bmi ( kg / m ) , smoking status ( current , past , or never ) , alcohol use ( never / rarely or one or more drinks per month ) , family history of diabetes ( yes or no ) , physical activity ( estimated energy expenditure from leisure activities of < 1,000 or 1,000 kcal per week ) , menopausal status and hormone therapy ( uncertain menopausal status , premenopausal , or postmenopausal including current , past , or never users of hormone therapy ) , history of hypertension ( yes or no ) , history of hyperlipidemia ( yes or no ) , and baseline dietary intakes of folate , vitamin b6 , or vitamin b12 ( tertiles for each ) . all analyses were conducted with sas version 9 ( sas institute , cary , nc ) , and a two - sided test with a significance level of = 0.05 ( p < 0.05 ) was used . during a median follow - up period of 7.3 years , 504 women were diagnosed with type 2 diabetes among 4,252 nondiabetic participants who underwent randomization in the wafacs . there were no statistically significant differences in baseline characteristics between the folic acid / b vitamin active treatment group and its corresponding placebo group ( table 1 ) . overall , there was no significant effect of folic acid / b vitamins on the development of type 2 diabetes compared with the placebo group ( table 2 and fig . in total , there were 245 incident cases ( 11.5% ) in the active treatment group and 259 ( 12.2% ) in the placebo group ( 172.5/10,000 person - years vs. 184.8/10,000 person - years ) , corresponding to an overall rr of 0.94 ( 95% ci 0.791.11 ; p = 0.46 ) after controlling for age and antioxidant treatment assignments ( table 2 ) . figure 2 shows the cumulative incidence of type 2 diabetes events among women in the treatment and placebo groups by year of follow - up . there appeared to be a trend toward a modest reduction in risk of type 2 diabetes for the treatment group versus placebo group over the follow - up period , but the log - rank test for the overall difference was not statistically significant ( p for log - rank test = 0.44 ) ( fig . rrs of self - reported type 2 diabetes by randomized folic acid and b vitamins intervention in the wafacs * adjusted for age and other randomized assignments ( vitamins c and e and -carotene ) . additionally adjusted for baseline variables , including bmi , smoking status , postmenopausal hormone use , multivitamin use , alcohol intake , coffee intake , physical activity , and family history of diabetes . cumulative incidence of self - reported type 2 diabetes by randomized treatment assignment ( active treatment versus placebo ) in the wafacs . in sensitivity analyses to minimize potential bias due to the inclusion of undiagnosed diabetes at baseline , folic acid / b vitamin treatment was not significantly associated with risk of type 2 diabetes when excluding those cases that occurred in the first 2 years ( rr 0.98 [ 95% ci 0.801.21 ] ; p = 0.87 ) . in a separate analysis where women were censored at the time they stopped taking at least two - thirds of their study pills or started to use outside folic acid / b vitamin supplements on 4 days per month , the results were unchanged after further adjustment for diabetes risk factors including bmi , physical activity , family history of diabetes , smoking , postmenopausal hormone use , multivitamin use , alcohol intake , and coffee consumption . to determine whether certain subgroups of women were at particularly high or low risk for type 2 diabetes with folic acid / b vitamin treatment , we conducted multiple subgroup analyses stratified by several prespecified diabetes risk factors ( fig . overall , there was no evidence that any of these factors modified the treatment effect on the risk of type 2 diabetes . similarly , neither baseline dietary folate intake nor intake of vitamin b6 or vitamin b12 modified the treatment effect . there was a significant reduction in risk of type 2 diabetes among women who had a family history of diabetes ( rr 0.77 [ 95% ci 0.600.99 ] ; p = 0.04 ) but not among those who had no such family history ( 1.09 [ 0.851.41 ] ; p = 0.49 ) , and the interaction was marginally significant ( p = 0.06 ) . however , these results from such subgroup analyses should be treated with caution , since they could be explained by chance alone due to multiple comparisons or imbalance of diabetes risk factors at baseline in small subgroups . in total , there were 245 incident cases ( 11.5% ) in the active treatment group and 259 ( 12.2% ) in the placebo group ( 172.5/10,000 person - years vs. 184.8/10,000 person - years ) , corresponding to an overall rr of 0.94 ( 95% ci 0.791.11 ; p = 0.46 ) after controlling for age and antioxidant treatment assignments ( table 2 ) . figure 2 shows the cumulative incidence of type 2 diabetes events among women in the treatment and placebo groups by year of follow - up . there appeared to be a trend toward a modest reduction in risk of type 2 diabetes for the treatment group versus placebo group over the follow - up period , but the log - rank test for the overall difference was not statistically significant ( p for log - rank test = 0.44 ) ( fig . rrs of self - reported type 2 diabetes by randomized intervention ( active treatment versus placebo ) within subgroups in the wafacs . rrs of self - reported type 2 diabetes by homocysteine - lowering intervention according to other randomized treatment assignment groups in the wafacs * adjusted for age and two randomized assignments other than stratified groups . in this large , randomized , double - blind , placebo - controlled trial with 7.3 years of treatment among 4,252 women at high risk for cvd , we found no significant effect of homocysteine - lowering treatment by a combination pill of folic acid and vitamins b6 and b12 on risk of type 2 diabetes . there remained no evidence for a treatment effect in a sensitivity analysis restricted to women compliant with their study pills over the follow - up period . our study provides the first randomized trial data regarding the long - term effect of folic acid / b vitamin supplementation on the risk of type 2 diabetes , although our findings remain to be corroborated by future research . it has been hypothesized that b vitamins may help reduce the risk of type 2 diabetes by ameliorating metabolic abnormalities , such as oxidative damage , inflammation , and endothelial dysfunction , which characterize all phases of insulin resistance and pancreatic -cell function and are implicated in the development and progression of type 2 diabetes . in contrast , direct evidence from randomized trials of b vitamin supplementation for type 2 diabetes has been very limited . some ( 2023 ) but not all ( 30 ) secondary prevention trials have suggested that folic acid supplementation may be effective in the improvement of oxidative stress ( 23 ) and endothelial dysfunction ( 2022 ) in patients with type 2 diabetes . to our knowledge , no large clinical trials have specifically examined homocysteine - lowering interventions on the primary prevention of type 2 diabetes . in the present study , we provide evidence that folic acid and b vitamins have a neutral effect on the risk of type 2 diabetes among nondiabetic women ; this is consistent with the absence of benefit from this intervention in lowering risk of cardiovascular events ( 31 ) . given the efficacy of the intervention in reducing homocysteine levels , our trial casts doubt on the etiologic role of hyperhomocysteinemia in the development of type 2 diabetes . population and to the randomized treatment with folic acid and b vitamins in our trial , albert et al . ( 25 ) have conducted an analysis of baseline and follow - up folate and homocysteine levels in a randomly chosen subpopulation ( 150 in the active group and 150 in the placebo group ) of wafacs participants . in contrast , our folic acid / b vitamin intervention lowered homocysteine levels by 18.5% ( 2.27 this reduction in homocysteine levels , however , was not associated with protection against the development of type 2 diabetes in this randomized trial . it is unknown whether a greater magnitude of homocysteine - lowering would have conferred protection against diabetes . genetic variations in enzymes involved in homocysteine metabolism may modulate the effect of treatment on risk of type 2 diabetes via their effects on circulating homocysteine levels ( 34 ) . interindividual genetic variability in our study population is an unlikely explanation for our null findings , however , because genetic factors should have been comparable in the active treatment and placebo groups by the randomization procedure . there may be subgroups of individuals with b vitamin deficiencies or genetic variants in the pathway of homocysteine metabolism that could particularly benefit from homocysteine - lowering therapy . of interest , our prespecified subgroup analyses showed a trend toward decreased risk associated with folic acid / vitamins b6 and b12 among women with a family history of diabetes , but these findings may have been due to chance and need to be confirmed in future investigations . in sensitivity analyses restricted to women compliant with their study pills , second , the use of a combination pill did not allow us to investigate the effects of individual components or potential interactions among them in relation to type 2 diabetes . third , misclassification in the self - reported diagnosis of type 2 diabetes is another concern . fourth , we did not measure homocysteine and folate levels in all participants to assess a modifying effect of baseline levels ; however , we found no evidence of benefits of b vitamin therapy among individuals with different levels of dietary folate , vitamin b6 , and vitamin b12 intakes at baseline . fifth , confounding by extraneous risk factors can not be completely excluded , although the baseline characteristics were well balanced in the treatment and placebo group , as expected in a large - scale trial with effective randomization . finally , our results based on women at high risk for cvd may not be generalizable to men or to the general population . in conclusion , in this study within a large , randomized , placebo - controlled trial among over 5,400 women at high risk for cvd , we observed no apparent benefit or harm of folic acid and vitamins b6 and b12 supplementation on the risk of type 2 diabetes over 7 years of treatment .

diabetes mellitus ( dm ) is a metabolic and multifactorial syndrome with disordered metabolism and hyperglycemia . on the etiological basis , factors which contributed to the dm and hyperglycemia are reduced secretion of insulin , inherited or acquired insulin deficiency , ineffectiveness of insulin , and low glucose utilization with high production of glucose . some risk factors which contributed to long - term complications for dm are diet , overweight , obesity , smoking , alcohol , level of physical activity , hormones , some medical treatments , viruses , vascular or cardiovascular disease , atherosclerosis , heart conditions , stroke , kidney disease , eye diseases , nerve damage , impaired thinking , infections and wounds , cancer , musculoskeletal disorders , pregnancy complications , emotional difficulties , insulin shock , diabetic ketoacidosis , and hyperosmolar hyperglycemic nonketotic state . people can lower the controllable risk factors like exercise and diet through improved health habits and can reduce their risk of developing diabetes [ 1 , 2 ] . there are several types of dm which exist and are caused by genetic or environmental factors and lifestyle choices . type 1 dm is juvenile diabetes and is called as insulin dependent diabetes mellitus ( iddm ) in which the pancreas fails to produce insulin due to autoimmune beta - cell destruction . it is usually diagnosed in young age like in children , adolescents , and young adults . type 2 dm is adult - onset diabetes and is called as noninsulin dependent diabetes mellitus ( niddm ) which results from the body tissues and cells inability to respond properly to the action of insulin . there are some other abnormalities like genetic and metabolic abnormalities which are produced in response to insulin action and secretion . type 2 dm usually occurs in adulthood and develops more with the age and sometimes it is also observed in children and some adolescents having obesity . there are some other types of dm in which specific genetic defects , metabolic and mitochondrial abnormalities , and some conditions that impair glucose tolerance are included [ 1 , 2 ] . diabetes is one of the most widely occurring human ailments and the world wide prevalence has risen over the past two decades . according to new publications of some health agencies like world health organization ( who ) and international diabetes federation ( idf ) , diabetes becomes an epidemic which is not controlled like other major diseases , for example , cancer and cardiovascular diseases , and becomes sixth leading cause of death worldwide . type 2 dm is much more common than type 1 dm and makes up about 90% of all cases of diabetes . the incidence of this disease in any developed or developing country is difficult to judge . it is quite obvious that the disease is multiplying geometrically more due to genetic and environmental factors . it has been used singly and in the compound form as a member of b complex family . it has important role in carbohydrates and fat metabolism and is essential for normal growth and development of the human body . it occurs as a part of our diet and is present in some diets like cereals , fortified bread , rice , nuts , meats , and legumes . recommended intake of thiamine for men is 1.4 mg / day , for woman is 1.1 mg / day , for pregnant women is 1.5 mg / day , and for breast feeding is 1.6 mg / day . 100 g corn flakes or 3 - 4 dl soya milk or 300 g ham covers the daily need . its deficiency results in a disease called beri beri in which cardiovascular , neurological , and dermatological complications arise . thiamine deficiency was treated with 50100 mg of thiamine per day for several days followed by 510 mg of oral thiamin per day which was given in parenteral . treatment is successful in about 50% of patients and replacement of other vitamins as needed . however aside of this restricted use and as a general tonic , it has never been administered as therapy for the diseases for many years . biomarker is a substance which is used as an indicator for pathological state of disease and a characteristic that is estimated and evaluated for normal , pathological , and pharmacologic responses to a therapeutic intervention . biomarkers can be used in laboratory for drug discovery , diagnosing , classification , and grading the severity of disease in both laboratory and clinical settings . biomarkers can also be used for the identification , characterization , and expression of the proteins for biological systems . there are some variations in the patterns of protein expression in normal healthy controls as compared with diseased person . detection of these differences in protein levels is an important area of research in the field of proteomic study . some of the disease areas of protein biomarkers are , for example , cancer , diabetes , and cardiovascular and neurological diseases . the protein biomarkers are very useful for diagnosis and prognosis of acute and chronic type of mortality in patients having diabetes , various forms of cancer , and other syndromes ( graves and haystead , 2002 ) . to identify the protein biomarkers , the proteomic approaches were used that can be exploited for potential diagnostic and prognostic biomarkers in the short and long term of different diseases . proteomics is the large - scale study of protein with their structures , functions , and information coded in a cell which is expressed and regulated at the protein level to achieve the function of an organism . clinical proteomics aims to discover protein biomarkers which may be a potential target for pharmaceutical field , disease diagnosis , and risk assessment . the goals of proteomics are to apply the proteomic technologies in the clinical research , public health , and environmental , agricultural , and veterinary research . there has been a great interest in the proteomic analysis of plasma and serum for the identification and characterization of protein biomarkers of different diseases . in the modern era for the protein identification , identification and characterization of low molecular weight proteins in the human plasma / serum and protein profile of human urine have been done by the proteomics . there are many conventional and advanced proteomic technologies that separate the proteins or peptides prior to mass spectrometry ( ms ) analysis . ever since o ' ferral introduced the high resolution two - dimensional gel electrophoresis system in 1975 , which became the most commonly used technique to analyze protein components of complex mixtures like bacterial extracts and blood plasma . in many ways , the two - dimensional gel electrophoresis was commonly used as a first step for protein identification by mass spectrometry . this approach has made it possible to carry out global protein analysis of living organisms that helps in examining the proteome . despite considerable reproducibility , this technique could not gain the attention of researchers mainly due to the limitations of this being a labor intensive and qualitative method . advanced technology in the proteomic filed for the protein separation is the commercial instrument proteomelab pf2d from beckman coulter . this is the 2d liquid chromatography system ( 2d - lc ) which separates proteins in the first dimension ( chromatographic focusing ) on the basis of isoelectric points ( pi ) of protein followed by the second dimension that is reversed phase high performance liquid chromatography ( rp - hplc ) , where proteins are further fractionated on the basis of hydrophobicity . the assessment , optimization , and separation from the biofluids like plasma , serum , urine , cerebrospinal fluid , and saliva have been observed by the pf2d . the identification and characterization of protein biomarkers have been achieved by the proteomics coupled with mass spectrometry ( ms ) analysis . ms technology is highly sensitive and is the most important developments in identification of the proteins . for the detection and characterization of various biomolecules like proteins , peptides , oligosaccharides , and oligonucleotides , the ms technology was used with the molecular mass range between 400 and 350,000 dalton . it detects very low quantities of sample up to 10 to 10 mole with an accuracy of 0.10.01% . matrix assistant laser desorption ionization / time of flight ( maldi - tof ms ) is relatively simple to operate . it has good mass accuracy with increased resolution and high sensitivity and is used in the field of proteomics very widely to identify and characterize the protein biomarkers through peptide mass fingerprinting that can be helpful to identify proteins involved in global diseases like diabetes and others . in the method of ms , selected proteins of choice were digested with a specific proteolytic enzyme and the resulting peptides were analyzed for further studies . the information obtained from this can be used for identification and confirmation of the proteins by searching different protein databases . the process of identification and characterization of disease protein biomarkers was summarized and shown in figure 1 . so , this disease takes an ever - increasing proportion of national health care budgets . without primary prevention there is also a need of introducing cost - effective treatment strategies to control this epidemic . in the following sections , identification , purification , characterization of protein biomarkers for early diagnosis of pathological states like diabetes mellitus , and role of thiamine on the levels of these marker proteins in diabetes type 2 were described . research work has been done for the search of protein biomarkers for monitoring and predicting the diabetes mellitus . several modern techniques mainly involving protein characterization by mass spectrometry and proteomic profiling of plasma / serum samples by proteomics have also been described . animal studies with high dose thiamine therapy have been shown to reduce diabetic nephropathy ( microalbuminuria ) and lipid disturbances . therefore a clinical trial of high dose thiamine therapy has been planned in our type 2 diabetic pakistani population with a hope to find protein biomarker and role of high dose thiamine on the levels of these biomarkers in the patients having diabetes type 2 . . this can cause severe acute and chronic complications like forming brain damage to amputations and heart disease . dysregulation of multiple glucoregulatory hormones ( insulin and glucagons ) that maintain glucose homeostasis result in the form of diabetes . the imbalances in these hormones lead to long term elevated levels of glucose and some of microvascular and macrovascular complications like retinopathy , nephropathy , and neuropathy . the blood sugar at normal levels in the human body is maintained and brought by a regulatory mechanism which is very effective and efficient . in this mechanism , the main organs are liver , autonomic nervous system , and certain glands of internal secretion called endocrine glands . to maintain the homeostasis the important and dominant tissues are liver which respond to the signals that indicate low or high levels of blood glucose . the vital function of liver is to produce glucose for the circulation in the blood . both low and high levels of blood glucose triggered the hormonal responses which initiate the pathways to restore the glucose homeostasis . reduced levels of blood glucose trigger release of glucagons from the beta - cells of pancreas , while the increased levels of blood glucose trigger release of insulin from the pancreatic beta - cells . dm results from the dysregulation of multiple glucoregulatory hormones ( insulin and glucagons ) . any defect in glucoregulatory hormones production leads to improper regulation of glucose in the blood and results in diabetes this is the glucose homeostasis in which blood glucose levels maintained in the body and dysregulation cause hyper- and hypoglycemia as shown in figure 2 . insulin is a peptide hormone synthesized as precursor of polypeptide preproinsulin with single chain 86 amino acids and is secreted by the beta - cells of the islets of langerhans of pancreatic cells . it facilitates the glucose uptake in the cells , regulates the carbohydrate , lipid , and protein metabolisms , and promotes the cell division and growth . beta - cells of pancreases secrete 0.251.5 units of insulin per hour during the state of fasting . any defect in insulin production leads to improper regulation of glucose in the blood and results in diabetes . similarly , postprandial glucagon secretion is abnormally increased in the patients with type 1 and type 2 diabetes . it has also played important role in postprandial hyperglycemia in the patients with diabetes mellitus . there are different types of glucose transporters called glut1 , glut2 , glut3 , and glut4 . the glucose metabolism in the beta - cells changes the ion channel activity which leads to secretion of insulin . the sulfonylurea receptor ( sur ) is the binding site for the drugs that act as secretagogues for the hormone insulin . glucose stimulation of insulin and its transport also initiate by the glut2 into the beta - cell . the metabolism of glucose-6-phosphate via glycolysis produces the atp which inhibits the activity of an atp - sensitive potassium channel . inhibition of this channel initiates the beta - cell membrane depolarization and opens calcium channels that stimulate insulin secretion as shown in figure 3 . dearrangement in the normal secretary patterns is one of the earliest signs of beta - cell dysfunction in diabetes . low insulin levels increase the production of glucose by promoting the hepatic gluconeogenesis and glycogenolysis in the fasting state . glucagons stimulate the glycogenolysis and gluconeogenesis by the liver and the renal medulla . reduced insulin levels declare the glycogen synthesis and low glucose uptake in insulin sensitive tissues . postprandially , glucose load elicits an increase in insulin and decrease in glucagons leading to a reversal of these processes . the main potion of postprandial glucose is utilized by skeletal muscle and insulin stimulated glucose uptake . type 2 dm is a heterogeneous group of disorders and is characterized by the resistance and impaired secretion of insulin and high levels of glucose production . there are some metabolic and genetic defects in the action or secretion of insulin which give rise to hyperglycemia in type 2 dm . obesity is very common in type 2 diabetes where the adipocytes cells secret a number of biological proteins ( leptin , tnf - alpha , resistin , and adiponectin ) that modulate insulin secretion or action and may contribute to insulin resistance . the pancreatic islets are unable to sustain the hyperinsulinemia state in the progression of insulin resistance and hyperinsulinemia in certain individuals . reduced insulin secretion and increased hepatic glucose production lead to hyperglycemia in fasting diabetes . ultimately the beta - cell failure may increase the release of inflammatory protein markers such as interleukin-6 ( il-6 ) and c - reactive protein ( crp ) in type 2 dm . the molecular mechanism of insulin resistance can be explained briefly as levels of insulin receptors and tyrosine kinase activity in skeletal muscles are reduced . polymorphism in insulin receptor substrates ( irs ) may also be linked with intolerance of glucose , which raised the possibility that polymorphism in different postreceptors molecules combined and created a state of insulin resistance . the pathogenesis of insulin resistance is focused on signaling defect of a phosphatidylinositol-3-kinase ( pi-3 kinase ) . among other abnormalities many docking proteins bind to these proteins and stimulated the metabolic action of insulin and pi-3-kinase pathway . as a result , insulin gives rise to elevation in glucose transport via pi-3-kinase pathway and stimulates the translocation of intracellular vesicles which has glucose transporters as glut4 to the plasma membrane as shown in figure 4 . there are some pathways of insulin signal transduction which are not resistant to effect of insulin like the cell growth / differentiation and mitogenic activated protein ( map ) kinase pathways . as a result , hyperinsulinemia elevated the insulin action through these pathways and accelerated the diabetes and other conditions like atherosclerosis . it also promotes the production of glucose by the liver and impairs the function of beta - cells in the case of obese type 2 dm . there are varying reports on the role of protein biomarker for the identification of different diseases like cancer , diabetes , and others . there are some proteins which may be up- and downregulated in the serum / plasma and urine of diabetes mellitus especially in type 2 . the protein biomarkers are very helpful for predicting long - term mortality in patients with diabetes , cancer , and coronary syndromes . searching for novel biomarkers can be done using tissues and/or biofluids ( blood , serum , plasma , and urine ) . the urine is an ideal biofluid for biomarker discovery in kidney diseases and diabetes mellitus . urine samples obtained from patients with other diseases or disorders that have clinical , biochemical , and metabolic profiles similar to those of the disease of interest must be included as the other controls . recently , thongboonkerd has extensively applied proteomics to biomarker discovery in several diseases with the hope of finding novel biomarkers for earliest diagnosis of the diseases at their very beginning phase and for prediction of therapeutic response , survival , and recurrence . a two - dimensional liquid phase chromatographic separation followed by the mass spectrometry method is involved for proteomics studies and biomarker identification of different diseases . proteomic analyses using prefractionation strategies were used to identify the biomarker for early detection , diagnosis , prognosis , tumor responses , and disease recurrence for predicting and monitoring biological markers . proteome analysis of human serum proteins and efficient prefractionation of low abundance proteins in human plasma were done . the construction of two - dimensional map was demonstrated which displayed nearly 3700 chromatographically separated proteins which included 235 more distinct proteins . complex protein mixture analysis by an all - liquid - phase 2d mapping technique has been done by proteomelab pf 2d that is a novel protein profiling and mapping technology . it is also a new powerful way to analyze complex protein mixture in tissues and cells . detection of electrophoretically derived protein mass based on ms / ms was used as additional constraints in proteomic analysis of human serum . the proteomics is a valid approach to screen for novel protein biomarkers in animal and human models of obesity and type 2 diabetes . proteins are reported as biomarkers in the biofluids , tissues , and cells especially in type 2 dm . one of them is apolipoprotein a1 ( apoa - i ) , which is the main component of high density lipoproteins ( hdl ) present in plasma . in case of excretion , hdl promotes cholesterol efflux from tissues to the liver for excretion ( yui et al . , 1998 ) . the level of apoa-1 is positively correlated with hdl cholesterol in the serum and negatively correlated with low density lipoproteins ( ldl ) cholesterol . have studied the apolipoprotein a - ii ( apoa - ii ) that is in forms of 20% of hdl cholesterol and in human it is present about two - thirds of hdl in humans . apolipoprotein h ( apo - h ) is also called 2-glycoprotein i. it is a plasma glycoprotein present in circulating and free protein or associated with lipoproteins . it has an important role in blood coagulation and clearance of apoptotic bodies from the circulation . the apoa - i - ciii - aiv levels in type 2 dm and coronary heart disease were studied . it is also called bad cholesterol and is responsible for carrying cholesterol to tissues . in 2002 , bach - ngohou and coworkers studied the apolipoprotein e ( apo - e ) which is important for the normal catabolism of triglyceride - rich lipoproteins . the serum levels of some protein biomarkers like clusterin and apolipoprotein j were elevated significantly in type 2 dm and during development of coronary heart disease or at myocardial infarction . observed the diabetic macular edema after proteomic analysis of vitreous and recorded six proteins including pigment epithelium derived factor ( pedf ) , apoa-4 , apoa-1 , thyroid hormone receptor interactor 11 ( trip-11 ) , retinol binding protein 4 ( rbp4 ) , and vitamin d binding protein ( vdbp ) and only apo h is present in nondiabetic controls . borth in 1992 studied the alpha 2-macroglobulin ( 2-m ) which is an inhibitor for the proteinase enzyme in the blood and tissue . it acts as a binding protein for numerous cytokines and growth factors and also acts as a leptin - binding protein in human plasma . measurement of levels of protein human 1-microglobulin ( 1 m ) was studied in the serum and urine of patients with various liver diseases . transthyretin and its miracle function and pathogenesis were also observed in one recent research work . in addition , protein biomarker for diabetic nephropathy such as microalbuminuria is also a known predictor for the coronary heart and peripheral vascular diseases and high risk of mortality in patients of type 2 dm . in 1989 , semenkovich and his coworkers observed that the lipoprotein lipase is an important enzyme for lipid homeostasis in humans . lipoprotein lipase is the primary enzyme that converts lipoprotein triglyceride ( tg ) to free fatty acids . tumor necrosis factor- ( tnf- ) is a pleiotropic cytokine and has role in immunity and inflammation and during chronic illness . this obesity - related hormone is a molecule which regulates the energy balance and body weight . c - reactive protein ( crp ) is a protein of acute phase condition and a strong biomarker of inflammation in the progression of various diseases like coronary heart disease , cancer , diabetes , and others . genetic variation and levels of crp and incidence of diabetes especially risk of developing type 2 diabetes mellitus have been studies . the concentration of crp in the blood of normal healthy control human beings ranges from 0 to 1.0 mg / dl . in the acute phase condition of inflammation lindsay and coworkers demonstrated that some of the proteins are downregulated in dm likeone of the adipokines adiponectin in indian population . studied that some protein biomarkers of diabetes are associated in young diabetic patients with oxidative stress and antioxidant status . proteomic analysis of proteins was done and involved in insulin resistance and type 2 dm with the b3-adrenergic receptor . a number of proteins , including mitochondrial atp synthase , desmin , actin , and myosin , are found carbonylated . 2002 have described that the elevated levels of acute - phase proteins and plasminogen activator inhibitor-1 ( pai-1 ) in type 2 diabetes with insulin resistance were observed . in type 2 dm , the intramuscular heat shock protein 72 and heme oxygenase-1 mrna were decreased . in literature , diabetes is associated with pancreatic cancer in 80% cases . tumor - derived peptide with an s - calcium binding protein is involved in the pancreatic cancer associated with diabetes which are the studies of bassoa and coworkers . recently , obesity and its related proteins like leptin were studied in the diabetic population of pakistan . list of identified proteins in human serum of patients having diabetes mellitus is given in table 1 . urinary protein profiling can reveal changes in excretion rates of specific proteins that can have predictive value in the clinical arena , for example , in the early diagnosis of disease , classification of disease with regard to likely therapeutic responses , assessment of prognosis , and monitoring response to therapy . yang et al . in 2005 have described that rbp4 protein is an important biomarker for insulin resistance in diabetes . in one such study which was carried out by metz and coworkers , the five protein biomarkers were identified as 2-fold upregulated in diabetes which were alpha-2-glycoprotein ( zinc ) , corticosteroid - binding globulin , and lumican . the clusterin and serotransferrin were 2-fold downregulated in diabetic samples compared to control . for urinary biomarkers in diabetic nephropathy , proteomic analyses identified some potential protein biomarkers . list of urinary proteins that are identified in diabetic patients with or without nephropathy are listed in table 2 . in 2007 , rao et al . have observed that there were seven proteins which were upregulated with increasing albuminuria and four proteins were downregulated . after some years , the proteomic analysis was done for the identification of human salivary biomarkers for type-2 diabetes . recently , in 2009 , jiang and his coworkers studied the identification of urinary soluble e - cadherin as a novel biomarker for diabetic nephropathy . observed that haptoglobins are a group of serum proteins ; originally identified and diagnostic values were determined . the goals of present research work were the identification and characterization of protein biomarkers for early diagnosis and prognosis of pathological states of diabetes mellitus type 2 and effects of high dose thiamine on the levels of marker proteins . these investigations shall be helpful in assessing the biochemical alterations in the pakistani diabetic population , which should contribute to the development of treatment plans for this disease which is one of the most widely occurring and debilitating diseases . results from this research will also contribute to the identification of protein markers of diabetes mellitus type 2 and thus development of novel diagnostic procedures for early detection of this complication in potential diabetics in our population . the findings from present research work will assist in planning preventive and effective treatment strategies for diabetic patients by identification of protein biomarkers and high dose thiamine as nutritional supplement .

in the present research work , the levels of protein biomarkers specific to diabetes mellitus type 2 in the pakistani population using proteomic technology have been identified and characterized and effect of high dose thiamine has been seen on the levels of these marker proteins . diabetic patients and normal healthy controls were recruited from the sheikh zayed hospital , lahore , pakistan . total biochemical assays and proteins were estimated by modern proteomic techniques . some proteins were up- and downregulated in diabetic samples as compared to control and decreased after thiamine therapy , while other protein markers did not show a significant change after the thiamine therapy . the effect of high dose thiamine on the levels of these identified protein biomarkers in the human urine has also been observed . assessment of the levels of these biomarkers will be helpful in not only early diagnosis but also prognosis of diabetes mellitus type 2 .

1. Introduction 2. Review of the Literature

diabetes mellitus ( dm ) is a metabolic and multifactorial syndrome with disordered metabolism and hyperglycemia . on the etiological basis , factors which contributed to the dm and hyperglycemia are reduced secretion of insulin , inherited or acquired insulin deficiency , ineffectiveness of insulin , and low glucose utilization with high production of glucose . type 1 dm is juvenile diabetes and is called as insulin dependent diabetes mellitus ( iddm ) in which the pancreas fails to produce insulin due to autoimmune beta - cell destruction . type 2 dm is adult - onset diabetes and is called as noninsulin dependent diabetes mellitus ( niddm ) which results from the body tissues and cells inability to respond properly to the action of insulin . there are some other abnormalities like genetic and metabolic abnormalities which are produced in response to insulin action and secretion . type 2 dm usually occurs in adulthood and develops more with the age and sometimes it is also observed in children and some adolescents having obesity . diabetes is one of the most widely occurring human ailments and the world wide prevalence has risen over the past two decades . according to new publications of some health agencies like world health organization ( who ) and international diabetes federation ( idf ) , diabetes becomes an epidemic which is not controlled like other major diseases , for example , cancer and cardiovascular diseases , and becomes sixth leading cause of death worldwide . type 2 dm is much more common than type 1 dm and makes up about 90% of all cases of diabetes . it is quite obvious that the disease is multiplying geometrically more due to genetic and environmental factors . it has been used singly and in the compound form as a member of b complex family . it has important role in carbohydrates and fat metabolism and is essential for normal growth and development of the human body . recommended intake of thiamine for men is 1.4 mg / day , for woman is 1.1 mg / day , for pregnant women is 1.5 mg / day , and for breast feeding is 1.6 mg / day . treatment is successful in about 50% of patients and replacement of other vitamins as needed . biomarker is a substance which is used as an indicator for pathological state of disease and a characteristic that is estimated and evaluated for normal , pathological , and pharmacologic responses to a therapeutic intervention . biomarkers can be used in laboratory for drug discovery , diagnosing , classification , and grading the severity of disease in both laboratory and clinical settings . biomarkers can also be used for the identification , characterization , and expression of the proteins for biological systems . there are some variations in the patterns of protein expression in normal healthy controls as compared with diseased person . detection of these differences in protein levels is an important area of research in the field of proteomic study . some of the disease areas of protein biomarkers are , for example , cancer , diabetes , and cardiovascular and neurological diseases . the protein biomarkers are very useful for diagnosis and prognosis of acute and chronic type of mortality in patients having diabetes , various forms of cancer , and other syndromes ( graves and haystead , 2002 ) . to identify the protein biomarkers , the proteomic approaches were used that can be exploited for potential diagnostic and prognostic biomarkers in the short and long term of different diseases . proteomics is the large - scale study of protein with their structures , functions , and information coded in a cell which is expressed and regulated at the protein level to achieve the function of an organism . clinical proteomics aims to discover protein biomarkers which may be a potential target for pharmaceutical field , disease diagnosis , and risk assessment . the goals of proteomics are to apply the proteomic technologies in the clinical research , public health , and environmental , agricultural , and veterinary research . there has been a great interest in the proteomic analysis of plasma and serum for the identification and characterization of protein biomarkers of different diseases . in the modern era for the protein identification , identification and characterization of low molecular weight proteins in the human plasma / serum and protein profile of human urine have been done by the proteomics . in many ways , the two - dimensional gel electrophoresis was commonly used as a first step for protein identification by mass spectrometry . this approach has made it possible to carry out global protein analysis of living organisms that helps in examining the proteome . advanced technology in the proteomic filed for the protein separation is the commercial instrument proteomelab pf2d from beckman coulter . this is the 2d liquid chromatography system ( 2d - lc ) which separates proteins in the first dimension ( chromatographic focusing ) on the basis of isoelectric points ( pi ) of protein followed by the second dimension that is reversed phase high performance liquid chromatography ( rp - hplc ) , where proteins are further fractionated on the basis of hydrophobicity . the assessment , optimization , and separation from the biofluids like plasma , serum , urine , cerebrospinal fluid , and saliva have been observed by the pf2d . the identification and characterization of protein biomarkers have been achieved by the proteomics coupled with mass spectrometry ( ms ) analysis . ms technology is highly sensitive and is the most important developments in identification of the proteins . for the detection and characterization of various biomolecules like proteins , peptides , oligosaccharides , and oligonucleotides , the ms technology was used with the molecular mass range between 400 and 350,000 dalton . it detects very low quantities of sample up to 10 to 10 mole with an accuracy of 0.10.01% . it has good mass accuracy with increased resolution and high sensitivity and is used in the field of proteomics very widely to identify and characterize the protein biomarkers through peptide mass fingerprinting that can be helpful to identify proteins involved in global diseases like diabetes and others . in the method of ms , selected proteins of choice were digested with a specific proteolytic enzyme and the resulting peptides were analyzed for further studies . the information obtained from this can be used for identification and confirmation of the proteins by searching different protein databases . the process of identification and characterization of disease protein biomarkers was summarized and shown in figure 1 . so , this disease takes an ever - increasing proportion of national health care budgets . without primary prevention there is also a need of introducing cost - effective treatment strategies to control this epidemic . in the following sections , identification , purification , characterization of protein biomarkers for early diagnosis of pathological states like diabetes mellitus , and role of thiamine on the levels of these marker proteins in diabetes type 2 were described . research work has been done for the search of protein biomarkers for monitoring and predicting the diabetes mellitus . several modern techniques mainly involving protein characterization by mass spectrometry and proteomic profiling of plasma / serum samples by proteomics have also been described . animal studies with high dose thiamine therapy have been shown to reduce diabetic nephropathy ( microalbuminuria ) and lipid disturbances . therefore a clinical trial of high dose thiamine therapy has been planned in our type 2 diabetic pakistani population with a hope to find protein biomarker and role of high dose thiamine on the levels of these biomarkers in the patients having diabetes type 2 . dysregulation of multiple glucoregulatory hormones ( insulin and glucagons ) that maintain glucose homeostasis result in the form of diabetes . the imbalances in these hormones lead to long term elevated levels of glucose and some of microvascular and macrovascular complications like retinopathy , nephropathy , and neuropathy . the blood sugar at normal levels in the human body is maintained and brought by a regulatory mechanism which is very effective and efficient . in this mechanism , the main organs are liver , autonomic nervous system , and certain glands of internal secretion called endocrine glands . to maintain the homeostasis the important and dominant tissues are liver which respond to the signals that indicate low or high levels of blood glucose . the vital function of liver is to produce glucose for the circulation in the blood . both low and high levels of blood glucose triggered the hormonal responses which initiate the pathways to restore the glucose homeostasis . reduced levels of blood glucose trigger release of glucagons from the beta - cells of pancreas , while the increased levels of blood glucose trigger release of insulin from the pancreatic beta - cells . dm results from the dysregulation of multiple glucoregulatory hormones ( insulin and glucagons ) . any defect in glucoregulatory hormones production leads to improper regulation of glucose in the blood and results in diabetes this is the glucose homeostasis in which blood glucose levels maintained in the body and dysregulation cause hyper- and hypoglycemia as shown in figure 2 . insulin is a peptide hormone synthesized as precursor of polypeptide preproinsulin with single chain 86 amino acids and is secreted by the beta - cells of the islets of langerhans of pancreatic cells . it facilitates the glucose uptake in the cells , regulates the carbohydrate , lipid , and protein metabolisms , and promotes the cell division and growth . any defect in insulin production leads to improper regulation of glucose in the blood and results in diabetes . similarly , postprandial glucagon secretion is abnormally increased in the patients with type 1 and type 2 diabetes . it has also played important role in postprandial hyperglycemia in the patients with diabetes mellitus . there are different types of glucose transporters called glut1 , glut2 , glut3 , and glut4 . the glucose metabolism in the beta - cells changes the ion channel activity which leads to secretion of insulin . the sulfonylurea receptor ( sur ) is the binding site for the drugs that act as secretagogues for the hormone insulin . the metabolism of glucose-6-phosphate via glycolysis produces the atp which inhibits the activity of an atp - sensitive potassium channel . inhibition of this channel initiates the beta - cell membrane depolarization and opens calcium channels that stimulate insulin secretion as shown in figure 3 . dearrangement in the normal secretary patterns is one of the earliest signs of beta - cell dysfunction in diabetes . low insulin levels increase the production of glucose by promoting the hepatic gluconeogenesis and glycogenolysis in the fasting state . postprandially , glucose load elicits an increase in insulin and decrease in glucagons leading to a reversal of these processes . type 2 dm is a heterogeneous group of disorders and is characterized by the resistance and impaired secretion of insulin and high levels of glucose production . there are some metabolic and genetic defects in the action or secretion of insulin which give rise to hyperglycemia in type 2 dm . obesity is very common in type 2 diabetes where the adipocytes cells secret a number of biological proteins ( leptin , tnf - alpha , resistin , and adiponectin ) that modulate insulin secretion or action and may contribute to insulin resistance . the pancreatic islets are unable to sustain the hyperinsulinemia state in the progression of insulin resistance and hyperinsulinemia in certain individuals . reduced insulin secretion and increased hepatic glucose production lead to hyperglycemia in fasting diabetes . ultimately the beta - cell failure may increase the release of inflammatory protein markers such as interleukin-6 ( il-6 ) and c - reactive protein ( crp ) in type 2 dm . the molecular mechanism of insulin resistance can be explained briefly as levels of insulin receptors and tyrosine kinase activity in skeletal muscles are reduced . polymorphism in insulin receptor substrates ( irs ) may also be linked with intolerance of glucose , which raised the possibility that polymorphism in different postreceptors molecules combined and created a state of insulin resistance . the pathogenesis of insulin resistance is focused on signaling defect of a phosphatidylinositol-3-kinase ( pi-3 kinase ) . there are some pathways of insulin signal transduction which are not resistant to effect of insulin like the cell growth / differentiation and mitogenic activated protein ( map ) kinase pathways . it also promotes the production of glucose by the liver and impairs the function of beta - cells in the case of obese type 2 dm . there are varying reports on the role of protein biomarker for the identification of different diseases like cancer , diabetes , and others . there are some proteins which may be up- and downregulated in the serum / plasma and urine of diabetes mellitus especially in type 2 . the protein biomarkers are very helpful for predicting long - term mortality in patients with diabetes , cancer , and coronary syndromes . the urine is an ideal biofluid for biomarker discovery in kidney diseases and diabetes mellitus . urine samples obtained from patients with other diseases or disorders that have clinical , biochemical , and metabolic profiles similar to those of the disease of interest must be included as the other controls . recently , thongboonkerd has extensively applied proteomics to biomarker discovery in several diseases with the hope of finding novel biomarkers for earliest diagnosis of the diseases at their very beginning phase and for prediction of therapeutic response , survival , and recurrence . a two - dimensional liquid phase chromatographic separation followed by the mass spectrometry method is involved for proteomics studies and biomarker identification of different diseases . proteome analysis of human serum proteins and efficient prefractionation of low abundance proteins in human plasma were done . complex protein mixture analysis by an all - liquid - phase 2d mapping technique has been done by proteomelab pf 2d that is a novel protein profiling and mapping technology . the proteomics is a valid approach to screen for novel protein biomarkers in animal and human models of obesity and type 2 diabetes . proteins are reported as biomarkers in the biofluids , tissues , and cells especially in type 2 dm . one of them is apolipoprotein a1 ( apoa - i ) , which is the main component of high density lipoproteins ( hdl ) present in plasma . in case of excretion , hdl promotes cholesterol efflux from tissues to the liver for excretion ( yui et al . the level of apoa-1 is positively correlated with hdl cholesterol in the serum and negatively correlated with low density lipoproteins ( ldl ) cholesterol . it has an important role in blood coagulation and clearance of apoptotic bodies from the circulation . the apoa - i - ciii - aiv levels in type 2 dm and coronary heart disease were studied . it is also called bad cholesterol and is responsible for carrying cholesterol to tissues . the serum levels of some protein biomarkers like clusterin and apolipoprotein j were elevated significantly in type 2 dm and during development of coronary heart disease or at myocardial infarction . observed the diabetic macular edema after proteomic analysis of vitreous and recorded six proteins including pigment epithelium derived factor ( pedf ) , apoa-4 , apoa-1 , thyroid hormone receptor interactor 11 ( trip-11 ) , retinol binding protein 4 ( rbp4 ) , and vitamin d binding protein ( vdbp ) and only apo h is present in nondiabetic controls . borth in 1992 studied the alpha 2-macroglobulin ( 2-m ) which is an inhibitor for the proteinase enzyme in the blood and tissue . measurement of levels of protein human 1-microglobulin ( 1 m ) was studied in the serum and urine of patients with various liver diseases . transthyretin and its miracle function and pathogenesis were also observed in one recent research work . in addition , protein biomarker for diabetic nephropathy such as microalbuminuria is also a known predictor for the coronary heart and peripheral vascular diseases and high risk of mortality in patients of type 2 dm . in 1989 , semenkovich and his coworkers observed that the lipoprotein lipase is an important enzyme for lipid homeostasis in humans . c - reactive protein ( crp ) is a protein of acute phase condition and a strong biomarker of inflammation in the progression of various diseases like coronary heart disease , cancer , diabetes , and others . genetic variation and levels of crp and incidence of diabetes especially risk of developing type 2 diabetes mellitus have been studies . the concentration of crp in the blood of normal healthy control human beings ranges from 0 to 1.0 mg / dl . in the acute phase condition of inflammation lindsay and coworkers demonstrated that some of the proteins are downregulated in dm likeone of the adipokines adiponectin in indian population . studied that some protein biomarkers of diabetes are associated in young diabetic patients with oxidative stress and antioxidant status . proteomic analysis of proteins was done and involved in insulin resistance and type 2 dm with the b3-adrenergic receptor . 2002 have described that the elevated levels of acute - phase proteins and plasminogen activator inhibitor-1 ( pai-1 ) in type 2 diabetes with insulin resistance were observed . in type 2 dm , the intramuscular heat shock protein 72 and heme oxygenase-1 mrna were decreased . tumor - derived peptide with an s - calcium binding protein is involved in the pancreatic cancer associated with diabetes which are the studies of bassoa and coworkers . recently , obesity and its related proteins like leptin were studied in the diabetic population of pakistan . list of identified proteins in human serum of patients having diabetes mellitus is given in table 1 . urinary protein profiling can reveal changes in excretion rates of specific proteins that can have predictive value in the clinical arena , for example , in the early diagnosis of disease , classification of disease with regard to likely therapeutic responses , assessment of prognosis , and monitoring response to therapy . in 2005 have described that rbp4 protein is an important biomarker for insulin resistance in diabetes . in one such study which was carried out by metz and coworkers , the five protein biomarkers were identified as 2-fold upregulated in diabetes which were alpha-2-glycoprotein ( zinc ) , corticosteroid - binding globulin , and lumican . the clusterin and serotransferrin were 2-fold downregulated in diabetic samples compared to control . for urinary biomarkers in diabetic nephropathy , proteomic analyses identified some potential protein biomarkers . list of urinary proteins that are identified in diabetic patients with or without nephropathy are listed in table 2 . have observed that there were seven proteins which were upregulated with increasing albuminuria and four proteins were downregulated . after some years , the proteomic analysis was done for the identification of human salivary biomarkers for type-2 diabetes . recently , in 2009 , jiang and his coworkers studied the identification of urinary soluble e - cadherin as a novel biomarker for diabetic nephropathy . observed that haptoglobins are a group of serum proteins ; originally identified and diagnostic values were determined . the goals of present research work were the identification and characterization of protein biomarkers for early diagnosis and prognosis of pathological states of diabetes mellitus type 2 and effects of high dose thiamine on the levels of marker proteins . these investigations shall be helpful in assessing the biochemical alterations in the pakistani diabetic population , which should contribute to the development of treatment plans for this disease which is one of the most widely occurring and debilitating diseases . results from this research will also contribute to the identification of protein markers of diabetes mellitus type 2 and thus development of novel diagnostic procedures for early detection of this complication in potential diabetics in our population . the findings from present research work will assist in planning preventive and effective treatment strategies for diabetic patients by identification of protein biomarkers and high dose thiamine as nutritional supplement .

anxiety and depression have strong and independent associations with the mental health domain in cancer patients . studies depict that cancer patients with a low level of optimism and a high level of pessimism are at risk of higher levels of anxiety and depression in addition to lowered health - related quality of life . further , anxiety , lack of positive support , detrimental interactions , threat of cancer , disease stage , age , and gender are observed to significantly predict the patient 's mental health and survival . patients in an interpersonal / love / familial relationship during cancer were most satisfied with support , and experienced the highest self - esteem and mental health ; however , cancer patients who were unemployed , economically disadvantaged , or living alone were at risk for mood and mental health difficulties . a number of qualitative studies in the last few years have also discussed specific mental health issues , emotions and coping of cancer patients . religion is seen to help cancer patients to cope better ( in terms of support , hope , and meaning ) with their disease . findings suggest that facets of spirituality ( the ability to find meaning and peace in life ) are an influential contributor to favorable adjustment during cancer . qualitative studies have shown that consistency and individuality characterized the role of religion and spirituality in cancer survivorship across themes such as impact of cancer on religion / spirituality , meaning - making , prayer , religious / spiritual role of others , and facing death . in india , apart from other traditional methods ( taking medications , indulging in exercise and activities to divert one 's attention ) , spiritual methods of coping ( prayer and meditation , adopting a positive attitude ) were the most frequently used mainstream coping strategy by the patients suffering from head and neck cancers . patients suffering from head and neck cancers are observed to have a relatively high risk of developing emotional disturbances after diagnosis and treatment . these emotional concerns can be best understood and explored through the method of content analysis or qualitative data . though a number of qualitative studies have been conducted in the last few years in the field of psychosocial oncology , none have looked at the emotions experienced and the coping of head and neck cancer patients . further , the researchers believed that it would be interesting to understand the psychosocial oncology - related issues ( such as the emotions and coping of patients ) in the context of a developing and spiritual - oriented country such as india . this study is part of a larger main study titled life after cancer : a psychosocial impact , which used a mixed methodology : a quantitative and qualitative paradigm . a total of 75 head and neck cancer patients from the tata memorial hospital ( tmh ) , a tertiary care hospital for cancer care in mumbai , maharashtra , india were studied . as a majority of the head and neck cancers are squamous cell carcinomas ( tumors that develop in the tissue lining the hollow of the body organs ) , consecutive patients suffering from squamous cell carcinomas of the oral cavity , hypopharynx , oropharynx , and larynx who were within the first 15 days post surgery were selected ( the institute sees on an average 4000 cases per year ) . only new patients ( no relapse cases ) in the age group of 30 - 60 years , with a minimum of 2 months of symptomatic phase ( before surgery ) , who could speak english or hindi were included in the study . the sociodemographic data of the patients who participated in the study were compiled [ table 1 ] . socio - demographic data of patients ( n = 75 ) * mean ( sd ) ; bpl : below poverty line earning less than rs 1700/- per month ; @income greater than rs 15,000 per month , taxable by the government of india an interview schedule was formulated based on the aim of the study , literature review , and discussion with two experts in the field . it aimed to elicit the emotions experienced by the patients after hearing their diagnosis of cancer and the coping strategies used by them to deal with these emotions ( the interview schedule is available from the authors on request ) . the interview schedule was written in two languages , english and hindi , for the convenience of administration and the hindi version was back - translated into english to check for concurrent validity . patients who were unable to speak ( some postoperative patients with cancer of the larynx ; n = 11 ) were asked to write down their responses on a sheet of paper . the study protocol was reviewed and approved by the erstwhile department of medical and psychiatric social work and the institute ethics committee at the tata institute of social sciences , mumbai , maharashtra , india . written informed consent of the patients to participate in the study was obtained . the researcher met the participants in their wards and conducted in - depth interviews with the help of the interview schedule . the time spent in conducting the interview ranged 0.5 - 2 h depending on the capacity of the patient to speak after surgery . the researcher took down verbatim notes of the patients responses ( written transcription ) in a separate response sheet . the patients were slow in their communication and could speak only a few words ( due to surgery ) ; thus the data generated were not enormous and the researcher was able to complete the recordings efficiently . a majority of the patients ( those who found it difficult to speak after surgery ) used gestures and facial expressions to communicate information . if the interviewer did not understand the interpretation of these cues , she clarified the interpretation of the same with the respondent during the interview itself , by asking the respondent to write his / her responses or by clarifying what he / she was saying with the family members . thus , only the interpretation of the nonverbal cues that were approved by the respondent was documented as part of the data collection . reflexivity was exercised as the questions asked were fixed and structured as per the interview schedule . therefore , the use of interview schedules did not offer much scope to the interviewer to include reflexivity in the analysis or bring in her reflections . the data thus collected were scrutinized by the second author ( the supervisor of the interviewer ) after each interview ( especially in the pilot study ) to correct any biases observed during the interviewing process . this was further corroborated by the information collected using the quantitative paradigm . while the information pertained to coping with the side effects of medication and cancer pain , part of this information was triangulated with the qualitative data . a content analysis of each transcribed interview schedule was conducted . as the data generated were not extensive [ as patients were slow in their communication and could speak only a few words ( due to surgery ) ] , only a manual thematic analysis was possible . the researcher then made a list of the common themes and subthemes across all interviews . a conceptual framework of the emotions experienced by the patients and the coping strategies employed by them were developed . the data collected were divided into two main parts : emotions experienced by the patients after the diagnosis of cancer andcoping strategies employed by the patients to deal with their emotions . each part generated several themes and subthemes , which were substantiated adequately by patient quotations . emotions experienced by the patients after the diagnosis of cancer and coping strategies employed by the patients to deal with their emotions . each part generated several themes and subthemes , which were substantiated adequately by patient quotations . a total of 75 head and neck cancer patients from the tata memorial hospital ( tmh ) , a tertiary care hospital for cancer care in mumbai , maharashtra , india were studied . as a majority of the head and neck cancers are squamous cell carcinomas ( tumors that develop in the tissue lining the hollow of the body organs ) , consecutive patients suffering from squamous cell carcinomas of the oral cavity , hypopharynx , oropharynx , and larynx who were within the first 15 days post surgery were selected ( the institute sees on an average 4000 cases per year ) . only new patients ( no relapse cases ) in the age group of 30 - 60 years , with a minimum of 2 months of symptomatic phase ( before surgery ) , who could speak english or hindi were included in the study . the sociodemographic data of the patients who participated in the study were compiled [ table 1 ] . socio - demographic data of patients ( n = 75 ) * mean ( sd ) ; bpl : below poverty line earning less than rs 1700/- per month ; @income greater than rs 15,000 per month , taxable by the government of india an interview schedule was formulated based on the aim of the study , literature review , and discussion with two experts in the field . it aimed to elicit the emotions experienced by the patients after hearing their diagnosis of cancer and the coping strategies used by them to deal with these emotions ( the interview schedule is available from the authors on request ) . the interview schedule was written in two languages , english and hindi , for the convenience of administration and the hindi version was back - translated into english to check for concurrent validity . patients who were unable to speak ( some postoperative patients with cancer of the larynx ; n = 11 ) were asked to write down their responses on a sheet of paper . the study protocol was reviewed and approved by the erstwhile department of medical and psychiatric social work and the institute ethics committee at the tata institute of social sciences , mumbai , maharashtra , india . the researcher met the participants in their wards and conducted in - depth interviews with the help of the interview schedule . the time spent in conducting the interview ranged 0.5 - 2 h depending on the capacity of the patient to speak after surgery . the researcher took down verbatim notes of the patients responses ( written transcription ) in a separate response sheet . the patients were slow in their communication and could speak only a few words ( due to surgery ) ; thus the data generated were not enormous and the researcher was able to complete the recordings efficiently . a majority of the patients ( those who found it difficult to speak after surgery ) used gestures and facial expressions to communicate information . if the interviewer did not understand the interpretation of these cues , she clarified the interpretation of the same with the respondent during the interview itself , by asking the respondent to write his / her responses or by clarifying what he / she was saying with the family members . thus , only the interpretation of the nonverbal cues that were approved by the respondent was documented as part of the data collection . reflexivity was exercised as the questions asked were fixed and structured as per the interview schedule . therefore , the use of interview schedules did not offer much scope to the interviewer to include reflexivity in the analysis or bring in her reflections . the data thus collected were scrutinized by the second author ( the supervisor of the interviewer ) after each interview ( especially in the pilot study ) to correct any biases observed during the interviewing process . this was further corroborated by the information collected using the quantitative paradigm . while the information pertained to coping with the side effects of medication and cancer pain , part of this information was triangulated with the qualitative data . a content analysis of each transcribed interview schedule was conducted . as the data generated were not extensive [ as patients were slow in their communication and could speak only a few words ( due to surgery ) ] , only a manual thematic analysis was possible . the researcher then made a list of the common themes and subthemes across all interviews . a conceptual framework of the emotions experienced by the patients and the coping strategies employed by them were developed . the data collected were divided into two main parts : emotions experienced by the patients after the diagnosis of cancer andcoping strategies employed by the patients to deal with their emotions . each part generated several themes and subthemes , which were substantiated adequately by patient quotations . emotions experienced by the patients after the diagnosis of cancer and coping strategies employed by the patients to deal with their emotions . each part generated several themes and subthemes , which were substantiated adequately by patient quotations . the patients experienced varied emotions on realizing that they were suffering from cancer , the cause of which could be mainly attributed to three themes : knowledge / discovery of their illness;duration of untreated illness , andobject of blame . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness:i felt very sad , upset , and scared when i was told that i had cancer . ( 47-year - old mr . ( 51-year - old m.)some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f.)i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . sks.)i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n.)i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . dp.)some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members:i still do not know about the seriousness of the illness of cancer . my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s.)those people who decided to mentally fight the illness felt that they could recover.i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m.)i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o.)emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . na.)because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . us.)the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . f.)i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . ss.)some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities:i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . d.)i am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . mm.)very few patients stated that timely treatment was sought and provided:i started treatment very early , immediately after my first symptoms were noticed . hence , i have been very lucky and i am happy with my current condition . d.)emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness.my fate has played a role and has brought me here to this hospital with this diagnosis of cancer r.)i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . bm.)others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . because of my own self my bidi and alcohol habits i have got cancer . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg.)as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . knowledge / discovery of their illness ; duration of untreated illness , and emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness:i felt very sad , upset , and scared when i was told that i had cancer . ( 51-year - old m.)some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f.)i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . sks.)i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n.)i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . dp.)some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members:i still do not know about the seriousness of the illness of cancer . my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s.)those people who decided to mentally fight the illness felt that they could recover.i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m.)i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o.)emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . na.)because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . us.)the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . f.)i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . ss.)some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities:i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . mm.)very few patients stated that timely treatment was sought and provided:i started treatment very early , immediately after my first symptoms were noticed . hence , i have been very lucky and i am happy with my current condition . d.)emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness.my fate has played a role and has brought me here to this hospital with this diagnosis of cancer r.)i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . bm.)others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg.)as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness : i felt very sad , upset , and scared when i was told that i had cancer . c. ) i often still cry when i think that i have cancer . ( 51-year - old m. ) some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . uncertainty in the future , costs of treatment , and operative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f. ) i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n. ) i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members : my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s. ) those people who decided to mentally fight the illness felt that they could recover . i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m. ) i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o. ) emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor . emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . poor and slow response of the patient to other treatments ( chemotherapy or radiation ) , inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . ( 47-year - old mr . na . ) because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . ( 54-year - old mrs . us . ) the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . v. ) i felt very anxious because of the prolonged length of the treatment . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities : i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . d. ) i am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . hence , i have been very lucky and i am happy with my current condition . d. ) emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness . my fate has played a role and has brought me here to this hospital with this diagnosis of cancer i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . blamed themselves ( for using the substances ) , blamed the substances , or blamed the government ( for not banning the substances ) for their present illness . because of my own self my bidi and alcohol habits i have got cancer . i have only myself to blame for the illness . ( 49-year - old mr . p. ) tobacco is the main cause of the illness . it should be banned . d. ) the government is to be blamed for the illness of cancer in the society . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg . ) as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . there were mainly four themes that depicted the manner in which the patients coped with their emotions . these themes were : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . hence , i did not feel upset and managed by telling myself that everything will be alright . rkc.)my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . ( 49-year - old mr . p.)life is full of hardships , which every man has to deal with by himself . t.)participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . i have a lot of faith in my god and i know that everything will be alright soon . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i have full faith in my doctor 's treatment and i know that i will get well soon . ( 42-year - old mr . ns.)the role of spirituality in coping is seen in the above narratives . when this is reiterated by professionals , this method of coping is further reinforced.ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c.)reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m.)as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s.)the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f.)my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . hence , i did not feel upset and managed by telling myself that everything will be alright . rkc.)my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . ( 49-year - old mr . p.)life is full of hardships , which every man has to deal with by himself . t.)participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . i have a lot of faith in my god and i know that everything will be alright soon . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i have full faith in my doctor 's treatment and i know that i will get well soon . ( 42-year - old mr . ns.)the role of spirituality in coping is seen in the above narratives . when this is reiterated by professionals , this method of coping is further reinforced.ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c.)reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m.)as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s.)the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f.)my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery . hence , i did not feel upset and managed by telling myself that everything will be alright . rkc . ) my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . life is full of hardships , which every man has to deal with by himself . t. ) participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients . developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness . i have a lot of faith in my god and i know that everything will be alright soon . d. ) i have a lot of faith in my god and my doctor . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i am in the hands of one of the best doctors in this field . i have full faith in my doctor 's treatment and i know that i will get well soon . ( 42-year - old mr . the role of spirituality in coping is seen in the above narratives . when this is reiterated by professionals , this method of coping ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . they coped with the illness by sharing their emotions with them . to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c. ) reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m. ) as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s. ) the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india . indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention . i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f. ) my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . the patients experienced varied emotions on realizing that they were suffering from cancer , the cause of which could be mainly attributed to three themes : knowledge / discovery of their illness;duration of untreated illness , andobject of blame . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness:i felt very sad , upset , and scared when i was told that i had cancer . ( 47-year - old mr . ( 51-year - old m.)some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f.)i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . sks.)i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n.)i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . dp.)some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members:i still do not know about the seriousness of the illness of cancer . my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s.)those people who decided to mentally fight the illness felt that they could recover.i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m.)i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o.)emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . na.)because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . us.)the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . f.)i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . ss.)some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities:i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . d.)i am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . mm.)very few patients stated that timely treatment was sought and provided:i started treatment very early , immediately after my first symptoms were noticed . hence , i have been very lucky and i am happy with my current condition . d.)emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness.my fate has played a role and has brought me here to this hospital with this diagnosis of cancer r.)i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . bm.)others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . because of my own self my bidi and alcohol habits i have got cancer . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg.)as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . knowledge / discovery of their illness ; duration of untreated illness , and emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness:i felt very sad , upset , and scared when i was told that i had cancer . ( 51-year - old m.)some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f.)i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . sks.)i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n.)i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . dp.)some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members:i still do not know about the seriousness of the illness of cancer . my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s.)those people who decided to mentally fight the illness felt that they could recover.i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m.)i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o.)emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . na.)because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . us.)the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . f.)i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . ss.)some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities:i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . mm.)very few patients stated that timely treatment was sought and provided:i started treatment very early , immediately after my first symptoms were noticed . hence , i have been very lucky and i am happy with my current condition . d.)emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness.my fate has played a role and has brought me here to this hospital with this diagnosis of cancer r.)i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . bm.)others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg.)as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . the immediate reaction after becoming aware of their illness was often one of sadness : i felt very sad , upset , and scared when i was told that i had cancer . c. ) i often still cry when i think that i have cancer . ( 51-year - old m. ) some patients were fearful or anxious about : uncertainty in the future , death , costs of treatment , andoperative procedures when they heard about their diagnosis . uncertainty in the future , costs of treatment , and operative procedures when they heard about their diagnosis . i thought i could never be cured , would suffer all through my life , and die . f. ) i felt very tense and anxious when i was told about my diagnosis , as i did not know what lay in the future . i thought something will happen to me ; i believed it was life - threatening " . i was scared when i heard that i was diagnosed with cancer , as i have seen my mother die due to this disease."(59-year - old mr . n. ) i was scared when i heard that i had a diagnosis of cancer , more because of the costs of treatment . i was not sure if i could afford the costs of the treatment and get well . some patients did not get mentally affected or emotionally disturbed about their diagnosis of cancer as the details of the illness were not disclosed to them by their doctor or family member . others , who knew about their diagnosis , were fearful of disclosing the same to their family members : my daughter however , knew everything about the illness . ( 55-year - old mrs . i believe my doctor will cure me and i will follow his instructions / treatment . s. ) those people who decided to mentally fight the illness felt that they could recover . i never felt bad when i was told that i was diagnosed with cancer . so when they told me i had cancer , i did not worry too much . my astrologer has told me that i will live a long life . m. ) i have been very positive about my treatment , from the day i was diagnosed with cancer . i could freely talk about my illness to everyone in the ward and discuss with them the causes and treatment options . o. ) emotional reactions and response on discovery or being told about the diagnosis of cancer varied from sadness , anxiety , fear of death and the inevitable , uncertainty about the future , and being disturbed . collusion as strategy was observed when people chose not to divulge the diagnosis to their family members or their loved ones . further , those who withheld the diagnosis for some time ultimately decided to disclose the information to their family members . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor . emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . this theme was defined in the study as the time from the disclosure of the diagnosis till the time the patient was surgically operated for cancer . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . poor and slow response of the patient to other treatments ( chemotherapy or radiation ) , inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . the treatment process is taking too long because of which i often get disillusioned and lose hope that i will ever get well soon . ( 47-year - old mr . na . ) because i was experiencing increasing pain by the day , i was upset that the treatment was not done earlier . ( 54-year - old mrs . us . ) the operation was stalled for 4 months as i did not have the money to pay for the treatment . once i was able to get the money , i got admitted to the hospital and was operated . v. ) i felt very anxious because of the prolonged length of the treatment . the further the date of my surgery got postponed , the more i was unsure that i would survive till then . i often wanted the treatment to end fast so that i could go home to my family . i delayed the treatment by 1 month , as i did not want to get operated . i tried convincing the doctor to opt for treatments other than surgery as i was scared that my face will get disfigured " . some patients were anxious to complete their treatment as they were eager to get back to their routine lives with their families , start work , and resume their responsibilities : i was anxious to complete treatment early so that i could go home and take care of my responsibilities and the family . d. ) i am dependent on my son for the costs of treatment and hospital stay . due to the long duration of the treatment and increasing costs , my son is often upset with me and this makes me feel sad . i want to get well fast so that i can go home , work as before , and earn my living . hence , i have been very lucky and i am happy with my current condition . d. ) emotions associated with the object of blame : a third theme that was connected to the patients emotions was their object of blame , which was expressed as anger toward their fate for their current illness . my fate has played a role and has brought me here to this hospital with this diagnosis of cancer i believe i have got this illness because of my past karma past life 's bad deeds . i must have definitely done something bad in my past life due to which i am suffering now . others who were addicted to substances ( nicotine , alcohol , gutka , and other addictive substances ) either expressed anger and : blamed themselves ( for using the substances),blamed the substances , orblamed the government ( for not banning the substances ) for their present illness . blamed themselves ( for using the substances ) , blamed the substances , or blamed the government ( for not banning the substances ) for their present illness . because of my own self my bidi and alcohol habits i have got cancer . i have only myself to blame for the illness . ( 49-year - old mr . p. ) tobacco is the main cause of the illness . it should be banned . d. ) the government is to be blamed for the illness of cancer in the society . if they had banned or taxed tobacco heavily , people would have been deterred from using it . ( 42-year - old rg . ) as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . there were mainly four themes that depicted the manner in which the patients coped with their emotions . these themes were : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . hence , i did not feel upset and managed by telling myself that everything will be alright . rkc.)my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . p.)life is full of hardships , which every man has to deal with by himself . t.)participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . i have a lot of faith in my god and i know that everything will be alright soon . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i have full faith in my doctor 's treatment and i know that i will get well soon . ( 42-year - old mr . ns.)the role of spirituality in coping is seen in the above narratives . when this is reiterated by professionals , this method of coping is further reinforced.ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c.)reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m.)as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s.)the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f.)my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . hence , i did not feel upset and managed by telling myself that everything will be alright . ( 42-year - old mr . rkc.)my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . p.)life is full of hardships , which every man has to deal with by himself . t.)participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . i have a lot of faith in my god and i know that everything will be alright soon . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i have full faith in my doctor 's treatment and i know that i will get well soon . ns.)the role of spirituality in coping is seen in the above narratives . when this is reiterated by professionals , this method of coping is further reinforced.ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c.)reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m.)as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s.)the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f.)my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery . , i did not feel upset and managed by telling myself that everything will be alright . rkc . ) my strong positive attitude and my will power have helped me to maintain a balanced outlook and are helping me to handle my emotions towards my illness . ( 49-year - old mr . life is full of hardships , which every man has to deal with by himself . t. ) participants demonstrated a strong will power to be able to deal with the illness and used metaphors that aided in coping . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients . developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness . i have a lot of faith in my god and i know that everything will be alright soon . d. ) i have a lot of faith in my god and my doctor . they both are my sources of strength , and unquestionable faith in them has helped me cope with my emotions about the illness . i am in the hands of one of the best doctors in this field . i have full faith in my doctor 's treatment and i know that i will get well soon . ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . c. ) reassurances from my family and my doctor that i will get well helped me to overcome my tension and worries a bit . m. ) as my family is in the village and i have come alone to mumbai for my treatment , i share my emotions with my copatients in the ward and feel better . s. ) the above quotes depict that the main sources of emotional support for the participants were their family members and in some cases their doctor . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india . indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention . i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . f. ) my family is in the village and i have no one to talk to here at the hospital . hence , i divert my mind , by consoling and counseling other fellow patients in the ward . i laugh and joke around with them so that they can forget about their illness at least when they are with me.(35-year - old mr . the content analysis of the qualitative results of the study puts forth a conceptual framework for understanding the emotions experienced and the coping strategies of head and neck cancer patients in india after diagnosis [ figure 1 ] . one 's knowledge about his / her illness of cancer and the duration of untreated illness of cancer affects the emotions of a survivor . he / she experiences emotions of sadness , anxiety , loss of hope , fear , and blame . depending on his / her family support system , inner resilience , and religious leanings ( faith in god ) , the survivor is able to cope with his / her illness through strategies such as : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention . conceptual framework of emotions and coping strategies of patients suffering from head and neck cancer though a number of studies have discussed the varied psychosocial issues experienced by the patients and the contexts in which they arise , this study particularly highlights the negative emotions of the patients to two main themes : being told about the illness and the duration of untreated illness . divulging the news or discovery of the illness is equivalent to the concept of breaking the bad news and emotions experienced by the patients after the doctors or their families have disclosed the however , understanding the phenomenon of the patient 's knowing is essential for any health care professional working with cancer patients in order to understand and provide for their psychosocial needs . the strong family ties and lineages act as a support system as well as a buffer for any individual , especially in the case of patients of chronic ailments such as cancer . most patients in india are able to ventilate their emotions to their family members and/or relatives who stand by them through their treatment . studies reiterate that ventilation of one 's emotions is an effective method of coping , which predicts the distress levels and reduces medical visits for cancer - related morbidities . encouraging this culturally accepted coping method could help patients in india to deal better with their diagnosis of cancer . in india , people have a strong religious orientation , which is often linked to their god . most of the religious scriptures ( e.g. , bhagavad gita ) reiterate that it is easier for people to cope when there is a grounded sense of god because it helps them to surrender to the supreme / unknown and feel secure . some of the alternate therapies such as pranic healing , reiki , art of living , and yoga use aspects of prayer and meditation to help the person practice alone in the process of healing . these religious practices and beliefs are also seen to improve the inner resilience of cancer patients , apart from being effective coping mechanisms . the object of blame was considered as a separate theme for a better understanding of the emotions of cancer patients in this study as the concept of blame is inbuilt in the karma theory ( as explained in the ancient indian scriptures such as the bhagavad gita ) , which states that one reaps the consequences of one 's actions and one suffers from a disease such as cancer because of his / her misdeeds , either in this life or in a previous existence . in such a scenario , the patient either blames himself / herself , others , or his / her fate , which facilitates acceptance of the disease by the survivor easily . while this concept may seem to be limiting , it also helps to project the blame to an unknown but sure source or object , thus freeing the individual to focus on dealing with the present reality ( a type of defense mechanism ) . it can be inferred , therefore , that such a strategy can either help in coping if used in a moderate amount or be considered as negative coping ( if excessively used ) . palliative care professionals need to be aware of these cultural and religious ideologies while providing psychosocial care to indian cancer patients . the largely negative emotions expressed by the patient could be attributed to the fact that all had undergone surgery recently and the surgical process itself could have impacted their emotional and mental health . the dynamics and interplay between the internal and external resources of the patient had a bearing on the coping strategies used by the patient . it is further interesting to note that all the patients in spite of the condition of their disease were able to cope by using at least one strategy to deal with their emotional and mental state . no patient was observed as getting overwhelmed by his / her emotions or by stating that he / she wanted to die . further , none of them had been provided any psychosocial intervention yet to help them deal with their condition , as they had undergone surgery only a few days prior to the interview . this research finding brings out a very important inference , which can be used in the oncological care for patients with head and neck cancers : in this context , focus on psychosocial intervention for the promotion of effective and healthy coping strategies is essential , especially soon after surgery . as a well - being promotion intervention , it is useful to design strategies that locate the distress , detract from it , and project it in a suitably acceptable and conducive manner and then move on to replace the distress with deflective interventions with an aim to build and strengthen the resilience in the individual suffering from head and neck cancers . though the generalization of the results of this study is limited by the inclusion criteria which was fairly homogenous and being country specific , the researchers believe that the results of this study are a significant contribution to the field of psychosocial oncology . further , the conceptual framework of this study could form a base for future quantitative studies that want to test the efficacy of any need - based psychosocial program for inpatients with head and neck cancers . this result however , needs to be tested outside india as the context of the emotions experienced by inpatients , the type of emotions expressed as well as the coping strategies could differ from culture to culture based on their underlying mores , ideologies , faiths , religions , and beliefs that have a bearing on the coping of patients in that culture . the authors received no financial support for the research , authorship , and/or publication of this article . the authors declared no potential conflict of interest with respect to the research , authorship , and/or publication of this article . the authors received no financial support for the research , authorship , and/or publication of this article . the authors declared no potential conflict of interest with respect to the research , authorship , and/or publication of this article .

background and rationale : patients suffering with head and neck cancers are observed to have a relatively high risk of developing emotional disturbances after diagnosis and treatment . these emotional concerns can be best understood and explored through the method of content analysis or qualitative data . though a number of qualitative studies have been conducted in the last few years in the field of psychosocial oncology , none have looked at the emotions experienced and the coping by head and neck cancer patients.materials and methods : seventy - five new cases of postsurgery patients of head and neck cancers were qualitatively interviewed regarding the emotions experienced and coping strategies after diagnosis.results:qualitative content analysis of the in - depth interviews brought out that patients experienced varied emotions on realizing that they were suffering from cancer , the cause of which could be mainly attributed to three themes : 1 ) knowledge of their illness ; 2 ) duration of untreated illness ; and 3 ) object of blame . they coped with their emotions by either : 1 ) inculcating a positive attitude and faith in the doctor / treatment , 2 ) ventilating their emotions with family and friends , or 3 ) indulging in activities to divert attention.conclusion:the results brought out a conceptual framework , which showed that an in - depth understanding of the emotions their root cause , coping strategies , and spiritual and cultural orientations of the cancer survivor is essential to develop any effective intervention program in india .

Background and Rationale Materials and Methods Sample Interview schedule Procedure and ethics Data analysis Results Emotions experienced by the patients Coping strategies of the patients Discussion Financial support and sponsorship Conflicts of interest

further , anxiety , lack of positive support , detrimental interactions , threat of cancer , disease stage , age , and gender are observed to significantly predict the patient 's mental health and survival . a number of qualitative studies in the last few years have also discussed specific mental health issues , emotions and coping of cancer patients . in india , apart from other traditional methods ( taking medications , indulging in exercise and activities to divert one 's attention ) , spiritual methods of coping ( prayer and meditation , adopting a positive attitude ) were the most frequently used mainstream coping strategy by the patients suffering from head and neck cancers . patients suffering from head and neck cancers are observed to have a relatively high risk of developing emotional disturbances after diagnosis and treatment . these emotional concerns can be best understood and explored through the method of content analysis or qualitative data . though a number of qualitative studies have been conducted in the last few years in the field of psychosocial oncology , none have looked at the emotions experienced and the coping of head and neck cancer patients . further , the researchers believed that it would be interesting to understand the psychosocial oncology - related issues ( such as the emotions and coping of patients ) in the context of a developing and spiritual - oriented country such as india . a total of 75 head and neck cancer patients from the tata memorial hospital ( tmh ) , a tertiary care hospital for cancer care in mumbai , maharashtra , india were studied . as a majority of the head and neck cancers are squamous cell carcinomas ( tumors that develop in the tissue lining the hollow of the body organs ) , consecutive patients suffering from squamous cell carcinomas of the oral cavity , hypopharynx , oropharynx , and larynx who were within the first 15 days post surgery were selected ( the institute sees on an average 4000 cases per year ) . socio - demographic data of patients ( n = 75 ) * mean ( sd ) ; bpl : below poverty line earning less than rs 1700/- per month ; @income greater than rs 15,000 per month , taxable by the government of india an interview schedule was formulated based on the aim of the study , literature review , and discussion with two experts in the field . it aimed to elicit the emotions experienced by the patients after hearing their diagnosis of cancer and the coping strategies used by them to deal with these emotions ( the interview schedule is available from the authors on request ) . the researcher met the participants in their wards and conducted in - depth interviews with the help of the interview schedule . a conceptual framework of the emotions experienced by the patients and the coping strategies employed by them were developed . emotions experienced by the patients after the diagnosis of cancer and coping strategies employed by the patients to deal with their emotions . as a majority of the head and neck cancers are squamous cell carcinomas ( tumors that develop in the tissue lining the hollow of the body organs ) , consecutive patients suffering from squamous cell carcinomas of the oral cavity , hypopharynx , oropharynx , and larynx who were within the first 15 days post surgery were selected ( the institute sees on an average 4000 cases per year ) . socio - demographic data of patients ( n = 75 ) * mean ( sd ) ; bpl : below poverty line earning less than rs 1700/- per month ; @income greater than rs 15,000 per month , taxable by the government of india an interview schedule was formulated based on the aim of the study , literature review , and discussion with two experts in the field . it aimed to elicit the emotions experienced by the patients after hearing their diagnosis of cancer and the coping strategies used by them to deal with these emotions ( the interview schedule is available from the authors on request ) . the researcher met the participants in their wards and conducted in - depth interviews with the help of the interview schedule . a conceptual framework of the emotions experienced by the patients and the coping strategies employed by them were developed . emotions experienced by the patients after the diagnosis of cancer and coping strategies employed by the patients to deal with their emotions . the patients experienced varied emotions on realizing that they were suffering from cancer , the cause of which could be mainly attributed to three themes : knowledge / discovery of their illness;duration of untreated illness , andobject of blame . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . knowledge / discovery of their illness ; duration of untreated illness , and emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . these themes were : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . when this is reiterated by professionals , this method of coping is further reinforced.ventilating emotions with family / friends and their responses : a majority of the participants had their families and relatives / friends to support and reassure them . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness . indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention . the patients experienced varied emotions on realizing that they were suffering from cancer , the cause of which could be mainly attributed to three themes : knowledge / discovery of their illness;duration of untreated illness , andobject of blame . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . )as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . knowledge / discovery of their illness ; duration of untreated illness , and emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . while the disclosure of the diagnosis affected some participants , it also strengthened the resolve of others who had buffer support of the family , and faith in god and in their treating doctor.emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . )as seen above , the common object of blame for the illness is either god / fate , own self , substances that are addictive , or the state for not adhering to stringent norms for the sale of such addictive substances . emotions arising from knowledge / discovery of their illness : the most important theme which kept repeating across all interviews was the emotions experienced as a result of the patient 's knowledge of his / her illnesses . emotions associated with the duration of untreated illness : another theme that was prominently connected to the emotions of the patients was the duration of untreated illness . it was observed that longer the duration of the illness , more were the negative emotions experienced by the patients such as anxiety , sadness , and loss of hope . the duration of untreated illness was prolonged either due to the : poor and slow response of the patient to other treatments ( chemotherapy or radiation),inability of the patient to afford the treatment / operation , postponement of the date of surgery , the patient 's fear of undergoing the operation . these themes were : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention : inculcating a positive attitude : to help them cope with the illness of cancer , some patients inculcated a strong positive attitude within themselves about their recovery.i believe that there is no point in brooding over the illness . earlier studies too have depicted that metaphorical talk about cancer reflects enduring metaphorical patterns of thought in the patients.developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness.i cope with my emotions on my own by saying my prayers . they coped with the illness by sharing their emotions with them.to help reduce my sadness , i used to talk to my family members , as they always instill hope and courage in me . this use of human resources and social networks ( especially one 's family ) to deal with ones emotions is characteristic of the strong indian family system in india.indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention.i tried to divert my mind to other activities such as knitting and prayer so that i would not get any negative emotions and thoughts . developing a strong faith in the doctor / god : the strong faith that they will be taken care of by their doctor and their god helped some patients to cope with their illness . indulging in activities to divert attention : patients who did not want to make their families worry and those who were alone during their treatment process often kept their emotions to themselves and consciously indulged in activities to divert their attention . the content analysis of the qualitative results of the study puts forth a conceptual framework for understanding the emotions experienced and the coping strategies of head and neck cancer patients in india after diagnosis [ figure 1 ] . one 's knowledge about his / her illness of cancer and the duration of untreated illness of cancer affects the emotions of a survivor . depending on his / her family support system , inner resilience , and religious leanings ( faith in god ) , the survivor is able to cope with his / her illness through strategies such as : inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , andindulging in activities to divert attention . inculcating a positive attitude , developing a strong faith in the doctor / god , ventilating emotions with family / friends and their responses , and indulging in activities to divert attention . conceptual framework of emotions and coping strategies of patients suffering from head and neck cancer though a number of studies have discussed the varied psychosocial issues experienced by the patients and the contexts in which they arise , this study particularly highlights the negative emotions of the patients to two main themes : being told about the illness and the duration of untreated illness . the object of blame was considered as a separate theme for a better understanding of the emotions of cancer patients in this study as the concept of blame is inbuilt in the karma theory ( as explained in the ancient indian scriptures such as the bhagavad gita ) , which states that one reaps the consequences of one 's actions and one suffers from a disease such as cancer because of his / her misdeeds , either in this life or in a previous existence . in such a scenario , the patient either blames himself / herself , others , or his / her fate , which facilitates acceptance of the disease by the survivor easily . this research finding brings out a very important inference , which can be used in the oncological care for patients with head and neck cancers : in this context , focus on psychosocial intervention for the promotion of effective and healthy coping strategies is essential , especially soon after surgery . as a well - being promotion intervention , it is useful to design strategies that locate the distress , detract from it , and project it in a suitably acceptable and conducive manner and then move on to replace the distress with deflective interventions with an aim to build and strengthen the resilience in the individual suffering from head and neck cancers . though the generalization of the results of this study is limited by the inclusion criteria which was fairly homogenous and being country specific , the researchers believe that the results of this study are a significant contribution to the field of psychosocial oncology . further , the conceptual framework of this study could form a base for future quantitative studies that want to test the efficacy of any need - based psychosocial program for inpatients with head and neck cancers . this result however , needs to be tested outside india as the context of the emotions experienced by inpatients , the type of emotions expressed as well as the coping strategies could differ from culture to culture based on their underlying mores , ideologies , faiths , religions , and beliefs that have a bearing on the coping of patients in that culture .

impairments in attention , cognitive control , and speed of information processing have been frequently observed after traumatic brain injury ( tbi ; stuss et al . , 1989 ; spikman et al . , deficits in these processes , which are considered to involve large - scale neural interactions ( mesulam , 1998 ) , may arise in part due to the diffuse underlying neuropathology present after tbi . while focal lesions play a significant role in functional outcome , the presence of diffuse axonal injury ( dai ) is more ubiquitous after tbi ( gentleman et al . , 1995 ) , and profound cognitive deficits have been observed in patients with dai in the absence of focal lesions ( scheid et al . , 2006 ) . in dai , rapid deceleration of the head results in disruption of ionic homeostasis , cytoskeletal misalignment , swelling of the axonal segment , and ultimate deafferentation of the axon from its synaptic field ( povlishock and christman , 1995 ; maxwell et al . , 1997 ) , ultimately causing volume loss throughout the cerebrum , especially in patients with moderate - to - severe injury ( levine et al . , 2008 ) . prefrontal cortical regions may be especially vulnerable due to their extensive reentrant connections with most cortical and subcortical regions ( petrides and pandya , 1999 ) . it has been suggested that functional recovery of the cognitive and behavioral sequelae of tbi may involve restoration of integration between frontal areas and modality - specific posterior regions ( chen et al . , 2006 ) . functional neuroimaging studies of attention and working memory have generally shown more widespread activation in tbi patients as compared to healthy subjects , particularly in frontal and temporal parietal areas ( christodoulou et al . , 2001 ; levine et al . , 2002 ; mcallister et al . , 2006 ; kim et al . , 2008 ; turner and levine , 2008 ) . this increase in the extent of neural activity after tbi is similar to patterns that have been observed during aging , where older participants show increased bilateral activity across a range of memory and attentional tasks in prefrontal cortex ( cabeza et al . , 2002 , 2004 ; de chastelaine et al . , 2011 ) as well as posterior parietal cortices ( huang et al . , 2011 ; vallesi et al . , 2011 ) greater recruitment of regions homologous to areas used for cognitive processes in controls has also been found ( christodoulou et al . , 2001 ; levine et al . , 2002 ; maruishi et al . , 2007 , for exception see perlstein et al . , most previous studies have been confounded methodologically either by the fact that tbi patients with and without focal lesions were mixed within their samples or because differences in behavioral performance between tbi patients and controls made it difficult to disentangle brain activity underlying performance effects from neural changes resulting from head injury ( levine et al . , 2006 ) . neuroimaging studies have also often used clinical neuropsychological tests of attention ( e.g. , stroop task , pasat ) that can have limited construct validity ( spikman et al . , 2001 ) and where there are limited data concerning functional neuroanatomy supporting these tests in healthy subjects . here we assessed neural activity patterns in patients with moderate - to - severe tbi with evidence of dai as the primary neuropathology and without significant focal lesions . we used a visual feature integration task designed to measure various attentional processes ( stuss et al . , 1989 ) . the task included conditions of increasing complexity and difficulty : a single - feature condition , where subjects had to respond to a target shape consisting of a specific combination of components and ignore other distracter shapes , requiring only one component to be kept in mind , and a multi - feature condition where subjects could only respond correctly to the target shape if all three components were kept in mind . the task also included a condition designed to assess focused attention , in which subjects avoided potential distraction by redundant information . tbi patients previously studied had slower reaction times ( rts ) in both single- and multi - feature conditions of the task compared to controls ( stuss et al . , 1989 ) . in addition , tbi patients demonstrated more variability in rt across all three conditions than in comparison subjects ( stuss et al . , 1994 ) . thus , one major advantage to this study is the use of an experimentally validated test to examine the neural correlates of attention in tbi ( stuss et al . , 2002 ) . given the diffuse neuropathology of our patient sample , we predicted increases in distributed alterations in neural activity following tbi in association with performance of this task , particularly in prefrontal regions . we used a multivariate neuroimage analysis method , partial least squares ( pls ; mcintosh et al . , 2004 ) , which is more sensitive to such distributed effects than traditional univariate neuroimaging analysis approaches as it emphasizes the interdependencies among neural regions . eight patients ( four males ) with moderate - to - severe tbi were recruited . all subjects were right - handed , native english speakers and were screened for previous neurological injury , major medical conditions affecting cognition , and history of psychiatric illness . ten healthy comparison subjects ( three males ) were recruited for functional imaging comparison ; two other groups helped characterize tbi patients neuropsychological status and structural neuroimaging data ( see table 1 ) . traumatic brain injury patient demographics , acute injury characteristics , structural neuroimaging data , and neuropsychological test data . demographics are compared to the functional imaging control subjects ( n = 10 ) . structural neuroimaging data are compared to age - matched healthy adults ( n = 12 ; levine et al . , 2008 ) . neuropsychological data are compared to age- , education- , and socioeconomic - matched healthy adults ( n = 27 ; turner and levine , 2008 ) . data from all three groups are included in the column control in the table . traumatic brain injury patients were recruited from consecutive admissions as part of the toronto tbi study ( fujiwara et al . , 2008 ; levine et al . , 2008 ; all patients had sustained a tbi as a result of a motor vehicle accident and were in the chronic stage of recovery at the time of study participation ( see table 1 ) . despite their significant tbis , all patients demonstrated good functional recovery as evidenced by a return to pre - injury employment or academic status . injury severity was determined by gcs as documented by the trauma team leader s score at the time of discharge from the trauma unit , corresponding to the recommended 6-h gcs score ( teasdale and jennett , 1974 ) . severity in one case ( 1054 ) was upgraded from that indicated by the gcs due to extended post - traumatic amnesia . seven of the eight patients underwent a separate structural mri protocol ( for more details see levine et al . , 2008 ) . interpretation by a board - certified radiologist specializing in tbi indicated evidence of dai - related neuropathology ( hemosiderin deposits ) localized to the frontal lobes ( six patients ) , parietal lobes ( four patients ) , the temporal lobe ( two patient ) , the corpus callosum ( two patients ) , the basal ganglia ( one patient ) , and the thalamus ( one patient ) . no patient had lesions greater than 3 mm in diameter , except patient 1047 who had a lesion in the r temporal pole , with a volume of 0.713 l 10 . this patient was retained in the sample as the lesion was relatively small and in a region remote from areas crucial to processing of the feature integration task . whole - brain volumetric measures of gray matter , white matter , and csf following our published image analysis methods ( levine et al . , 2008 ) showed evidence of atrophy relative to age- and education - matched comparison subjects . taken together , the radiological interpretation and significant volume loss in the tbi patients are consistent with dai . all eight patients underwent neuropsychological testing . neuropsychological test data were compared with a local normative sample of age , education , and socioeconomically matched control subjects . tbi patients demonstrated average to high average performance on the verbal subtask of the shipley institute of living scale ( zachary , 1986 ) . tbi patients also performed normally on other neuropsychological tests of attention and executive functioning , including ( with one exception ) a task explicitly measuring executive control within working memory . the experimental design was based on a previous behavioral study that examined the relationship between rt and varying degrees of complexity on a feature integration task in subjects with head injury ( stuss et al . , 1989 ) . subjects were shown geometric shapes while scanning during one of three conditions . in each condition , the stimuli were presented at random and considered either subjects were instructed to press one button to the target stimulus and a different button to the non - target stimulus ( see below for details ) . the stimuli had three different components ( shape , color , and line orientation within the shape ) , and each of these could appear in one of four possible states ( see figure 1 for examples ) : depiction of the single - feature , multi - feature , and redundant reaction time conditions . for each condition , examples of a target and of non - targets are shown where relevant . single - feature condition : one of the four geometric shapes selected as the target was presented randomly . both target and non - target shapes shared the same color and line orientation components so that the stimuli were complex , but subjects had to make their choice based solely on shape . redundant condition : the stimuli were characterized by the same components as in the multi - feature and single - feature conditions , but no state specific to the target could ever appear in the non - target . for example if the target was a red circle with vertical lines , no non - target appeared as red , a circle , or with vertical lines . hence the stimuli were as complex as in the single- and multi - feature conditions , but most of the information was redundant . stimuli were intermixed with visual fixation on a cross , which was taken as the baseline condition for brain activity . the timing of the stimuli corresponded to an event - related design so that the hemodynamic response function ( hrf ) could be optimally separated out for each stimulus . stimuli were presented in the following manner : participants rested their index and middle fingers on a two - button pad . at the beginning of each recording block , a target stimulus was presented for 6 s. four seconds after the target stimulus disappeared , a series of 48 test stimuli were presented at inter - stimulus intervals varying between 2.5 and 7.5 s. each test stimulus remained on screen for 2 s or until a response was made . thus , there were a total of 36 target trials and 108 non - target trials in each condition . mean rt and variability [ defined as coefficient of variation ( sd / mean ) , cvrt ] measures were calculated for each condition for the non - target trials . non - target trials were analyzed separately as they required more complex attentional processing demands than the target trials . by definition , the target trials match on all possible features , whereas the non - target trials have to be analyzed on a feature - by - feature basis to determine the match . in addition , because the non - target trials share features with the targets in the single- and multi - feature conditions , correct response to the non - targets requires inhibition of the response to the target which may be triggered by these shared features . as there were only 36 target trials , these were not analyzed separately due to lack of power . control and tbi groups were compared on mean rt , cvrt , and accuracy on the task using independent samples t - tests . rt measurements were not available for one patient and one control subject due to technical difficulties . scanning was performed on a 3.0-t mri system ( signa 3t94 hardware , vh3m3 software ; general electric healthcare , waukesha , wi , usa ) . before functional scanning , standard high - resolution three - dimensional t1-weighted fast spoiled gradient ( fspgr ) echo pulse sequence was used to acquire 124 axial anatomic mri slices . functional scans were obtained using a single - shot t2-weighted pulse sequence with spiral in out , achieving 26 axial slices ( tr / te of 2000/30 ms , flip angle of 70 , acquisition matrix = 64 64 , fov of 20 cm , and 3.1 mm 3.1 mm 5 mm voxel resolution ) . images were preprocessed using the analysis of functional neuroimages ( afni ; cox , 1996 ) and statistical parametric mapping ( spm99 ) software ( wellcome department of cognitive neurology , london , uk ) . the functional time series was despiked , and six parameter rigid body motion correction was done by coregistering volumes to a reference functional scan using afni ( cox and jesmanowicz , 1999 ) . subsequently , using spm99 , images were spatially transformed to standard mni space ( evans et al . , the resulting voxel size was 4 mm 4 mm 4 mm . the data were then smoothed spatially with an 8-mm full - width half- maximum gaussian filter . the effect of any global differences in fmri signal intensity between individual subjects was removed by calculating the percentage signal change for each voxel during the active conditions as compared to the signal intensity during visual fixation for each subject within each run . the preprocessed fmri data were then analyzed using a pls approach ( mcintosh and lobaugh , 2004 ) . specifically , pls is a set of multivariate analysis techniques that can be used to identify a new set of variables ( called latent variables , lvs ) that relate any set of independent measures , such as the experimental design ( as in this study ) or activity in a seed region , to a set of dependent measures , in this case , brain activity of each subject group ( mcintosh et al . , 1996 ) . pls carries out the computation of the optimal least squares fit to cross - block correlation between the independent and dependent measures . in comparison to principal component analysis ( pca ) , pls has the advantage that solutions are constrained to the relevant experimental manipulations ( mcintosh and lobaugh , 2004 ) . relative to a more traditional general linear model ( glm ) analysis , pls is more powerful to detect distributed changes because it analyzes the patterns of covariance across all voxels simultaneously rather than assessing each voxel s activity as independent from the rest of the brain s activity . in addition , pls uses a bootstrapping estimation technique that emphasizes the reliability of voxel activation over signal strength alone , which allows the inclusion of consistently activating voxels , which may have been excluded in the glm analysis into the overall brain activity pattern . pls data are interpreted by relating the pattern of reliably active voxels to a set of brain scores , which are determined for each task condition and subject group . the brain scores are similar to the factor scores in a pca and indicate how strongly the subject groups express the brain activity pattern . if , for example , the brain score for a certain condition is positive , then the subject group expresses the pattern reliably for that condition in all voxels that have positive values above threshold . through this technique , we were able to assess changes in distributed activity occurring after the diffuse injuries caused by tbi . all the three task conditions ( single - feature , multi - feature , and redundant ) and baseline were included in this analysis . for each condition , the hrf of each voxel was defined as the intensity difference from trial onset during seven consecutive post - stimulus temporal lags ( lag = 2 s or 1 tr ) averaged across trials . no assumption was made about the shape of hrf , allowing investigation of changes in task - related activity at different lags along the whole temporal window of 14 s. the data matrix containing all voxels and associated activity values for the seven consecutive post - stimulus temporal lags ( columns ) for all conditions and subjects in each group ( rows ) was mean - centered column - wise with respect to overall grand average . singular value decomposition ( svd ) was then applied to this matrix to generate mutually orthogonal lvs , with decreasing order of magnitude , analogous to pca . an lv can be thought of as the optimal values linking the brain activity data with the task design . each lv consisted of : ( i ) a singular value , ( ii ) a pattern of design scores , which provided a set of contrasts across task conditions , and ( iii ) a singular image , which showed how the spatial distribution of activity across the brain relates to the task conditions and subject groups . the weighted linear combinations that related the brain activity measurements to group and task design were the salience values , which reflected how strongly each given voxel contributed to an lv . a brain score was generated for each subject by multiplying the salience values by the raw voxel data . this reflected how much a subject expressed the pattern of salience values across voxels and temporal lags . similarly , salience values were generated for the design , which reflected how strongly each task condition contributed to the lv . design scores were generated in a similar fashion as the brain scores using the design saliences . they can be thought of as a set of contrasts that code the effects resulting from the svd . interpretation of the relation between the polarity of the saliences in the singular image and the direction of hrf change in the areas reliably activated in each lv requires relating the saliences to the group brain scores . for instance , positive saliences would indicate areas that are relatively more active in conditions with positive brain scores . conversely , negative saliences would indicate areas that are relatively more active in conditions with negative brain scores ( see figure 3 below , for an example ) . the significance for each lv as a whole was determined by 500 permutation tests ( edgington , 1980 ) , in which the observations were randomly reordered without replacement to calculate the probability of each lv having occurred by chance . the stability of each voxel s contribution to the latent variable was determined through bootstrap resampling ( sampson et al . voxels were considered reliable if they had a ratio of salience value to se ( hereafter referred to as the bootstrap ratio , which can be interpreted similarly to a z score if the distribution for a given voxel is normal ; efron and tibshirani , 1986 ) . for each lag , clusters with at least 50 contiguous voxels with a bootstrap ratio + 2.5 or at least 250 contiguous voxels with a bootstrap ratio 2.5 ( equivalent to a confidence interval of 99% ) were considered to be reliable . as will be seen in the results , the asymmetric cluster size thresholds for positive and negative bootstrap ratios were dictated by large group differences in activity patterns . coordinates of the voxel with the peak bootstrap ratio within each cluster were obtained in mni space . the approximate gyral locations and brodmann areas were then identified using the talairach daemon tool ( lancaster et al . , 2000 ) . we ran two supplementary pls analyses examining the relationship between brain activity across voxels and experimental conditions separately in both control and tbi patient groups . reliability and significance were assessed in the same way as the overall pls analysis described above . we then computed the intersection between each individual group analysis with the overall analysis by identifying voxels that had robust activity ( defined by a bootstrap ratio 2.5 ) in both analyses . this allowed us to identify voxels in which there was a convergence of activity in one subject group with the activity in the overall analysis and helped determine which subject group was driving the overall activity pattern . supplementary analyses were done on the hemodynamic response signal obtained from selected clusters of activity that were identified by the task pls to confirm the effects obtained between groups and across task conditions . two - way analysis of variances ( anova ) looking at the effects of group and task condition were conducted on clusters with both positive and negative saliences . eight patients ( four males ) with moderate - to - severe tbi were recruited . all subjects were right - handed , native english speakers and were screened for previous neurological injury , major medical conditions affecting cognition , and history of psychiatric illness . ten healthy comparison subjects ( three males ) were recruited for functional imaging comparison ; two other groups helped characterize tbi patients neuropsychological status and structural neuroimaging data ( see table 1 ) . traumatic brain injury patient demographics , acute injury characteristics , structural neuroimaging data , and neuropsychological test data . demographics are compared to the functional imaging control subjects ( n = 10 ) . structural neuroimaging data are compared to age - matched healthy adults ( n = 12 ; levine et al . , 2008 ) . neuropsychological data are compared to age- , education- , and socioeconomic - matched healthy adults ( n = 27 ; turner and levine , 2008 ) . data from all three groups are included in the column control in the table . traumatic brain injury patients were recruited from consecutive admissions as part of the toronto tbi study ( fujiwara et al . , 2008 ; levine et al . , 2008 ; all patients had sustained a tbi as a result of a motor vehicle accident and were in the chronic stage of recovery at the time of study participation ( see table 1 ) . despite their significant tbis , all patients demonstrated good functional recovery as evidenced by a return to pre - injury employment or academic status . injury severity was determined by gcs as documented by the trauma team leader s score at the time of discharge from the trauma unit , corresponding to the recommended 6-h gcs score ( teasdale and jennett , 1974 ) . severity in one case ( 1054 ) was upgraded from that indicated by the gcs due to extended post - traumatic amnesia . seven of the eight patients underwent a separate structural mri protocol ( for more details see levine et al . , 2008 ) . interpretation by a board - certified radiologist specializing in tbi indicated evidence of dai - related neuropathology ( hemosiderin deposits ) localized to the frontal lobes ( six patients ) , parietal lobes ( four patients ) , the temporal lobe ( two patient ) , the corpus callosum ( two patients ) , the basal ganglia ( one patient ) , and the thalamus ( one patient ) . no patient had lesions greater than 3 mm in diameter , except patient 1047 who had a lesion in the r temporal pole , with a volume of 0.713 l 10 . this patient was retained in the sample as the lesion was relatively small and in a region remote from areas crucial to processing of the feature integration task . whole - brain volumetric measures of gray matter , white matter , and csf following our published image analysis methods ( levine et al . , 2008 ) showed evidence of atrophy relative to age- and education - matched comparison subjects . taken together , the radiological interpretation and significant volume loss in the tbi patients are consistent with dai . all eight patients underwent neuropsychological testing . neuropsychological test data were compared with a local normative sample of age , education , and socioeconomically matched control subjects . tbi patients demonstrated average to high average performance on the verbal subtask of the shipley institute of living scale ( zachary , 1986 ) . tbi patients also performed normally on other neuropsychological tests of attention and executive functioning , including ( with one exception ) a task explicitly measuring executive control within working memory . the experimental design was based on a previous behavioral study that examined the relationship between rt and varying degrees of complexity on a feature integration task in subjects with head injury ( stuss et al . , 1989 ) . subjects were shown geometric shapes while scanning during one of three conditions . in each condition , the stimuli were presented at random and considered either subjects were instructed to press one button to the target stimulus and a different button to the non - target stimulus ( see below for details ) . the stimuli had three different components ( shape , color , and line orientation within the shape ) , and each of these could appear in one of four possible states ( see figure 1 for examples ) : depiction of the single - feature , multi - feature , and redundant reaction time conditions . for each condition , examples of a target and of non - targets are shown where relevant . single - feature condition : one of the four geometric shapes selected as the target was presented randomly . both target and non - target shapes shared the same color and line orientation components so that the stimuli were complex , but subjects had to make their choice based solely on shape . redundant condition : the stimuli were characterized by the same components as in the multi - feature and single - feature conditions , but no state specific to the target could ever appear in the non - target . for example if the target was a red circle with vertical lines , no non - target appeared as red , a circle , or with vertical lines . hence the stimuli were as complex as in the single- and multi - feature conditions , but most of the information was redundant . stimuli were intermixed with visual fixation on a cross , which was taken as the baseline condition for brain activity . the timing of the stimuli corresponded to an event - related design so that the hemodynamic response function ( hrf ) could be optimally separated out for each stimulus . stimuli were presented in the following manner : participants rested their index and middle fingers on a two - button pad . at the beginning of each recording block , a target stimulus was presented for 6 s. four seconds after the target stimulus disappeared , a series of 48 test stimuli were presented at inter - stimulus intervals varying between 2.5 and 7.5 s. each test stimulus remained on screen for 2 s or until a response was made . thus , there were a total of 36 target trials and 108 non - target trials in each condition . mean rt and variability [ defined as coefficient of variation ( sd / mean ) , cvrt ] measures were calculated for each condition for the non - target trials . non - target trials were analyzed separately as they required more complex attentional processing demands than the target trials . by definition , the target trials match on all possible features , whereas the non - target trials have to be analyzed on a feature - by - feature basis to determine the match . in addition , because the non - target trials share features with the targets in the single- and multi - feature conditions , correct response to the non - targets requires inhibition of the response to the target which may be triggered by these shared features . as there were only 36 target trials , these were not analyzed separately due to lack of power . control and tbi groups were compared on mean rt , cvrt , and accuracy on the task using independent samples t - tests . rt measurements were not available for one patient and one control subject due to technical difficulties . scanning was performed on a 3.0-t mri system ( signa 3t94 hardware , vh3m3 software ; general electric healthcare , waukesha , wi , usa ) . before functional scanning , standard high - resolution three - dimensional t1-weighted fast spoiled gradient ( fspgr ) echo pulse sequence was used to acquire 124 axial anatomic mri slices . functional scans were obtained using a single - shot t2-weighted pulse sequence with spiral in out , achieving 26 axial slices ( tr / te of 2000/30 ms , flip angle of 70 , acquisition matrix = 64 64 , fov of 20 cm , and 3.1 mm 3.1 mm 5 mm voxel resolution ) . images were preprocessed using the analysis of functional neuroimages ( afni ; cox , 1996 ) and statistical parametric mapping ( spm99 ) software ( wellcome department of cognitive neurology , london , uk ) . the functional time series was despiked , and six parameter rigid body motion correction was done by coregistering volumes to a reference functional scan using afni ( cox and jesmanowicz , 1999 ) . the difference in the timing of slice acquisition subsequently , using spm99 , images were spatially transformed to standard mni space ( evans et al . , the resulting voxel size was 4 mm 4 mm 4 mm . the data were then smoothed spatially with an 8-mm full - width half- maximum gaussian filter . the effect of any global differences in fmri signal intensity between individual subjects was removed by calculating the percentage signal change for each voxel during the active conditions as compared to the signal intensity during visual fixation for each subject within each run . the preprocessed fmri data were then analyzed using a pls approach ( mcintosh and lobaugh , 2004 ) . specifically , pls is a set of multivariate analysis techniques that can be used to identify a new set of variables ( called latent variables , lvs ) that relate any set of independent measures , such as the experimental design ( as in this study ) or activity in a seed region , to a set of dependent measures , in this case , brain activity of each subject group ( mcintosh et al . , 1996 ) . pls carries out the computation of the optimal least squares fit to cross - block correlation between the independent and dependent measures . in comparison to principal component analysis ( pca ) , pls has the advantage that solutions are constrained to the relevant experimental manipulations ( mcintosh and lobaugh , 2004 ) . relative to a more traditional general linear model ( glm ) analysis , pls is more powerful to detect distributed changes because it analyzes the patterns of covariance across all voxels simultaneously rather than assessing each voxel s activity as independent from the rest of the brain s activity . in addition , pls uses a bootstrapping estimation technique that emphasizes the reliability of voxel activation over signal strength alone , which allows the inclusion of consistently activating voxels , which may have been excluded in the glm analysis into the overall brain activity pattern . pls data are interpreted by relating the pattern of reliably active voxels to a set of brain scores , which are determined for each task condition and subject group . the brain scores are similar to the factor scores in a pca and indicate how strongly the subject groups express the brain activity pattern . if , for example , the brain score for a certain condition is positive , then the subject group expresses the pattern reliably for that condition in all voxels that have positive values above threshold . through this technique , we were able to assess changes in distributed activity occurring after the diffuse injuries caused by tbi . all the three task conditions ( single - feature , multi - feature , and redundant ) and baseline were included in this analysis . for each condition , the hrf of each voxel was defined as the intensity difference from trial onset during seven consecutive post - stimulus temporal lags ( lag = 2 s or 1 tr ) averaged across trials . no assumption was made about the shape of hrf , allowing investigation of changes in task - related activity at different lags along the whole temporal window of 14 s. the data matrix containing all voxels and associated activity values for the seven consecutive post - stimulus temporal lags ( columns ) for all conditions and subjects in each group ( rows ) was mean - centered column - wise with respect to overall grand average . singular value decomposition ( svd ) was then applied to this matrix to generate mutually orthogonal lvs , with decreasing order of magnitude , analogous to pca . an lv can be thought of as the optimal values linking the brain activity data with the task design . each lv consisted of : ( i ) a singular value , ( ii ) a pattern of design scores , which provided a set of contrasts across task conditions , and ( iii ) a singular image , which showed how the spatial distribution of activity across the brain relates to the task conditions and subject groups . the weighted linear combinations that related the brain activity measurements to group and task design were the salience values , which reflected how strongly each given voxel contributed to an lv . a brain score was generated for each subject by multiplying the salience values by the raw voxel data . this reflected how much a subject expressed the pattern of salience values across voxels and temporal lags . similarly , salience values were generated for the design , which reflected how strongly each task condition contributed to the lv . design scores were generated in a similar fashion as the brain scores using the design saliences . they can be thought of as a set of contrasts that code the effects resulting from the svd . interpretation of the relation between the polarity of the saliences in the singular image and the direction of hrf change in the areas reliably activated in each lv requires relating the saliences to the group brain scores . for instance , positive saliences would indicate areas that are relatively more active in conditions with positive brain scores . conversely , negative saliences would indicate areas that are relatively more active in conditions with negative brain scores ( see figure 3 below , for an example ) . the significance for each lv as a whole was determined by 500 permutation tests ( edgington , 1980 ) , in which the observations were randomly reordered without replacement to calculate the probability of each lv having occurred by chance . the stability of each voxel s contribution to the latent variable was determined through bootstrap resampling ( sampson et al . , 1989 voxels were considered reliable if they had a ratio of salience value to se ( hereafter referred to as the bootstrap ratio , which can be interpreted similarly to a z score if the distribution for a given voxel is normal ; efron and tibshirani , 1986 ) . for each lag , clusters with at least 50 contiguous voxels with a bootstrap ratio + 2.5 or at least 250 contiguous voxels with a bootstrap ratio 2.5 ( equivalent to a confidence interval of 99% ) were considered to be reliable . as will be seen in the results , the asymmetric cluster size thresholds for positive and negative bootstrap ratios coordinates of the voxel with the peak bootstrap ratio within each cluster were obtained in mni space . the approximate gyral locations and brodmann areas were then identified using the talairach daemon tool ( lancaster et al . , 2000 ) . we ran two supplementary pls analyses examining the relationship between brain activity across voxels and experimental conditions separately in both control and tbi patient groups . reliability and significance were assessed in the same way as the overall pls analysis described above . we then computed the intersection between each individual group analysis with the overall analysis by identifying voxels that had robust activity ( defined by a bootstrap ratio 2.5 ) in both analyses . this allowed us to identify voxels in which there was a convergence of activity in one subject group with the activity in the overall analysis and helped determine which subject group was driving the overall activity pattern . supplementary analyses were done on the hemodynamic response signal obtained from selected clusters of activity that were identified by the task pls to confirm the effects obtained between groups and across task conditions . two - way analysis of variances ( anova ) looking at the effects of group and task condition were conducted on clusters with both positive and negative saliences . behaviorally , tbi patients and controls differed significantly on mean rt for all three task conditions [ single - feature : f(1,14 ) = 11.893 , p < 0.01 , cohen s d = 1.75 ; multi - feature : f(1,14 ) = 23.230 , p < 0.001 , cohen s d = 2.40 ; redundant : f(1,14 ) = 5.065 , p < 0.05 , cohen s d = 1.12 ] . for cvrt , there was a significant group difference for the multi - feature condition [ f(1,14 ) = 7.529 , p < 0.05 , cohen s d = 1.41 ] . the group difference in cvrt fell just short of statistical significance for the single - feature condition [ f(1,14 ) = 4.290 , p = 0.057 cohen s d = 1.09 ] . there was no significant difference for the redundant condition [ f(1,14 ) = 1.901 , p = n.s . finally , there was a significant group difference in accuracy on the multi - feature condition [ f(1,14 ) = 5.134 , p < 0.05 , cohen s d = 1.08 ] , but not the single - feature [ f(1,14 ) < 1 , p = n.s . , cohen s d = 0.29 ] or redundant conditions [ f(1,14 ) = 2.418 , p = n.s . , cohen s d = 0.73 ] . see figure 2 for the group behavioral results . mean reaction time ( rt ) ( a ) , coefficient of variation on reaction time ( cvrt ) ( b ) , and ( c ) accuracy for each task condition for functional imaging control subjects and tbi patients . * = significant differences between controls and tbi ( p < 0.05 ) . * * = significant differences between control and tbi ( p < 0.01 ) . the task pls analysis on the fmri data revealed one significant lv ( explained cross - block covariance = 29.94% , p < 0.024 ) . the group brain scores with 95% confidence intervals for the lv are shown in figure 3a . the lv showed similar activity patterns for tbi patients and controls , but these activity patterns were expressed for different conditions by each group : the multi - feature condition for controls and the single - feature condition and baseline in tbi patients . the lv also showed additional activity patterns in tbi patients for the multi - feature and redundant conditions . the clusters with positive and negative saliences are listed in table 2 , respectively , and are shown in figure 3b . regions with positive saliences show greater activity for the multi - feature condition in controls and the single - feature condition and baseline in tbi patients . these regions included cerebellum , thalamus , ventral premotor cortex , middle and posterior cingulate , dorsolateral prefrontal cortex ( dlpfc ) , anterior prefrontal cortex ( apfc ) , and the parahippocampal gyrus . regions with negative saliences show greater activity for the multi - feature and redundant conditions in tbi patients only and included a widespread set of regions in frontal , parietal , and occipital cortices . ( a ) brain scores for the significant latent variable ( lv ) from the task pls analysis . the average brain scores for each group and condition are considered to reliably contribute to the lv if the confidence interval does not cross 0 . the brain scores are similar to factor scores and indicate how strongly an individual subject expresses the patterns on the latent variable . the score is the dot product of the subject s raw brain activity data and the singular image from the latent variable showing the pattern of brain activity that is most related to the group and task differences ( mcintosh et al . , 2004 ) . ( b ) singular image showing reliable clusters ( number of voxels 50 for positive saliences , number of voxels 250 for negative saliences , bootstrap ratio 2.5 ) . the brain activity patterns occur five lags ( 10 s ) after stimulus onset time . the x axis shows the location of the axial slice with a coordinate along the z axis in mni atlas space . warm colors indicate clusters with positive bootstrap ratios , which were more active in group and task conditions with positive brain scores in ( a ) . cool colors indicate clusters with negative bootstrap ratios , which were more active for group and task conditions with negative brain scores . the bootstrap ratio map is superimposed on the average anatomical scans from all 18 subjects . reliable clusters identified for the significant lv in the task pls analysis ( bootstrap ratios lag refers to the time period , in trs of 2 s each , after stimulus onset during which the peak bootstrap ratio occurred . cluster region and ba indicate the locations and brodmann areas determined from talairach and tournoux ( 1988 ) . a designation for cross - reference to regions active in lag 5 and appearing in figure 3 is indicated . see figures 4a , b for the results of the two - way anovas on the signal of selected voxels identified by the task pls . ( a ) regions with positive saliences show a stronger hemodynamic response for tbi patients in the single - feature and baseline conditions relative to the other two conditions . = marginally significantly greater response than in single - feature and baseline conditions . ( b ) selected regions with negative saliences show a greater hemodynamic response for tbi patients than controls on either the multi - feature or the redundant condition . * = significantly greater response in tbi patients than controls . the supplementary individual group task pls analyses did not reveal a significant lv ( p > 0.05 ) for either control or tbi patient groups . qualitative inspection of the intersection between the control task pls and the overall analysis revealed almost no overlap in activity patterns ( figure 5a ) . however there was overlap between tbi brain activity and the overall analysis in several regions including two large clusters in the left inferior parietal lobule ( figure 5b ) . several regions show overlapping activity , including two larger clusters in the left inferior parietal lobule , suggesting the brain activity patterns in the overall analysis are primarily being driven by the tbi group . design score plots show pattern from the single group analysis in blue and the overall group analysis in pink ( repeated from figure 3a ) , with design scores for the single - feature , multi - feature , redundant , and baseline conditions shown from left to right for each group . the design scores reflect how much the task condition contributes to the overall latent variable pattern for each group . our findings demonstrate differentiated functional neural activity underlying attentional processes in subjects with dai neuropathology due to tbi compared to neurologically normal individuals . previous studies examining executive control processes such as working memory and attention after tbi have been confounded by the fact that heterogeneous neuropathology subtypes were included in their samples ( kim et al . focal lesions were restricted to small hemosiderin deposits spread throughout the cortex , which reflected the presence of dai , except in the case of one subject who had a small contusion in an area remote from regions supporting attentional processes . on the whole , the subjects in our sample showed a predominance of dai as the underlying neuropathology without the involvement of focal lesions . evidence of dai is additionally found in the fact that our patients show whole - brain parenchymal ( gray + white matter ) volume loss compared to controls . the relatively pure neuropathology of our sample allowed us to draw more specific conclusions about the effect of dai on neural activity supporting attention than has been previously possible . previous studies with tbi subjects have shown activity changes that occur on a distributed level ( christodoulou et al . , 2001 ; mcallister et al . , 2006 ; kim et al . , 2008 ; levine et al . , 2008 ; some studies have identified altered patterns of activity compared to controls ( levine et al . , 2002 ; kim et al . , 2008 ) whereas other studies have shown load - related activity differences in tbi patients in areas devoted to task processing that are also active in controls ( perlstein et al . , 2004 ) . in the present study , we demonstrate an overlapping set of regions that are active for the task in both tbi patients and controls ; however , we also find a set of regions that are uniquely recruited by tbi patients for the more visually complex task conditions . tbi patients and controls both show activity in a set of regions composed of cerebellum , thalamus , ventral premotor cortex , middle and posterior cingulate , dlpfc , apfc , and the parahippocampal gyrus . these regions have been shown to constitute large - scale networks involved in cognitive control and maintenance of task - relevant information , as well as play a role in spatial attention ( kim et al . , 1999 ) . a host of studies have ascribed top - down attentional control to the dlpfc ( e.g. , macdonald et al . , 2000 ; koechlin et al . , 2003 ; more recent functional connectivity work has also implicated middle cingulate / precuneus as comprising a network including dlpfc that is involved in task - related control ( dosenbach et al . , 2007 ) . in addition , the posterior cingulate is related to the speed of spatial target detection ( mesulam et al . , 2001 ) . posterior cingulate / retrosplenial cortex has extensive anatomical connections to neocortical and hippocampal regions ( pandya et al . , 1981 ) , and therefore may show greater activity during attention to events that have acquired salience through experience . several fmri studies have shown the apfc and frontal operculum to be involved in maintenance of task sets ( braver et al . , 2003 ; dosenbach et al . , 2006 ; sakai and passingham , 2006 ) , which may be more generally related to the role of these regions in working memory ( wager and smith , 2003 ) . more recent work has described a cingulo - opercular network that includes the thalamus and apfc that shows sustained activity for the maintenance of task goals ( dosenbach et al . , 2007 ) . both networks related to top - down control have been shown to have connections with the cerebellum ( dosenbach et al . , 2008 ) , which is important in error monitoring ( fiez , 1996 ) . it has also been recently shown that tbi patients with increased severity as indicated by gcs score show increased activity in cingulate and thalamic structures during a task requiring cognitive control ( scheibel et al . , 2009 ) ; however , these data were taken from tbi patients in the acute phase of recovery . in addition , it has been shown that older participants have augmented activity in regions related to cognitive control during more complex task processing demands ( vallesi et al . , 2011 ) , which may reflect some commonalities in neural reorganization in neuropathologies causing damage that is diffuse in nature . our results demonstrate that activity in regions thought to mediate cognitive control persists into the chronic stage of tbi for relatively moderate task demands . we did not find activity in the anterior cingulate and inferior parietal regions that are often associated with attentional control networks . this may be due to the fact that the analysis only identified overlapping clusters in tbi patients and controls , and these regions may show spatially differentiated activity patterns between the subject groups . we show that these brain activity patterns related to task processing are activated for the multi - feature condition in controls and the single - feature condition in tbi patients . that brain activity for relatively moderate working memory task demands in tbi patients is similar to processing of increased task demands in control subjects . in mcallister et al . ( 2001 ) study , parametrically increasing task load resulted in dose - dependent increases of activity in working memory - related regions for control subjects . however , tbi patients showed augmented activity during moderate processing loads that plateaued as task difficulty increased . here our findings show that control subjects recruit the neural regions described above while integrating three features of the target shape and maintaining this information throughout the task , whereas tbi patients require the same attentional resources in order to maintain only the shape of the target , which they are able to do with a similar level of accuracy compared to controls ( for an analysis of the functional connectivity underlying this pattern in patients with tbi and dai , some of which also participated in the present study , see turner et al . the fact that we were able to show this relationship in areas associated with cognitive control underscores the importance of using a task that is experimentally validated to measure attention as a construct when assessing the neural correlates of tbi . we additionally show that the tbi participants robustly recruit the same attentional resources involved in the single - feature condition during the baseline condition . importantly , this finding may inform previous research showing altered functional activity after tbi ( levine et al . , 2002 ) . while this prior study argued that altered functional activity may be due to cortical disinhibition resulting from the axonal deafferentation due to dai , the results could not easily disambiguate this from the possibility of cortical activity being instead driven by compensatory processes . our demonstration of inappropriate task - related activity occurring in the absence of a stimulus during the baseline trials appears to support the notion that altered functional activity in tbi is more likely due to cortical dysregulation and an inability to inhibit activity . furthermore , because the baseline trials were rapidly interspersed between the task trials , the mechanism of cortical dysregulation resulting in task - related activity persisting throughout the baseline may be an overall slowness in processing of all three task conditions for the tbi patients compared to the controls . although slowness in speed of information processing has been observed behaviorally after tbi ( see mathias and wheaton , 2007 for a review ) , there are no neuroimaging studies investigating a related delay in the hemodynamic response returning to baseline , and therefore this remains an open question . it has been suggested that a lack of focusing of activity after tbi reflects reduced efficiency of neural processing ( levine et al . our results demonstrate a widespread set of frontal , parietal , and occipital regions that are uniquely active in tbi patients during the multi - feature and redundant conditions . it is likely that this set of regions identified in tbi patients reflects decreased efficiency during task processing as would also seem to be suggested by the slowed task performance compared to controls . similar findings of more widespread activity in older participants has also been interpreted as indicative of neural inefficiency in cases when performance was matched to or worse than controls ( morcom et al . . both of these conditions present stimuli of increasing complexity compared to the single - feature condition . in addition , both conditions are more difficult than the single - feature condition because the multi - feature condition requires subjects to integrate more than one salient feature of the target shape , and , while the redundant condition task can be performed by only remembering one feature , subjects are required to suppress responses to distracting features of the non - target shapes . tbi patients often show increased difficulty with focused attention and distractibility ( levine et al . , 1998 ) , and this is reflected by the fact that the tbi patients made more errors than controls on both the multi - feature and redundant conditions , although this difference was only significant during the multi - feature conditions . our findings support previous studies showing more dispersed activity in tbi patients during tasks of working memory and attention ( christodoulou et al . , 2001 ; levine et al . , 2002 ; kim et al . , 2008 ) and strongly underscore the distributed nature of the dai . it is likely that rehabilitation may be accompanied by a reintegration of regions involved in frontoparietal networks ( chen et al . , 2006 ) . previous research has shown decrease in the volume and degree of activation in inferior and middle frontal and parietal regions accompanying improved performance in tbi patients after cognitive training ( kim et al . , 2008 ) . as noted above , this finding may be related to disinhibition due deafferentation of axons as a result of dai . in the presence of a complex task , however , the possibility of compensatory activity changes related to coping with increasing processing demands can not be ruled out . because all of the patients in the sample are in the chronic stage , it is feasible that neuroplastic changes occurred since the time of injury . indeed , axonal sprouting and synaptogenesis have been shown to take place during recovery ( povlishock , 1992 ) . finally , a comparison of the intersection between the individual control task pls and the overall group analysis vs. the intersection with the individual tbi task pls and the overall group analysis revealed that there was overlap mainly between tbi group activity and the overall analysis . this indicated that the regional patterns we identified were tapping into cognitive processing occurring mainly in the patient group after the injury . it is important to point out that most of the patients in the sample show relatively normal performance on neuropsychological tests of attention , working memory , and executive function . this is a common occurrence despite the fact that many tbi patients complain of having difficulty coping with activities of daily life . the insensitivity of traditional neuropsychological tests to the subjective deficits reported by tbi patients poses a problem in terms of diagnosis and treatment . detection of altered patterns of neural activity may provide a more fine - tuned measure of decreased organization and efficiency of cognitive processes and may be a beneficial addition to a clinician s standard diagnostic arsenal . 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 .

traumatic brain injury ( tbi ) patients typically respond more slowly and with more variability than controls during tasks of attention requiring speeded reaction time . these behavioral changes are attributable , at least in part , to diffuse axonal injury ( dai ) , which affects integrated processing in distributed systems . here we use a multivariate method sensitive to distributed neural activity to compare brain activity patterns of patients with chronic phase moderate to - severe tbi to those of controls during performance on a visual feature integration task assessing complex attentional processes that has previously shown sensitivity to tbi . the tbi patients were carefully screened to be free of large focal lesions that can affect performance and brain activation independently of dai . the task required subjects to hold either one or three features of a target in mind while suppressing responses to distracting information . in controls , the multi - feature condition activated a distributed network including limbic , prefrontal , and medial temporal structures . tbi patients engaged this same network in the single - feature and baseline conditions . in multi - feature presentations , tbi patients alone activated additional frontal , parietal , and occipital regions . these results are consistent with neuroimaging studies using tasks assessing different cognitive domains , where increased spread of brain activity changes was associated with tbi . our results also extend previous findings that brain activity for relatively moderate task demands in tbi patients is similar to that associated with of high task demands in controls .

Introduction Materials and Methods Subjects Visual feature-matching task fMRI scanning and analyses Results Discussion Conflict of Interest Statement

impairments in attention , cognitive control , and speed of information processing have been frequently observed after traumatic brain injury ( tbi ; stuss et al . , deficits in these processes , which are considered to involve large - scale neural interactions ( mesulam , 1998 ) , may arise in part due to the diffuse underlying neuropathology present after tbi . while focal lesions play a significant role in functional outcome , the presence of diffuse axonal injury ( dai ) is more ubiquitous after tbi ( gentleman et al . , 1995 ) , and profound cognitive deficits have been observed in patients with dai in the absence of focal lesions ( scheid et al . , 1997 ) , ultimately causing volume loss throughout the cerebrum , especially in patients with moderate - to - severe injury ( levine et al . functional neuroimaging studies of attention and working memory have generally shown more widespread activation in tbi patients as compared to healthy subjects , particularly in frontal and temporal parietal areas ( christodoulou et al . this increase in the extent of neural activity after tbi is similar to patterns that have been observed during aging , where older participants show increased bilateral activity across a range of memory and attentional tasks in prefrontal cortex ( cabeza et al . , most previous studies have been confounded methodologically either by the fact that tbi patients with and without focal lesions were mixed within their samples or because differences in behavioral performance between tbi patients and controls made it difficult to disentangle brain activity underlying performance effects from neural changes resulting from head injury ( levine et al . here we assessed neural activity patterns in patients with moderate - to - severe tbi with evidence of dai as the primary neuropathology and without significant focal lesions . we used a visual feature integration task designed to measure various attentional processes ( stuss et al . the task included conditions of increasing complexity and difficulty : a single - feature condition , where subjects had to respond to a target shape consisting of a specific combination of components and ignore other distracter shapes , requiring only one component to be kept in mind , and a multi - feature condition where subjects could only respond correctly to the target shape if all three components were kept in mind . tbi patients previously studied had slower reaction times ( rts ) in both single- and multi - feature conditions of the task compared to controls ( stuss et al . in addition , tbi patients demonstrated more variability in rt across all three conditions than in comparison subjects ( stuss et al . given the diffuse neuropathology of our patient sample , we predicted increases in distributed alterations in neural activity following tbi in association with performance of this task , particularly in prefrontal regions . , 2004 ) , which is more sensitive to such distributed effects than traditional univariate neuroimaging analysis approaches as it emphasizes the interdependencies among neural regions . eight patients ( four males ) with moderate - to - severe tbi were recruited . traumatic brain injury patient demographics , acute injury characteristics , structural neuroimaging data , and neuropsychological test data . traumatic brain injury patients were recruited from consecutive admissions as part of the toronto tbi study ( fujiwara et al . interpretation by a board - certified radiologist specializing in tbi indicated evidence of dai - related neuropathology ( hemosiderin deposits ) localized to the frontal lobes ( six patients ) , parietal lobes ( four patients ) , the temporal lobe ( two patient ) , the corpus callosum ( two patients ) , the basal ganglia ( one patient ) , and the thalamus ( one patient ) . this patient was retained in the sample as the lesion was relatively small and in a region remote from areas crucial to processing of the feature integration task . taken together , the radiological interpretation and significant volume loss in the tbi patients are consistent with dai . the experimental design was based on a previous behavioral study that examined the relationship between rt and varying degrees of complexity on a feature integration task in subjects with head injury ( stuss et al . the stimuli had three different components ( shape , color , and line orientation within the shape ) , and each of these could appear in one of four possible states ( see figure 1 for examples ) : depiction of the single - feature , multi - feature , and redundant reaction time conditions . single - feature condition : one of the four geometric shapes selected as the target was presented randomly . redundant condition : the stimuli were characterized by the same components as in the multi - feature and single - feature conditions , but no state specific to the target could ever appear in the non - target . hence the stimuli were as complex as in the single- and multi - feature conditions , but most of the information was redundant . stimuli were intermixed with visual fixation on a cross , which was taken as the baseline condition for brain activity . by definition , the target trials match on all possible features , whereas the non - target trials have to be analyzed on a feature - by - feature basis to determine the match . in addition , because the non - target trials share features with the targets in the single- and multi - feature conditions , correct response to the non - targets requires inhibition of the response to the target which may be triggered by these shared features . specifically , pls is a set of multivariate analysis techniques that can be used to identify a new set of variables ( called latent variables , lvs ) that relate any set of independent measures , such as the experimental design ( as in this study ) or activity in a seed region , to a set of dependent measures , in this case , brain activity of each subject group ( mcintosh et al . in addition , pls uses a bootstrapping estimation technique that emphasizes the reliability of voxel activation over signal strength alone , which allows the inclusion of consistently activating voxels , which may have been excluded in the glm analysis into the overall brain activity pattern . pls data are interpreted by relating the pattern of reliably active voxels to a set of brain scores , which are determined for each task condition and subject group . the brain scores are similar to the factor scores in a pca and indicate how strongly the subject groups express the brain activity pattern . all the three task conditions ( single - feature , multi - feature , and redundant ) and baseline were included in this analysis . an lv can be thought of as the optimal values linking the brain activity data with the task design . each lv consisted of : ( i ) a singular value , ( ii ) a pattern of design scores , which provided a set of contrasts across task conditions , and ( iii ) a singular image , which showed how the spatial distribution of activity across the brain relates to the task conditions and subject groups . as will be seen in the results , the asymmetric cluster size thresholds for positive and negative bootstrap ratios were dictated by large group differences in activity patterns . eight patients ( four males ) with moderate - to - severe tbi were recruited . traumatic brain injury patient demographics , acute injury characteristics , structural neuroimaging data , and neuropsychological test data . traumatic brain injury patients were recruited from consecutive admissions as part of the toronto tbi study ( fujiwara et al . , 2008 ; all patients had sustained a tbi as a result of a motor vehicle accident and were in the chronic stage of recovery at the time of study participation ( see table 1 ) . interpretation by a board - certified radiologist specializing in tbi indicated evidence of dai - related neuropathology ( hemosiderin deposits ) localized to the frontal lobes ( six patients ) , parietal lobes ( four patients ) , the temporal lobe ( two patient ) , the corpus callosum ( two patients ) , the basal ganglia ( one patient ) , and the thalamus ( one patient ) . this patient was retained in the sample as the lesion was relatively small and in a region remote from areas crucial to processing of the feature integration task . taken together , the radiological interpretation and significant volume loss in the tbi patients are consistent with dai . the experimental design was based on a previous behavioral study that examined the relationship between rt and varying degrees of complexity on a feature integration task in subjects with head injury ( stuss et al . the stimuli had three different components ( shape , color , and line orientation within the shape ) , and each of these could appear in one of four possible states ( see figure 1 for examples ) : depiction of the single - feature , multi - feature , and redundant reaction time conditions . single - feature condition : one of the four geometric shapes selected as the target was presented randomly . redundant condition : the stimuli were characterized by the same components as in the multi - feature and single - feature conditions , but no state specific to the target could ever appear in the non - target . hence the stimuli were as complex as in the single- and multi - feature conditions , but most of the information was redundant . stimuli were intermixed with visual fixation on a cross , which was taken as the baseline condition for brain activity . by definition , the target trials match on all possible features , whereas the non - target trials have to be analyzed on a feature - by - feature basis to determine the match . in addition , because the non - target trials share features with the targets in the single- and multi - feature conditions , correct response to the non - targets requires inhibition of the response to the target which may be triggered by these shared features . functional scans were obtained using a single - shot t2-weighted pulse sequence with spiral in out , achieving 26 axial slices ( tr / te of 2000/30 ms , flip angle of 70 , acquisition matrix = 64 64 , fov of 20 cm , and 3.1 mm 3.1 mm 5 mm voxel resolution ) . specifically , pls is a set of multivariate analysis techniques that can be used to identify a new set of variables ( called latent variables , lvs ) that relate any set of independent measures , such as the experimental design ( as in this study ) or activity in a seed region , to a set of dependent measures , in this case , brain activity of each subject group ( mcintosh et al . in addition , pls uses a bootstrapping estimation technique that emphasizes the reliability of voxel activation over signal strength alone , which allows the inclusion of consistently activating voxels , which may have been excluded in the glm analysis into the overall brain activity pattern . all the three task conditions ( single - feature , multi - feature , and redundant ) and baseline were included in this analysis . each lv consisted of : ( i ) a singular value , ( ii ) a pattern of design scores , which provided a set of contrasts across task conditions , and ( iii ) a singular image , which showed how the spatial distribution of activity across the brain relates to the task conditions and subject groups . behaviorally , tbi patients and controls differed significantly on mean rt for all three task conditions [ single - feature : f(1,14 ) = 11.893 , p < 0.01 , cohen s d = 1.75 ; multi - feature : f(1,14 ) = 23.230 , p < 0.001 , cohen s d = 2.40 ; redundant : f(1,14 ) = 5.065 , p < 0.05 , cohen s d = 1.12 ] . for cvrt , there was a significant group difference for the multi - feature condition [ f(1,14 ) = 7.529 , p < 0.05 , cohen s d = 1.41 ] . the group difference in cvrt fell just short of statistical significance for the single - feature condition [ f(1,14 ) = 4.290 , p = 0.057 cohen s d = 1.09 ] . finally , there was a significant group difference in accuracy on the multi - feature condition [ f(1,14 ) = 5.134 , p < 0.05 , cohen s d = 1.08 ] , but not the single - feature [ f(1,14 ) < 1 , p = n.s . mean reaction time ( rt ) ( a ) , coefficient of variation on reaction time ( cvrt ) ( b ) , and ( c ) accuracy for each task condition for functional imaging control subjects and tbi patients . the lv showed similar activity patterns for tbi patients and controls , but these activity patterns were expressed for different conditions by each group : the multi - feature condition for controls and the single - feature condition and baseline in tbi patients . the lv also showed additional activity patterns in tbi patients for the multi - feature and redundant conditions . regions with positive saliences show greater activity for the multi - feature condition in controls and the single - feature condition and baseline in tbi patients . these regions included cerebellum , thalamus , ventral premotor cortex , middle and posterior cingulate , dorsolateral prefrontal cortex ( dlpfc ) , anterior prefrontal cortex ( apfc ) , and the parahippocampal gyrus . regions with negative saliences show greater activity for the multi - feature and redundant conditions in tbi patients only and included a widespread set of regions in frontal , parietal , and occipital cortices . the score is the dot product of the subject s raw brain activity data and the singular image from the latent variable showing the pattern of brain activity that is most related to the group and task differences ( mcintosh et al . the brain activity patterns occur five lags ( 10 s ) after stimulus onset time . ( a ) regions with positive saliences show a stronger hemodynamic response for tbi patients in the single - feature and baseline conditions relative to the other two conditions . = marginally significantly greater response than in single - feature and baseline conditions . ( b ) selected regions with negative saliences show a greater hemodynamic response for tbi patients than controls on either the multi - feature or the redundant condition . * = significantly greater response in tbi patients than controls . however there was overlap between tbi brain activity and the overall analysis in several regions including two large clusters in the left inferior parietal lobule ( figure 5b ) . several regions show overlapping activity , including two larger clusters in the left inferior parietal lobule , suggesting the brain activity patterns in the overall analysis are primarily being driven by the tbi group . design score plots show pattern from the single group analysis in blue and the overall group analysis in pink ( repeated from figure 3a ) , with design scores for the single - feature , multi - feature , redundant , and baseline conditions shown from left to right for each group . our findings demonstrate differentiated functional neural activity underlying attentional processes in subjects with dai neuropathology due to tbi compared to neurologically normal individuals . focal lesions were restricted to small hemosiderin deposits spread throughout the cortex , which reflected the presence of dai , except in the case of one subject who had a small contusion in an area remote from regions supporting attentional processes . on the whole , the subjects in our sample showed a predominance of dai as the underlying neuropathology without the involvement of focal lesions . previous studies with tbi subjects have shown activity changes that occur on a distributed level ( christodoulou et al . , 2008 ) whereas other studies have shown load - related activity differences in tbi patients in areas devoted to task processing that are also active in controls ( perlstein et al . in the present study , we demonstrate an overlapping set of regions that are active for the task in both tbi patients and controls ; however , we also find a set of regions that are uniquely recruited by tbi patients for the more visually complex task conditions . , 2011 ) , which may reflect some commonalities in neural reorganization in neuropathologies causing damage that is diffuse in nature . our results demonstrate that activity in regions thought to mediate cognitive control persists into the chronic stage of tbi for relatively moderate task demands . this may be due to the fact that the analysis only identified overlapping clusters in tbi patients and controls , and these regions may show spatially differentiated activity patterns between the subject groups . we show that these brain activity patterns related to task processing are activated for the multi - feature condition in controls and the single - feature condition in tbi patients . that brain activity for relatively moderate working memory task demands in tbi patients is similar to processing of increased task demands in control subjects . however , tbi patients showed augmented activity during moderate processing loads that plateaued as task difficulty increased . here our findings show that control subjects recruit the neural regions described above while integrating three features of the target shape and maintaining this information throughout the task , whereas tbi patients require the same attentional resources in order to maintain only the shape of the target , which they are able to do with a similar level of accuracy compared to controls ( for an analysis of the functional connectivity underlying this pattern in patients with tbi and dai , some of which also participated in the present study , see turner et al . we additionally show that the tbi participants robustly recruit the same attentional resources involved in the single - feature condition during the baseline condition . our demonstration of inappropriate task - related activity occurring in the absence of a stimulus during the baseline trials appears to support the notion that altered functional activity in tbi is more likely due to cortical dysregulation and an inability to inhibit activity . furthermore , because the baseline trials were rapidly interspersed between the task trials , the mechanism of cortical dysregulation resulting in task - related activity persisting throughout the baseline may be an overall slowness in processing of all three task conditions for the tbi patients compared to the controls . although slowness in speed of information processing has been observed behaviorally after tbi ( see mathias and wheaton , 2007 for a review ) , there are no neuroimaging studies investigating a related delay in the hemodynamic response returning to baseline , and therefore this remains an open question . our results demonstrate a widespread set of frontal , parietal , and occipital regions that are uniquely active in tbi patients during the multi - feature and redundant conditions . it is likely that this set of regions identified in tbi patients reflects decreased efficiency during task processing as would also seem to be suggested by the slowed task performance compared to controls . both of these conditions present stimuli of increasing complexity compared to the single - feature condition . in addition , both conditions are more difficult than the single - feature condition because the multi - feature condition requires subjects to integrate more than one salient feature of the target shape , and , while the redundant condition task can be performed by only remembering one feature , subjects are required to suppress responses to distracting features of the non - target shapes . , 1998 ) , and this is reflected by the fact that the tbi patients made more errors than controls on both the multi - feature and redundant conditions , although this difference was only significant during the multi - feature conditions . our findings support previous studies showing more dispersed activity in tbi patients during tasks of working memory and attention ( christodoulou et al . previous research has shown decrease in the volume and degree of activation in inferior and middle frontal and parietal regions accompanying improved performance in tbi patients after cognitive training ( kim et al . in the presence of a complex task , however , the possibility of compensatory activity changes related to coping with increasing processing demands can not be ruled out . it is important to point out that most of the patients in the sample show relatively normal performance on neuropsychological tests of attention , working memory , and executive function . detection of altered patterns of neural activity may provide a more fine - tuned measure of decreased organization and efficiency of cognitive processes and may be a beneficial addition to a clinician s standard diagnostic arsenal .

cadmium ( cd ) is known to be a potent toxic metal and a significant environmental pollutant . studies concerning animals exposed to cadmium are of great interest since the presence of this metal in the environment has increased due to industrial activity and agricultural practices , thereby entering the food chain . in human populations , cadmium exposure occurs primarily through dietary sources and drinking water as well as cigarette smoking . cadmium exposure in mammals has been proved to cause bone problems and renal dysfunctions , alter reproductive functions and exert neurotoxic effects . it has been shown that cd is also an endocrine disruptor that may play a role in the aetiology of the pathologies that involve the hypothalamic - pituitary - testicular axis of mammals . cadmium mimics the function of steroid hormones and its potential role in the development of hormone - dependent cancers is widely discussed . studies carried out on rats also prove that cd is absorbed and retained in the pituitary gland , leading to a decrease in content of luteinizing hormone and alteration of gland functionality . in the mouse mus platythrix , cadmium induces hypertrophy and hyperplasia of pituitary gonadotrophs . in rat , cd modifies the lactotroph cell activity of the pituitary gland through biochemical , genomic and morphological changes and modifies plasma levels of luteinizing hormone ( lh ) and follicle - stimulating hormone ( fsh ) . lower concentrations of cdcl2 do not seem to influence the gonadotrope ( gth ) cells in the cyprinid puntius sarana . compared with other classes of vertebrates , reptiles are rarely used in studies on the possible toxic effects of heavy metals . however , they are important bioindicators because they are susceptible to the accumulation of persistent pollution which is identified as a major threat to reptile populations worldwide . although cd accumulation in various reptile organs has been studied , there has been very little experimental laboratory research on the effects of cadmium in reptiles . particularly lacking are effect - based studies in reptiles exposed to known concentrations of contaminants . although the physiological function of this metal is unknown , there is evidence to suggest that cadmium is a metallohormone with estrogenic and androgenic effects . as we have already reported elsewhere , in the lizard an acute treatment with a single high ( 20 g/10 g body mass ) intraperitoneal dose of cdcl2 not only induces apoptosis , especially in the rostral pars distalis , which appears irreversible , but also alters the normal endocrine function of the gland . once again in the lizard , we also reported that chronic exposure to cdcl2 affects the hormonal secretion of gth cells through an inhibitory effect . the aim of this paper was thus to analyse the effects of cadmium on acth and prl cells in the lizard podarcis sicula ( p. sicula ) exposed to chronic oral treatment for 120 days at an average dose ( 10 g/10 g body mass ) of cdcl2 . this study was performed on 30 adult females of p. sicula , captured near naples ( italy ) and kept under controlled conditions of light and temperature . cdcl2 was administered to the lizards in drinking water for four months at a daily dose of 10 g/10 g body mass while control lizards received cadmium - free water . groups of four treated and two control animals were killed at 10 , 30 , 60 , 90 and 120 days . experiments were performed in accordance with the guidelines for animal experimentation of the italian department of health under the supervision of a veterinarian , and organised to minimise stress and the number of lizards used . the pituitary gland is extremely small and almost completely enclosed in the sella turcica , which makes its removal difficult and may cause damage to the gland . after removal of the skullcap , the brains were fixed in bouin s solution for 48 h at room temperature and then decalcified in a solution of 5% edta in 10% formalin for 2530 days , dehydrated and enclosed in paraffin . this was the only procedure able to preserve not only the morphology but also the antigenicity of the cells . mallory s trichromic stain was used for the study of the general morphology while the immunohistochemical procedure was applied to identify and observe the adenohypophyseal cells . for immunohistochemical staining , the sections were processed according to the abc technique using the following heterologous antisera at specific working dilutions : anti - human prl ( 1/300 , signet laboratories , dedham , ma , usa ) and anti - synthetic acth1 - 24 ( 1/600 , biogenesis , poole , uk ) . visualization was carried out using the vectastain elite abc kit ( vector labs , inc . , burlingame , ca , usa ) and revealed by 3 mg 3,3-diaminobenzidine - tetrahydrochloride ( sigma , st . louis , mo , usa ) in 10 ml pbs and 150 l 3% h2o2 . antibody specificity was assessed by omitting the primary antisera and absorbing each antiserum with the specific hormone . the images were examined and acquired by a kontron electronic imaging system ks300 ( zeiss , oberkochen , germany ) . quantification of the two cellular types was carried out on at least 300 cells , with a visible nucleus , on serial sections per animal and relative to specific regions , namely the rostral , medial and caudal pars distalis and pars intermedia . the data obtained were pooled and analysed performing student s t - test to determine the significance ( p<0.05 ) between control and cadmium exposed groups . in all control specimens the pituitary gland appeared compact and extended in the cephalic - caudal direction in which the pars distalis ( pd ) was divided into a rostral part ( rpd ) , a caudal part ( cpd ) and a medial part ( mpd ) ( figure 1 a ) . the whole pd consisted of homogeneous vascularised cellular cordons with an evident basal lamina surrounding them and with the cells clearly identifiable by mallory stain ( figure 1 b ) . in the treated animals the pituitary gland tissue appeared atrophied in some areas , with wide irregular intercellular spaces , which appeared more extensive at 60 ( figure 1 c ) and evident also at 90 and 120 days . the gland also showed greater vascularisation ; a basal lamina surrounded the cellular cordons only partially and several cells appeared altered in shape ( figure 1 d ) . figure 1mallory stain . the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . ( d ) detail of panel c : the cellular cordons show a major vascularisation ( * ) , a basal lamina only partly surrounding them ( ) and several cells with altered shape ( ) . bar 140 m in a and c ; bar 40 m in b and d. mallory stain . ( a ) control lizard showing the extension of the gland . at the top the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . ( d ) detail of panel c : the cellular cordons show a major vascularisation ( * ) , a basal lamina only partly surrounding them ( ) and several cells with altered shape ( ) . bar 140 m in a and c ; bar 40 m in b and d. in the control lizards , acth and prl cells appeared clearly through immunohistochemical detection as distinct cellular populations with a specific distribution in the pd or pi ( pars intermedia ) . in treated specimens we observed the increase in occurrence and immunostain both for acth and for prl cells . acth and prl cells already at 10 days revealed a small increase , albeit with a different time course : prl cells appeared more abundant at 30 days from treatment , while acth were more copious at 60 days . in all these cells we also observed greater cytoplasmic immunostaining intensity . in control lizards prl cells were found essentially in the rpd ( 24.00.5% ) but also in the mpd ( 20.10.24% ) ( figure 2 a ) . they were generally isolated or clustered in small cellular cordons of three - five cells ( figure 2 b ) . prl cells appeared pyriform or ovoidal in shape , with an eccentric and ovoidal nucleus and a moderately dense cytoplasm ( figure 2 c ) . a few prl cells were also observed in the cpd ( 1.20.05% ) , but they were always absent in the pi . at 10 days of treatment prl cells had slightly increased in the rpd and mpd , but they were more numerous in the cpd ( table 1 ) . their occurrence peaked at 30 days from treatment ( figure 2 d ) : in the rpd ( 34.01.7% ) , mpd ( 39.81.2% ) and cpd ( 17.30.67% ) . in all these regions they also showed greater immunostaining intensity ( figure 2 e , f ) . at 60 and 90 days of treatment prl cells still appeared numerous , albeit with diminishing values , and at 120 days close to those of the control animals as reported in table 1 . ( a f ) prl cells ( in brown ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( b , c ) details of panel a showing these isolated cells ( b ) or organized into small cordons ( c ) . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g l ) acth cells ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. abc technique . ( a f ) prl cells ( in brown ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( b , c ) details of panel a showing these isolated cells ( b ) or organized into small cordons ( c ) . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. table 1percentage immunopositive prolactin cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd24.00.526.81.534.01.7 * 31.21.129.70.6624.81.1mpd20.10.2429.21.3539.81.225.41.04525.71.1123.10.9cpd1.20.058.30.56 * 17.30.675.10.33 * 3.90.42.10.2pi000000immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . immunopositive cells are expressed as mean sem ( % ) ; p<0.01 , compared with control groups . acth cells were observed in both the rpd ( 29.00.7% ) , and the mpd ( 12.00.13% ) , as well as in the pi ( 59.00.12% ) , but were absent in the cpd of the control specimens ( figure 2 g , i ) . acth cells were elongated in shape , with a generally central nucleus and higher cytoplasmic density in the pd ( figure 2 h ) compared with pi . at 10 days of treatment their occurrence appeared similar to that of control lizards , except for the presence of a few cells also in the cpd ( table 2 ) . acth cells increased markedly after 60 days of treatment with strong immunostaining of the cytoplasm in all the regions of the gland ( figure 2 j , k , l ) : in the rpd ( 40.31.11% ) , mpd ( 23.80.9% ) , pi ( 76.21.25% ) and cpd ( 16.10.85% ) . in the caudal region they appeared small and isolated cells , with no cordonal organization ( figure 2 l ) . at 90 and 120 days of treatment acth cells showed a similar distribution and morphology to the control ; in the cpd as well their occurrence had considerably decreased , appearing nearly absent at 120 days of treatment . table 2percentage immunopositive corticotropic cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd29.00.726.31.130.30.940.31.11 * 29.80.9529.30.13mpd12.00.1314.00.815.80.823.80.9 * 14.50.2112.80.69cpd00 1.50.363.70.18 * 16.10.855.10.6 * 0.350.3pi59.00.1260.11.8473.21.976.21.25 * 69.51.1163.21.18immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . immunopositive cells are expressed as mean sem ( % ) ; p<0.01 , compared with control groups . in all control specimens the pituitary gland appeared compact and extended in the cephalic - caudal direction in which the pars distalis ( pd ) was divided into a rostral part ( rpd ) , a caudal part ( cpd ) and a medial part ( mpd ) ( figure 1 a ) . the whole pd consisted of homogeneous vascularised cellular cordons with an evident basal lamina surrounding them and with the cells clearly identifiable by mallory stain ( figure 1 b ) . in the treated animals the pituitary gland tissue appeared atrophied in some areas , with wide irregular intercellular spaces , which appeared more extensive at 60 ( figure 1 c ) and evident also at 90 and 120 days . the gland also showed greater vascularisation ; a basal lamina surrounded the cellular cordons only partially and several cells appeared altered in shape ( figure 1 d ) . figure 1mallory stain . the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . ( d ) detail of panel c : the cellular cordons show a major vascularisation ( * ) , a basal lamina only partly surrounding them ( ) and several cells with altered shape ( ) . bar 140 m in a and c ; bar 40 m in b and d. mallory stain . ( a ) control lizard showing the extension of the gland . at the top the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . ( d ) detail of panel c : the cellular cordons show a major vascularisation ( * ) , a basal lamina only partly surrounding them ( ) and several cells with altered shape ( ) . bar 140 m in a and c ; bar 40 m in b and d. in the control lizards , acth and prl cells appeared clearly through immunohistochemical detection as distinct cellular populations with a specific distribution in the pd or pi ( pars intermedia ) . in treated specimens we observed the increase in occurrence and immunostain both for acth and for prl cells . acth and prl cells already at 10 days revealed a small increase , albeit with a different time course : prl cells appeared more abundant at 30 days from treatment , while acth were more copious at 60 days . in control lizards prl cells were found essentially in the rpd ( 24.00.5% ) but also in the mpd ( 20.10.24% ) ( figure 2 a ) . they were generally isolated or clustered in small cellular cordons of three - five cells ( figure 2 b ) . prl cells appeared pyriform or ovoidal in shape , with an eccentric and ovoidal nucleus and a moderately dense cytoplasm ( figure 2 c ) . a few prl cells were also observed in the cpd ( 1.20.05% ) , but they were always absent in the pi . at 10 days of treatment prl cells had slightly increased in the rpd and mpd , but they were more numerous in the cpd ( table 1 ) . their occurrence peaked at 30 days from treatment ( figure 2 d ) : in the rpd ( 34.01.7% ) , mpd ( 39.81.2% ) and cpd ( 17.30.67% ) . in all these regions they also showed greater immunostaining intensity ( figure 2 e , f ) . at 60 and 90 days of treatment prl cells still appeared numerous , albeit with diminishing values , and at 120 days close to those of the control animals as reported in table 1 . ( a f ) prl cells ( in brown ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( b , c ) details of panel a showing these isolated cells ( b ) or organized into small cordons ( c ) . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g l ) acth cells ( in brown ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. abc technique . ( a f ) prl cells ( in brown ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( b , c ) details of panel a showing these isolated cells ( b ) or organized into small cordons ( c ) . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. table 1percentage immunopositive prolactin cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd24.00.526.81.534.01.7 * 31.21.129.70.6624.81.1mpd20.10.2429.21.3539.81.225.41.04525.71.1123.10.9cpd1.20.058.30.56 * 17.30.675.10.33 * 3.90.42.10.2pi000000immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . immunopositive cells are expressed as mean sem ( % ) ; p<0.01 , compared with control groups . acth cells were observed in both the rpd ( 29.00.7% ) , and the mpd ( 12.00.13% ) , as well as in the pi ( 59.00.12% ) , but were absent in the cpd of the control specimens ( figure 2 g , i ) . acth cells were elongated in shape , with a generally central nucleus and higher cytoplasmic density in the pd ( figure 2 h ) compared with pi . at 10 days of treatment their occurrence appeared similar to that of control lizards , except for the presence of a few cells also in the cpd ( table 2 ) . acth cells increased markedly after 60 days of treatment with strong immunostaining of the cytoplasm in all the regions of the gland ( figure 2 j , k , l ) : in the rpd ( 40.31.11% ) , mpd ( 23.80.9% ) , pi ( 76.21.25% ) and cpd ( 16.10.85% ) . in the caudal region they appeared small and isolated cells , with no cordonal organization ( figure 2 l ) . at 90 and 120 days of treatment acth cells showed a similar distribution and morphology to the control ; in the cpd as well their occurrence had considerably decreased , appearing nearly absent at 120 days of treatment . table 2percentage immunopositive corticotropic cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd29.00.726.31.130.30.940.31.11 * 29.80.9529.30.13mpd12.00.1314.00.815.80.823.80.9 * 14.50.2112.80.69cpd00 1.50.363.70.18 * 16.10.855.10.6 * 0.350.3pi59.00.1260.11.8473.21.976.21.25 * 69.51.1163.21.18immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . immunopositive cells are expressed as mean sem ( % ) ; p<0.01 , compared with control groups . our findings indicate the toxic effect of cadmium upon the pituitary gland of the lizard p. sicula exposed to chronic oral treatment with an average cdcl2 dosage . cytotoxic action may be inferred both from the morphological alteration of the gland and from the dysregulatory process in acth and prl cells , effects which are nonetheless subject to different time courses . in terms of morphology , we observed the progressive disorganisation of hypophyseal tissue due to the appearance of atrophied areas with wide intercellular spaces , which were more expressed at 90 and 120 days of treatment . similar effects have been reported in other glandular tissues such as the thyroid of the catfish clarias batrachus and the testis of the cyprinid puntius sarana and of the monkey presbytis entellus entellus . however , in lizard exposed to acute treatment with a single high intraperitoneal dose of cdcl2 we previously noted that this metal induces apoptosis , as also reported in the anterior pituitary cells of the rat , and that this effect is irreversible . in this chronic treatment , apart from morphological damage , a parallel increase in the vessel network was also observed . the protraction of such tissue alterations and the enhanced vascularization of the pituitary gland agree with previous findings that chronic exposure to cd can lead to elevations in blood pressure . considerable evidence suggests that hypertensive effects of cd result from complex actions on both the vascular endothelium and vascular smooth muscle . the same increase in blood pressure could partly explain the alterations in the tissue architecture which we found in this gland . likewise , we also observed in the lizard the inhibitory action of cadmium in both acth and prl cells , just as we previously reported for gonadotrope cells . indeed , both these cell types during chronic treatment are more numerous and show marked immunoreactivity . however , we observed that the cellular increase peaked for prl at 30 days , while for acth it peaked after 60 days of treatment . this finding is also supported by a concomitant increase in immunostaining of the cytoplasmic granules of all these cells . the increase in occurrence and in cytoplasmic density is indicative of a hormonal accumulation in these cells due to the inhibiting effect induced by cadmium . it has been proved elsewhere that divalent cations , such as cd , inhibit in vitro release of gh and prl from bovine adenohypophyseal secretory granules . further , the inhibitory effect of cadmium on the hormonal secretion of many adenohypophyseal cells has been found in mammals by virtue of biochemical studies : the levels of lh and fsh in serum of rat and pig exposed to cadmium decrease . in mammals cd differentially affects the secretory mechanisms of the pituitary hormones : the effects of this metal are dosedependent only for prolactin and acth . in the fish puntius sarana , only high concentrations of cdcl2 influence the pituitary gonadotropins with a gradual accumulation of secretory granules . also in the lizard cd could well compete with calcium at pituitary level through the membrane channels or change intracellular calcium mobilization , as postulated for mammals , and inhibit hormone secretion . however , cd could also cause alterations in receptor binding and secretory mechanisms of pituitary hormones as reported by pillai et al . in female rats : cadmium generates free radicals which change the biophysical properties of the pituitary membranes with an inhibitory effect on hormone secretion . however , in the present study we observed that , unlike tissue alteration which persists in time , the inhibitory action both on acth and prl cells diminishes : at 120 days the occurrence of these cells returned to values similar to those observed in the control lizards . this reaction could be viewed as a probable adaptation to the toxic action of cadmium in time , when the cd dosage is at moderate levels . it may also be attributed to the activation of defence mechanisms such as the action of metallothionein , given that cd in chronic intoxication stimulates de novo synthesis of mts ; toxicity in the cells is assumed to start when cd ion loading exceeds the buffering capacity of intracellular mts . that said , the lizard appears to be a good experimental model for studying the action of heavy metals on the endocrine system .

we analyzed the effect of cadmium on corticotropic ( acth ) and prolactin ( prl ) cells in the pituitary gland of the podarcis sicula ( p. sicula ) lizard under chronic exposure to this metal . adult lizards were given cdcl2 in drinking water at the dose of 10 g/10 g body mass for 120 days . light microscopy was performed after histological and immunohistochemical staining , and the effects were followed at regular time intervals up to 120 days post - treatment . we detected substantial variations in the general morphology of the pituitary : unlike the control lizards in which the gland appeared compact , the treated lizards showed a glandular tissue with dilated spaces that were more extensive at 90 and 120 days . prl and acth cells showed an increase in occurrence and immunostaining intensity in treated lizards in comparison with the same cells of control animals . this cellular increase peaked for prl at 30 days in the rostral , medial and also caudal pars distalis of the gland . acth cells appeared to increase markedly after 60 days of treatment in both the pars distalis and the pars intermedia . again , at 60 days small , isolated acth cells were also found in the caudal pars distalis in which these cells were generally absent . however , at 120 days both these cellular types showed an occurrence , distribution and morphology similar to those observed in the control lizards . in lizards , protracted oral exposure to cadmium evidently involves an alteration of the normal morphology of the gland and an inhibitory effect of acth and prl cells , since they increase in occurrence and immunostaining . yet in time the inhibitory effect of cadmium on acth and prl cells falls back and their occurrence appears similar to that of the control lizard .

Introduction Materials and Methods Results General morphology Immunohistochemistry Prolactin cells Corticotropic cells Discussion

studies carried out on rats also prove that cd is absorbed and retained in the pituitary gland , leading to a decrease in content of luteinizing hormone and alteration of gland functionality . in rat , cd modifies the lactotroph cell activity of the pituitary gland through biochemical , genomic and morphological changes and modifies plasma levels of luteinizing hormone ( lh ) and follicle - stimulating hormone ( fsh ) . as we have already reported elsewhere , in the lizard an acute treatment with a single high ( 20 g/10 g body mass ) intraperitoneal dose of cdcl2 not only induces apoptosis , especially in the rostral pars distalis , which appears irreversible , but also alters the normal endocrine function of the gland . once again in the lizard , we also reported that chronic exposure to cdcl2 affects the hormonal secretion of gth cells through an inhibitory effect . the aim of this paper was thus to analyse the effects of cadmium on acth and prl cells in the lizard podarcis sicula ( p. sicula ) exposed to chronic oral treatment for 120 days at an average dose ( 10 g/10 g body mass ) of cdcl2 . cdcl2 was administered to the lizards in drinking water for four months at a daily dose of 10 g/10 g body mass while control lizards received cadmium - free water . groups of four treated and two control animals were killed at 10 , 30 , 60 , 90 and 120 days . experiments were performed in accordance with the guidelines for animal experimentation of the italian department of health under the supervision of a veterinarian , and organised to minimise stress and the number of lizards used . the pituitary gland is extremely small and almost completely enclosed in the sella turcica , which makes its removal difficult and may cause damage to the gland . for immunohistochemical staining , the sections were processed according to the abc technique using the following heterologous antisera at specific working dilutions : anti - human prl ( 1/300 , signet laboratories , dedham , ma , usa ) and anti - synthetic acth1 - 24 ( 1/600 , biogenesis , poole , uk ) . quantification of the two cellular types was carried out on at least 300 cells , with a visible nucleus , on serial sections per animal and relative to specific regions , namely the rostral , medial and caudal pars distalis and pars intermedia . in all control specimens the pituitary gland appeared compact and extended in the cephalic - caudal direction in which the pars distalis ( pd ) was divided into a rostral part ( rpd ) , a caudal part ( cpd ) and a medial part ( mpd ) ( figure 1 a ) . in the treated animals the pituitary gland tissue appeared atrophied in some areas , with wide irregular intercellular spaces , which appeared more extensive at 60 ( figure 1 c ) and evident also at 90 and 120 days . the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . at the top the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . bar 140 m in a and c ; bar 40 m in b and d. in the control lizards , acth and prl cells appeared clearly through immunohistochemical detection as distinct cellular populations with a specific distribution in the pd or pi ( pars intermedia ) . in treated specimens we observed the increase in occurrence and immunostain both for acth and for prl cells . acth and prl cells already at 10 days revealed a small increase , albeit with a different time course : prl cells appeared more abundant at 30 days from treatment , while acth were more copious at 60 days . in control lizards prl cells were found essentially in the rpd ( 24.00.5% ) but also in the mpd ( 20.10.24% ) ( figure 2 a ) . a few prl cells were also observed in the cpd ( 1.20.05% ) , but they were always absent in the pi . at 10 days of treatment prl cells had slightly increased in the rpd and mpd , but they were more numerous in the cpd ( table 1 ) . their occurrence peaked at 30 days from treatment ( figure 2 d ) : in the rpd ( 34.01.7% ) , mpd ( 39.81.2% ) and cpd ( 17.30.67% ) . at 60 and 90 days of treatment prl cells still appeared numerous , albeit with diminishing values , and at 120 days close to those of the control animals as reported in table 1 . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g l ) acth cells ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. table 1percentage immunopositive prolactin cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd24.00.526.81.534.01.7 * 31.21.129.70.6624.81.1mpd20.10.2429.21.3539.81.225.41.04525.71.1123.10.9cpd1.20.058.30.56 * 17.30.675.10.33 * 3.90.42.10.2pi000000immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . acth cells were observed in both the rpd ( 29.00.7% ) , and the mpd ( 12.00.13% ) , as well as in the pi ( 59.00.12% ) , but were absent in the cpd of the control specimens ( figure 2 g , i ) . at 10 days of treatment their occurrence appeared similar to that of control lizards , except for the presence of a few cells also in the cpd ( table 2 ) . acth cells increased markedly after 60 days of treatment with strong immunostaining of the cytoplasm in all the regions of the gland ( figure 2 j , k , l ) : in the rpd ( 40.31.11% ) , mpd ( 23.80.9% ) , pi ( 76.21.25% ) and cpd ( 16.10.85% ) . at 90 and 120 days of treatment acth cells showed a similar distribution and morphology to the control ; in the cpd as well their occurrence had considerably decreased , appearing nearly absent at 120 days of treatment . table 2percentage immunopositive corticotropic cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd29.00.726.31.130.30.940.31.11 * 29.80.9529.30.13mpd12.00.1314.00.815.80.823.80.9 * 14.50.2112.80.69cpd00 1.50.363.70.18 * 16.10.855.10.6 * 0.350.3pi59.00.1260.11.8473.21.976.21.25 * 69.51.1163.21.18immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . in all control specimens the pituitary gland appeared compact and extended in the cephalic - caudal direction in which the pars distalis ( pd ) was divided into a rostral part ( rpd ) , a caudal part ( cpd ) and a medial part ( mpd ) ( figure 1 a ) . in the treated animals the pituitary gland tissue appeared atrophied in some areas , with wide irregular intercellular spaces , which appeared more extensive at 60 ( figure 1 c ) and evident also at 90 and 120 days . the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . at the top the subdivision of the gland into rostral ( rpd ) , medial ( mpd ) , caudal ( cpd ) pars distalis and pars intermedia ( pi ) is reported . bar 140 m in a and c ; bar 40 m in b and d. in the control lizards , acth and prl cells appeared clearly through immunohistochemical detection as distinct cellular populations with a specific distribution in the pd or pi ( pars intermedia ) . in treated specimens we observed the increase in occurrence and immunostain both for acth and for prl cells . acth and prl cells already at 10 days revealed a small increase , albeit with a different time course : prl cells appeared more abundant at 30 days from treatment , while acth were more copious at 60 days . in control lizards prl cells were found essentially in the rpd ( 24.00.5% ) but also in the mpd ( 20.10.24% ) ( figure 2 a ) . a few prl cells were also observed in the cpd ( 1.20.05% ) , but they were always absent in the pi . at 10 days of treatment prl cells had slightly increased in the rpd and mpd , but they were more numerous in the cpd ( table 1 ) . their occurrence peaked at 30 days from treatment ( figure 2 d ) : in the rpd ( 34.01.7% ) , mpd ( 39.81.2% ) and cpd ( 17.30.67% ) . at 60 and 90 days of treatment prl cells still appeared numerous , albeit with diminishing values , and at 120 days close to those of the control animals as reported in table 1 . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . ( a ) control lizard , showing the occurrence of prl cells in the rpd and in the mpd . ( d ) treated lizard at 30 days , showing the increase in prl cells extending also into the cpd . ( e , f ) details of panel d showing the greater intensity of immunostain of the cytoplasm in the mpd ( e ) and the cpd ( f ) . ( g ) control lizard , showing the occurrence of acth cells in the rpd , the mpd and also in the pi . ( h , i ) details of panel g to show their elongated shape and the cytoplasm moderately marked in the rpd ( h ) and their absence in cpd ( i ) . ( j ) treated lizard at 60 days with an evident strong immunostain of acth cells , which also appears in the cpd . ( k , l ) details of panel j highlighting the marked immunostain of these cells in the rpd ( k ) , as well as their occurrence in the cpd ( l ) . bar 150 m in a , d , g , j ; bar 33 m in b , e , i , l ; bar 15 m in c , f ; bar 38 m in h , k. table 1percentage immunopositive prolactin cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd24.00.526.81.534.01.7 * 31.21.129.70.6624.81.1mpd20.10.2429.21.3539.81.225.41.04525.71.1123.10.9cpd1.20.058.30.56 * 17.30.675.10.33 * 3.90.42.10.2pi000000immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . acth cells were observed in both the rpd ( 29.00.7% ) , and the mpd ( 12.00.13% ) , as well as in the pi ( 59.00.12% ) , but were absent in the cpd of the control specimens ( figure 2 g , i ) . at 10 days of treatment their occurrence appeared similar to that of control lizards , except for the presence of a few cells also in the cpd ( table 2 ) . acth cells increased markedly after 60 days of treatment with strong immunostaining of the cytoplasm in all the regions of the gland ( figure 2 j , k , l ) : in the rpd ( 40.31.11% ) , mpd ( 23.80.9% ) , pi ( 76.21.25% ) and cpd ( 16.10.85% ) . at 90 and 120 days of treatment acth cells showed a similar distribution and morphology to the control ; in the cpd as well their occurrence had considerably decreased , appearing nearly absent at 120 days of treatment . table 2percentage immunopositive corticotropic cells in pituitary gland of control and treated lizards.control10 days30 days60 days90 days120 daysrpd29.00.726.31.130.30.940.31.11 * 29.80.9529.30.13mpd12.00.1314.00.815.80.823.80.9 * 14.50.2112.80.69cpd00 1.50.363.70.18 * 16.10.855.10.6 * 0.350.3pi59.00.1260.11.8473.21.976.21.25 * 69.51.1163.21.18immunopositive cells are expressed as mean sem ( % ) ; * p<0.05,p<0.01 , compared with control groups . our findings indicate the toxic effect of cadmium upon the pituitary gland of the lizard p. sicula exposed to chronic oral treatment with an average cdcl2 dosage . cytotoxic action may be inferred both from the morphological alteration of the gland and from the dysregulatory process in acth and prl cells , effects which are nonetheless subject to different time courses . in terms of morphology , we observed the progressive disorganisation of hypophyseal tissue due to the appearance of atrophied areas with wide intercellular spaces , which were more expressed at 90 and 120 days of treatment . however , in lizard exposed to acute treatment with a single high intraperitoneal dose of cdcl2 we previously noted that this metal induces apoptosis , as also reported in the anterior pituitary cells of the rat , and that this effect is irreversible . the protraction of such tissue alterations and the enhanced vascularization of the pituitary gland agree with previous findings that chronic exposure to cd can lead to elevations in blood pressure . the same increase in blood pressure could partly explain the alterations in the tissue architecture which we found in this gland . likewise , we also observed in the lizard the inhibitory action of cadmium in both acth and prl cells , just as we previously reported for gonadotrope cells . however , we observed that the cellular increase peaked for prl at 30 days , while for acth it peaked after 60 days of treatment . the increase in occurrence and in cytoplasmic density is indicative of a hormonal accumulation in these cells due to the inhibiting effect induced by cadmium . further , the inhibitory effect of cadmium on the hormonal secretion of many adenohypophyseal cells has been found in mammals by virtue of biochemical studies : the levels of lh and fsh in serum of rat and pig exposed to cadmium decrease . in mammals cd differentially affects the secretory mechanisms of the pituitary hormones : the effects of this metal are dosedependent only for prolactin and acth . in female rats : cadmium generates free radicals which change the biophysical properties of the pituitary membranes with an inhibitory effect on hormone secretion . however , in the present study we observed that , unlike tissue alteration which persists in time , the inhibitory action both on acth and prl cells diminishes : at 120 days the occurrence of these cells returned to values similar to those observed in the control lizards .

recombinant methods were used to synthesize a series of asymmetric peptide amphiphiles genetically fused to the carboxy - terminus of an elp ( table 1 , supporting information figure s1 ) . plasmids coding for the amphiphiles were transformed into escherichia coli , purified after expression by inverse transition cycling , lyophilized , and stored at 20 c for future use . all elp segments consist of the sequence ( vpgag)n , where the number of pentapeptide repeats herein , we refer to ( vpgag)40 as a40 , ( vpgag)80 as a80 , and ( vpgag)160 as a160 . the assembly domains consist of the sequence ( xgy)8 , where x represents a hydrophobic amino acid and y represents the number of glycine spacers ( y = 0 , 1 , or 2 ) . a c - terminal tyrosine was included to facilitate absorbance - based protein quantification ( supporting information figure s1 ) . following purification , matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ) was performed on each construct to confirm its molecular weight ( supporting information table s1 ) . each polymer amphiphile was analyzed by dynamic light scattering ( dls ) to determine the extent of self - assembly and the hydrodynamic radius of the self - assembled nanoparticle . amphiphiles with ( xgg)8 segments incorporating x = leu , ile , his , and tyr did not self - assemble and remained fully soluble ( rh 6 nm ; figure 1a , table 2 ) . interestingly , peptides incorporating leu and his at the x position within a ( xgg)8 displayed a minor secondary population ( an estimated 510% of total mass ) of self - assembled structures with the remainder unassembled ( supporting information figure s5 ) . in contrast , amphiphiles in - corporating phe ( f ) or trp ( w ) at the x position self - assembled into nanoparticles with hydrodynamic radii ranging from 43 to 81 nm ( table 2 ) . ( a ) a160-(igg)8 does not self - assemble and has a rh of 7 nm , whereas a160-(fgg)8 self - assembles into nanostructures with a rh of 42 nm . ( b ) a160-(yg)8 self - assembles into nanostructures with a rh of 73 nm that disassemble at ph 12 ( pkatyr = 10.1 ) because the deprotonation of tyrosine to tyrosinate greatly increases the hydrophilicity of the assembly domain , thereby disrupting the nanoparticle core . ( c ) a160-(fgg)8 constructs with a rh of 42 nm do not disassemble at high ph because phenylalanine residues do not become charged at high ph . surprisingly , we observed that the number of gly ( g ) residues within the assembly domain potently controls the self - assembly of these asymmetric amphiphiles . for instance , a160-(ygg)8 displays insufficient amphiphilicity to self - assemble and thus exhibits an rh of 6 nm ( table 2 ) . however , replacing the ( ygg)8 domain with a ( yg)8 or y8 domain results in self - assembly into nanoparticles with a rh of 71 and 58 nm , respectively ( table 2 ) . this effect is most likely due to the hydrophilicity of the gly residues ; as the number of glycine residues decreases from 2 to 1 or 0 , the overall hydrophobicity of the assembly domain surpasses the threshold necessary to trigger nanoparticle self - assembly . it is clear , however , that not all systems are affected by the number of glycine residues equally ; amphiphiles with more hydrophobic assembly domains comprised of phe self - assemble regardless of the number of gly spacers ( table 2 ) . in this case , the hydrophobic driving force for self - assembly is strong enough to outweigh the contribution of the gly residues . the aggregation number nagg , or the number of polypeptide chains per nanoparticle , was determined by static light scattering rg , radius of gyration , was determined by static light scattering . . indicates no data : static light scattering and trps measurements were not performed for nonself - assembling amphiphiles . no data : we were unable to measure the size of a160-y8 by trps . as the ionized form of tyr ( pkatyr = 10.1 ) is 10 times more hydrophilic than neutral tyrosine , the a160-(yg)8 construct offered a unique opportunity to confirm that self - assembly is primarily driven by the hydrophobicity of the assembly domain . upon increasing the ph to 12 , the vast majority of the a160-(yg)8 constructs disassembled into unimers ( figure 1b ) because ionizing the tyrosinate residues disrupted the stabilizing hydrophobic cohesion forces in the hydrophobic core . the a160-(fgg)8 constructs , which do not display a pka within this range , remained unaffected by the increase in ph ( figure 1c ) . for those amphiphiles that self - assembled , static light scattering was used to calculate the aggregation number nagg so as to match table 2 , the number of amphiphile chains per nanoparticle , and the radius of gyration ( rg ) . the nanoparticles displayed aggregation numbers ranging from 50 to 600 chains per nanoparticle ( table 2 ) and radii of gyration spanning 45.0 to 124.4 nm ( table 2 ) . the radius of gyration and the hydrodynamic radius were used to calculate the shape factor = rg / rh , which is characteristic of the morphology of the scatterer , where = 0.775 for solid spheres , = 1 for hollow spheres ( vesicles ) , and = 1.505 for random coils . for cylindrical structures , the relationship between and the elongation = l/2r has been determined as eq 1(35)1 despite the observed range in nanoparticle size , the shape factor was remarkably consistent between the self - assembling amphiphiles . varied between 1.31 and 1.43 , hence indicating a conserved nonspherical morphology and for a rodlike structure according to eq 1 to an axial ratio of 68 . the one exception , a160-y8 , displayed a value of 0.78 , indicating a different morphology . we also investigated the effect of chain length on nanoparticle morphology by examining constructs with an ( fgg)8 assembly domain and different lengths of the elp domain . we observed an increase in the aggregation number from 111 to 290 and 575 as the chain length decreased from 160 to 80 to 40 pentamers , respectively . surprisingly , only minimal fluctuations in size and morphology were observed with rg and rh shrinking slightly and increasing from 1.31 to 1.43 . tunable resistive pulse sensing , a particle - by - particle analysis technique that relies not on scattering but the physical ability of nanoparticles to pass through a narrow pore was also used as an independent measure of particle size . radii determined by trps were broadly in agreement with the hydrodynamic radii determined by dls ( table 2 , supporting information figure s3 ) . we have previously shown that the lcst phase behavior of elps is exquisitely sensitive to polypeptide composition , fused proteins or chemically conjugated small molecules , ionic strength , and solvent conditions and can be used to drive the self - assembly of gold nanoparticles . thus , it was not surprising to observe that the hydrophobicity of the ( xgg)8 assembly domain also affected the lcst phase behavior ( figure 2 ) . in accordance with previous publications characterizing elps fused to proteins or chemically conjugated to small molecules , the tt of the amphiphile decreased as the hydrophobicity of the fused peptide assembly domain increased . we have also shown that the lcst phase transition behavior of unimeric elps is concentration - dependent such that a higher concentration of an elp phase separates at a lower temperature . this inverse relationship between concentration and the transition temperature was observed for all nonself - assembling amphiphiles . in contrast , the tt of elps that display self - assembly showed virtually no dependence on elp concentration . we hypothesize that this is due to the high and constant local elp chain concentration within each self - assembled structure , even as the global solution concentration changes . in other words , the elp chains are entropically trapped once they self - assemble into micelles , and thus the entropic penalty for aggregation is smaller than for the free chains of elp unimers . the magnitude of this penalty no longer varies significantly with concentration because the structures remain assembled in a similar fashion across a wide range of concentrations . in fact , it is possible to separate the constructs into assembling ( x = phe or trp ) and nonassembling ( x = leu , ile , his , or tyr ) populations based solely on the thermal turbidimetry profile as a function of concentration ( figure 2 ) . tt of nonself - assembling amphiphiles ( control , leu , ile , his , and tyr ) display a strong concentration dependence , whereas self - assembling amphiphiles ( phe and trp ) display tt that are near - independent of concentration . fluorescence spectrophotometry using pyrene as a probe was performed to sample the hydrophobicity of the inner core of the self - assembled structures and to determine their critical aggregation concentration ( cac ) . as pyrene is released from the lipophilic core of an assembled nanoparticle to the aqueous environment as a result of nanoparticle disassembly , it experiences a shift in its characteristic fluorescence profile ( figure 3 ) . more specifically , the fluorescent intensity ratio at the 370373 nm peak to the 381384 nm peak ( i1/i3 ) increases sigmoidally as the hydrophilicity of the environment increases . because nonassembling constructs ( e.g. , a160-(ygg)8 ) do not provide a lipophilic environment at any concentration , they exhibit a fluorescence profile that is independent of amphiphile concentration ( figure 3 ) . additionally , the cac was used to calculate the free energy of micellization ( gmic ) given the relation gmic = rt ln(xcac ) ( where xcac is the cac expressed as a mole fraction ) . the amphiphile consisting of an a160 elp and a ( ygg)8 , assembly domains does not self - assemble and shows no concentration dependence in its i1/i3 ratio , whereas amphiphiles with ( fgg)8 and ( wgg)8 , assembly domains self - assemble into cylindrical micelles and show a two - state concentration - dependent behavior in their i1/i3 ratio . the ( fgg)8 core is more hydrophobic than the ( wgg)8 core , as indicated by its lower final i1/i3 value . self - assembling asymmetric amphiphiles display cacs ranging from 0.4 to 13 m and free energies of micellization ranging from 37.8 to 46.4 kj / mol ( table 3 ) . in addition , the minimum i1/i3 value can be used as a measure of the hydrophobicity of the core . asymmetric amphiphiles display minima ranging from 1.19 ( a40-(fgg)8 , most hydrophobic ) to 1.41 ( a160-(wgg)8 , least hydrophobic ) ( table 3 ) . surprisingly , the ( fgg)8 domain formed a core that was more hydrophobic than that of ( wgg)8 although tryptophan is considered more hydrophobic than phenylalanine . it is possible that the smaller phenylalanine side chains allow higher packing densities or promote enhanced stacking interactions . we also observed that decreasing the length of the elp domain from 160 to 40 pentapeptide repeats slightly increases the hydrophobicity and stability of the ( fgg)8 core as a function of elp chain length ( table 3 , supporting information figure s6 ) . despite clear evidence of self - assembled structures by light scattering ( table 2 ) and cryo - tem ( figure 4 ) , the pyrene fluorescence data for a160-y8 indicated that the fluor was in an aqueous environment over a range of concentrations , thus suggesting a lack of self - assembly . we hypothesize that this artifact may be due to the small size of the core , the low hydrophobicity of the core , or a combination of these two factors . cryo - tem experiments were performed to directly visualize the self - assembled structures in their near - native hydrated state . however , due to their relatively low electron density , polypeptide amphiphiles are challenging to image by cryo - tem as they exhibit lower contrast than many synthetic polymer amphiphiles . samples were imaged at a low voltage ( 80 kev ) to increase the contrast between the buffer and the polypeptide chains . although the hydrophobic cores have sufficient electron density to be clearly visualized by cryo - tem under these conditions , we were unable to visualize the hydrated elp corona in self - assembled structures or single elp chains ( unimers ) due to their low electron density and high degree of solvation . in contrast to conjugation - driven assembly , which only yields spherical micelles , these asymmetric amphiphiles spontaneously assemble into cylindrical micelles upon achieving sufficient hydrophobicity in the core ( figure 4a f ) . for instance , the a160 amphiphile with a terminal ( ygg)8 domain showed no self - assembly , but as the glycine fraction in the assembly domain was reduced with ( yg)8 and y8 assembly domains , amphiphiles spontaneously self - assembled into cylindrical micelles ( figure 4b c ) . we found that the gly content of the self - assembly domain controls the length of the micelles and hence their aspect ratio . although the radii remained approximately 11.5 nm for both the a160-(yg)8 and a160-y8 constructs , the length decreased from 119 nm for the ( yg)8 construct to 60 nm for the y8 construct , resulting in a decreased aspect ratio ( table 4 ) . the cryo - tem results were also consistent with results from light scattering . this change in aspect ratio is consistent with the values of 1.37 and 0.78 observed for a160-(yg)8 and a160-y8 , respectively ( table 2 ) . the value of 0.78 for a160-(yg)8 seemingly indicates a spherical morphology ; however , cryo - tem reveals that it is in fact a cylindrical morphology with a low aspect ratio . whereas the composition of the assembly domain played a role in controlling the aspect ratio of the nanoparticles , the length of the elp domain ( 160 , 80 , and 40 pentapeptides ) attached to the ( fgg)8 assembly domain only resulted in minor differences in the aspect ratio ( figure 4d f , table 4 ) . ( a ) constructs such as a160-(ygg)8 that do not self - assemble could not be visualized by cryo - tem because of their high levels of hydration and low densities . ( b , c ) changing the assembly domain from ( yg)8 ( b ) to y8 ( c ) causes a significant decrease in the length of the cylindrical micelles . ( d f ) a160-(fgg)8 ( d ) , a80-(fgg)8 ( e ) , and a40-(fgg)8 ( f ) self - assemble into cylindrical micelles with similar aspect ratios . scale bar represents 200 nm . data are reported as mean standard deviation with a minimum of 25 measurements . this apparent aspect ratio is defined only by the portion of the assembly that is visible by cryo - tem and as such differs from the true aspect ratio determined by scattering . to gain deeper insight into the nanometer scale structure of these self - assembled constructs , sans spectra of a160-(fgg)8 and a160-(yg)8 were acquired at concentrations ranging from 0.3 to 3 wt % in d2o ( figure 5 ) . the sans data span a q - range of 0.024 nm ( q : magnitude of the wave vector ) , which roughly translates to 1.5300 nm in real space . the spectra for both amphiphiles are very similar , and a number of structural features can be deduced directly from the spectra . the scattering intensity for both amphiphiles follows a q power law at mid to high q , which is typical for polymer chains . at q 0.15 nm the intensity increases abruptly , corresponding to a cross - sectional radius of 3.83q for cylinders ( 26 nm ) and then plateaus at lower q , hence indicating that the largest scattering dimension is approximately 150 nm . vsans ( very small - angle neutron scattering ) experiments were performed at lower q to confirm this size limit ( supporting information figure s7 ) . the concentration series data reveal strong repulsive interactions visible as a structure factor correlation peak . as they likely arise from excluded volume effects and given the low volume fraction of amphiphiles , this can only be explained by a high degree of solvation of the nanostructures . sans spectra and analytical model fits ( solid lines ) for a160-(fgg)8 ( a ) and a160-(yg)8 ( b ) . the q slope in the mid to high q region is characteristic of the polymer chains in the hydrophilic part of the nanostructures . the structure factor peak at low q is caused by repulsive interactions between structures . to obtain more reliable values for the size parameters of these micelles , next we analytically modeled the sans spectra . on the basis of cryo - tem data , we chose a model of isotropically oriented homogeneous cylindrical micelles that also incorporated scattering from individual polypeptide chains at smaller length scales ( see supporting information ) . to obtain a robust set of output parameters , we simultaneously fitted all spectra from a given amphiphile with the same parameter set , thus covering a wide concentration range . for a160-(fgg)8 and a160-(yg)8 the cylinder lengths obtained from the model are 174 and 164 nm and the radii are 24 and 21 nm , respectively , corresponding to an axial ratio of 4 , which is in good agreement with the light scattering data . the rg of the individual polymer chains ( the hydrophilic elp brush ) is 11 nm and the hydration of the micelles ( the volume fraction of the nanostructure occupied by water ) is 0.94 . however , in cryo - tem it is possible that many of the cylindrical micelles are tilted within the vitreous ice layer , leading to a lower observed length . it is also possible that the outer region of the core is too highly solvated to be observed by cryo - tem , thus explaining the larger radius observed by sans . we have observed that amphiphilic block copolymers comprised of a water - soluble polypeptide and an extremely short hydrophobic peptide self - assemble into cylindrical micelles . this observation is highly surprising and contrary to accepted theories of self - assembly of synthetic block copolymers because their extremely high hydrophilic weight fraction would predict either a lack of self - assembly or assembly into star - like micelles . we also observed that self - assembly is highly sensitive to the hydrophobicity of the assembly domain : less hydrophobic residues such as leu and ile do not drive self - assembly , whereas the more hydrophobic tyr , phe , and trp residues spontaneously form nanoparticles . self - assembly is also sensitive to the number of glycine residues between the hydrophobic residues . for instance , the ( ygg)8 domain was not sufficiently hydrophobic to drive self - assembly of a160 elp , whereas both the ( yg)8 and y8 domains drove self - assembly into cylindrical micelles . among the self - assembling constructs , the rg and rh determined by light scattering varied substantially ( from 46.1 to 124.4 nm and from 33.0 to 91.4 nm , respectively ) , while the shape factor ( rg / rh ) remained remarkably constant ( varying only between 1.31 and 1.43 ) for all but one amphiphile . cryo - tem revealed that all constructs other than a160-y8 self - assembled into similar cylindrical morphologies , and a160-y8 self - assembled into cylindrical micelles with a significantly lower aspect ratio . in addition to cryo - tem , sans provided a complementary ensemble measurement that confirmed the cylindrical morphology with core radii of 2123 nm and a length of 170 nm . all amphiphiles retained the stimulus - responsiveness of the parent elp chain with nonself - assembling constructs exhibiting a significant concentration dependence in their tt , while the self - assembling constructs displayed little to no concentration dependence in their tt . fluorescence spectroscopy using pyrene as a probe revealed that despite their similar morphologies , the cylindrical micelles formed cores with varying levels of hydrophobicity ( as measured by the characteristic fluorescence behavior of pyrene ) and displayed a range of cacs from 0.4 to 13.0 m . these low cacs correspond to relatively high values for the gibbs free energy of micellization of 4045 kj / mol , which indicate the strongly amphiphilic character of these compounds . our most unexpected finding is that asymmetric amphiphiles with hydrophilic fractions as high as 97% self - assemble into cylindrical micelles rather than spherical micelles . synthetic block copolymers canonically form spherical micelles when the hydrophilic weight fraction exceeds 60% , cylindrical micelles are favored between 45% and 55% , and vesicles are constrained to the region between 25% and 45% . the free energy of self - assembly and thus the morphology are determined primarily by the stretching of the core - forming block , the repulsion between the corona blocks , and the interfacial tension between the core and the solvent . surprisingly , within the sequence space sampled here the hydrophilic fraction does not seem to play a significant role in determining the morphology , as phenylalanine - based constructs with hydrophilic fractions of 97% ( chain length n = 160 ) , 94% ( n = 80 ) , and 88% ( n = 40 ) all assembled into similar cylindrical nanostructures as seen by cryo - tem and light scattering . however , the hydrophilic fraction significantly influenced the aggregation number with higher hydrophilic fractions leading to fewer polypeptide chains per nanoparticle . the self - assembly of these asymmetric amphiphiles also raises more fundamental questions about how the self - assembly of polypeptide chains differs from that of synthetic polymers . the nature of the hydrophobic interactions in the core of asymmetric amphiphiles is not yet well understood . given the aromatic groups present in tyr , phe , and trp side - chains , we believe that stacking of aromatic groups may play an important and as yet unexplored role in amphiphile self - assembly . the fact that the core diameter observed by sans is larger than the stretched length of the hydrophobic assembly domain also indicates that part of the hydrophilic elp chain may be collapsed onto the hydrophobic core . the slight increase in the hydrophobicity of the core as the elp block is shortened from 160 to 80 and 40 with an assembly domain of ( fgg)8 ( as measured by pyrene i1/i3 , table 3 ) is consistent with this hypothesis , as it indicates that the hydrophobic region may consist not only of the ( fgg)8 domain but also of a portion of the elp domain . in addition to their interesting material characteristics , this class of self - assembling amphiphiles should be useful for a number of biomedical applications . for instance , promoting self - assembly via a genetically encoded peptide instead of a chemically conjugated hydrophobe could allow for hydrophilic drugs to be repurposed into therapeutic nanoparticles . while water - soluble chemotherapeutics display improved pharmacokinetics and biodistribution relative to their hydrophobic counterparts , they are still plagued by rapid deactivation in serum , clearance from circulation , and nonspecific absorption into healthy tissues . in the case of hydrophobic therapeutics , these issues are typically ameliorated by the sequestration of the drug into a protected nanoparticle core . the design of triblock peptide polymers with an elp segment , followed by a drug attachment domain and an assembly domain , could result in self - assembly into stable nanostructures despite the conjugation of a hydrophilic therapeutic within the core . restriction enzymes and t4 dna ligase were purchased from new england biolabs ( ipswich , ma ) . all custom oligonucleotides were synthesized by integrated dna technologies inc . escherichia coli cells were purchased from edge biosystems ( gaithersburg , md ) . all escherichia coli cultures were grown in tbdry media purchased from mo bio laboratories , inc . the highly asymmetric amphiphiles were synthesized from synthetic oligomers using plasmid reconstruction recursive directional ligation . the asymmetric amphiphiles described in this chapter are of the form skgpg-(agvpg)n-(xgy)8-y , where the polypeptide length n ( 40 , 80 , or 160 ) , the number of glycine spacers y ( 0 , 1 , or 2 ) , and the identity of the hydrophobic residue x are systematically varied ( table 1 , supporting information figure s1 ) . each construct was expressed using a previously published hyperexpression protocol , which relies on the leakiness of the t7 promoter . fifty milliliter cultures were grown overnight and used to inoculate 1 l flasks of tbdry supplemented with 45 g / ml kanamycin . the flasks were then incubated at 37 c for 24 h and 190 rpm . each construct was purified using a modified inverse transition cycling ( itc ) protocol , where the solution was never heated above room temperature . briefly , the cell suspension was centrifuged at 3000 rpm for 10 min at 4 c , the cell pellet resuspended in pbs , and then lysed by sonication on ice for 3 min ( 10 s on , 40 s off ) ( masonix s-4000 ; farmingdale , ny ) . polyethylenimine 0.7% w / v was added to the lysate to precipitate nucleic acid contaminants . the solution was kept on ice , and 3 m nacl was added to isothermally trigger the phase transition of the elp . the coacervate was then centrifuged for 10 min at 14 000 g and 20 c , the supernatant was decanted and discarded , and the pellet was resuspended in phosphate buffer . this suspension was cooled to 4 c , and then centrifuged for 10 min at 14 000 and 4 c to remove any insoluble contaminants . turbidity profiles were obtained for each of the constructs by recording the optical density as a function of temperature ( 1 c / min ramp ) on a temperature controlled uv vis spectrophotometer ( cary 300 bio ; varian instruments ; palo alto , ca ) . dynamic light scattering was used to determine the hydrodynamic radius ( rh ) of the various constructs at 25 c and 25 m amphiphile concentration using a dynapro plate reader ( wyatt technology ; santa barbara , ca ) , following filtration through 0.22 m millex - gv filters ( millipore ; billerica , ma ) . the data was analyzed with a regularization fit for raleigh spheres . additional dynamic and static light scattering measurements were performed using an alv / cgs-3 goniometer system ( germany ) . samples for the alv / cgs-3 goniometer system were prepared in pbs at 25 m and filtered through 0.22 m millex - gv filters into a 10 mm disposable borosilicate glass tube ( fischer ) . measurements were obtained at 25 c for angles between 30150 at 5 increments with each angle consisting of 3 runs for 10 s. results were analyzed by partial zimm plot analysis using alv / dynamic and static fit and plot software . lyophilized samples were resuspended in pbs to a concentration of 50 m and filtered through 0.22 m millex - gv filters . forty microliters of the sample was loaded onto a qnano ( izon ; christchurch , new zealand ) instrument equipped with 50150 nm pores ( a160-(fgg)8 , a160-(fg)8 , a80-(fgg)8 , and a40-(fgg)8 ) or 100300 nm pores ( a160-(yg)8 , a160-f8 , and a160-(wgg)8 ) . samples were measured for at least 30 s and a minimum of 500 blockade events were recorded per sample . samples were calibrated against known standards ( izon ) for the same stretch and voltage . an assay to determine the critical aggregation concentration ( cac ) using pyrene as a probe was performed using a cary eclipse spectrophotometer equipped with a xenon flash lamp ( varian instruments ; palo alto , ca ) . one microliter of a stock solution of 12 mm pyrene ( fluka / sigma - aldrich ; st . both the stock solution and diluted pyrene solution in pbs were sonicated for 10 min prior to use . one to five milligrams of lyophilized elp was added to this solution , which was then used to create a dilution series for that elp . each sample was placed in a reduced volume cuvette and scanned ( ex , 334 ; em , 360380 ; ex slit 10 nm ; em slit 2.5 nm ) . pyrene fluorescence displays four peaks ; the intensity of the first ( i1 , 370373 nm ) and third peak ( i3 , 381384 nm ) were recorded . the ratio i1/i3 was plotted as a function of amphiphile concentration , and the cac was defined as the inflection point of the sigmoid of best fit . cryo - tem experiments were performed at duke university s shared materials instrumentation facility ( durham , nc ) and at the university of pennsylvania in the penn regional nanotechnology facility ( philadelphia , pa ) . at duke university , lacey holey carbon grids ( ted pella , redding , ca ) were glow discharged in a pelco easiglow cleaning system ( ted pella , redding , ca ) . a 3 l drop ( 200 m elp concentration ) was deposited onto the grid , blotted for 3 s with an offset of 3 mm , and vitrified in liquid ethane using the vitrobot mark iii ( fei , eindhoven , netherlands ) . prior to vitrification , the sample chamber was maintained at 22 c and 100% relative humidity to prevent sample evaporation . grids were transferred to a gatan 626 cryoholder ( gatan , pleasanton , ca ) and imaged with an fei tecnai g twin tem ( fei , eindhoven , netherlands ) , operating at 80 kev . at the university of pennsylvania , lacey formvar / carbon grids ( ted pella ) were washed in chloroform to remove the formvar template and carbon coated with a quorum q150 t es carbon coater ( quorum technologies , united kingdom ) . grids were cleaned with hydrogen / oxygen plasma for 15 s using the solarus advanced plasma system 950 ( gatan , pleasanton , ca ) . a 2 l drop ( 200 m elp concentration ) was deposited onto the grid and added to a gatan cp3 cryoplunger ( gatan , pleasanton , ca ) . grids were transferred to a gatan ct3500tr cryoholder ( gatan , pleasanton , ca ) and immediately inserted into a jeol 2010 tem ( jeol , tokyo , japan ) operating at 80 kev . sans experiments were conducted at the jlich center for neutron science at mlz in munich , germany , in duplicate ( two independent samples measured on two similar instruments : kws-1 and kws-2 ) . samples were poured into quartz cuvettes and acquisitions were performed at room temperature ( observed temperature : 21.5 1.5 c ) . d2o was used as a solvent to increase the contrast ( difference in scattering length densities , sld ) and decrease the incoherent background mainly caused by hydrogen . three configurations were used at a fixed wavelength of 0.46 nm ( with full width at half - maximum of 10% on kws-1 and 20% on kws-2 ) , changing the sample - to - detector distance ( 1 , 5 , and 20 m ) . data were reduced in the small - angle approximation using the bersans software program , correcting measured intensities for the transmission , dead - time , detector background ( with b4c as a neutron absorber at the sample position ) , sample background ( either the empty cuvette or the solvent ) and light water ( 1 mm neutron pathway ) for the flat field . the absolute scale was obtained from a tabulated value of a 1.5 mm sheet of plexiglas . very small - angle neutron scattering ( vsans ) data were measured on kws-3 at mlz , at a wavelength of 1.2 nm and a sample - to - detector distance of 9.5 m with a beam aperture of 2 2 mm , the detector being at the focal point of the beam . the scattering from a cuvette filled with d2o ( 2 mm pathway ) was subtracted . the slds were calculated with values from jacrot , assuming that all exchangeable protons were replaced by deuterons .

elastin - like polypeptides ( elps ) are a class of biopolymers consisting of the pentameric repeat ( vpgg)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble - to - insoluble phase transition in aqueous solution . we have discovered a remarkably simple approach to driving the spontaneous self - assembly of high molecular weight elps into nanostructures by genetically fusing a short 1.5 kda ( xgy)z assembly domain to one end of the elp . classical theories of self - assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles . surprisingly , when sufficiently hydrophobic amino acids ( x ) are presented in a periodic sequence such as ( fgg)8 or ( yg)8 , these highly asymmetric polypeptides self - assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag . these nanostructures were characterized by light scattering , tunable resistive pulse sensing , fluorescence spectrophotometry , and thermal turbidimetry , as well as by cryogenic transmission electron microscopy ( cryo - tem ) and small - angle neutron scattering ( sans ) . these short assembly domains provide a facile strategy to control the size , shape , and stability of stimuli responsive polypeptide nanostructures .

Results Discussion Materials and Methods

all elp segments consist of the sequence ( vpgag)n , where the number of pentapeptide repeats herein , we refer to ( vpgag)40 as a40 , ( vpgag)80 as a80 , and ( vpgag)160 as a160 . the assembly domains consist of the sequence ( xgy)8 , where x represents a hydrophobic amino acid and y represents the number of glycine spacers ( y = 0 , 1 , or 2 ) . each polymer amphiphile was analyzed by dynamic light scattering ( dls ) to determine the extent of self - assembly and the hydrodynamic radius of the self - assembled nanoparticle . amphiphiles with ( xgg)8 segments incorporating x = leu , ile , his , and tyr did not self - assemble and remained fully soluble ( rh 6 nm ; figure 1a , table 2 ) . ( a ) a160-(igg)8 does not self - assemble and has a rh of 7 nm , whereas a160-(fgg)8 self - assembles into nanostructures with a rh of 42 nm . ( b ) a160-(yg)8 self - assembles into nanostructures with a rh of 73 nm that disassemble at ph 12 ( pkatyr = 10.1 ) because the deprotonation of tyrosine to tyrosinate greatly increases the hydrophilicity of the assembly domain , thereby disrupting the nanoparticle core . surprisingly , we observed that the number of gly ( g ) residues within the assembly domain potently controls the self - assembly of these asymmetric amphiphiles . for instance , a160-(ygg)8 displays insufficient amphiphilicity to self - assemble and thus exhibits an rh of 6 nm ( table 2 ) . however , replacing the ( ygg)8 domain with a ( yg)8 or y8 domain results in self - assembly into nanoparticles with a rh of 71 and 58 nm , respectively ( table 2 ) . this effect is most likely due to the hydrophilicity of the gly residues ; as the number of glycine residues decreases from 2 to 1 or 0 , the overall hydrophobicity of the assembly domain surpasses the threshold necessary to trigger nanoparticle self - assembly . it is clear , however , that not all systems are affected by the number of glycine residues equally ; amphiphiles with more hydrophobic assembly domains comprised of phe self - assemble regardless of the number of gly spacers ( table 2 ) . in this case , the hydrophobic driving force for self - assembly is strong enough to outweigh the contribution of the gly residues . as the ionized form of tyr ( pkatyr = 10.1 ) is 10 times more hydrophilic than neutral tyrosine , the a160-(yg)8 construct offered a unique opportunity to confirm that self - assembly is primarily driven by the hydrophobicity of the assembly domain . for those amphiphiles that self - assembled , static light scattering was used to calculate the aggregation number nagg so as to match table 2 , the number of amphiphile chains per nanoparticle , and the radius of gyration ( rg ) . the radius of gyration and the hydrodynamic radius were used to calculate the shape factor = rg / rh , which is characteristic of the morphology of the scatterer , where = 0.775 for solid spheres , = 1 for hollow spheres ( vesicles ) , and = 1.505 for random coils . we also investigated the effect of chain length on nanoparticle morphology by examining constructs with an ( fgg)8 assembly domain and different lengths of the elp domain . tunable resistive pulse sensing , a particle - by - particle analysis technique that relies not on scattering but the physical ability of nanoparticles to pass through a narrow pore was also used as an independent measure of particle size . we have previously shown that the lcst phase behavior of elps is exquisitely sensitive to polypeptide composition , fused proteins or chemically conjugated small molecules , ionic strength , and solvent conditions and can be used to drive the self - assembly of gold nanoparticles . thus , it was not surprising to observe that the hydrophobicity of the ( xgg)8 assembly domain also affected the lcst phase behavior ( figure 2 ) . in accordance with previous publications characterizing elps fused to proteins or chemically conjugated to small molecules , the tt of the amphiphile decreased as the hydrophobicity of the fused peptide assembly domain increased . we have also shown that the lcst phase transition behavior of unimeric elps is concentration - dependent such that a higher concentration of an elp phase separates at a lower temperature . in contrast , the tt of elps that display self - assembly showed virtually no dependence on elp concentration . in other words , the elp chains are entropically trapped once they self - assemble into micelles , and thus the entropic penalty for aggregation is smaller than for the free chains of elp unimers . in fact , it is possible to separate the constructs into assembling ( x = phe or trp ) and nonassembling ( x = leu , ile , his , or tyr ) populations based solely on the thermal turbidimetry profile as a function of concentration ( figure 2 ) . tt of nonself - assembling amphiphiles ( control , leu , ile , his , and tyr ) display a strong concentration dependence , whereas self - assembling amphiphiles ( phe and trp ) display tt that are near - independent of concentration . fluorescence spectrophotometry using pyrene as a probe was performed to sample the hydrophobicity of the inner core of the self - assembled structures and to determine their critical aggregation concentration ( cac ) . the amphiphile consisting of an a160 elp and a ( ygg)8 , assembly domains does not self - assemble and shows no concentration dependence in its i1/i3 ratio , whereas amphiphiles with ( fgg)8 and ( wgg)8 , assembly domains self - assemble into cylindrical micelles and show a two - state concentration - dependent behavior in their i1/i3 ratio . in addition , the minimum i1/i3 value can be used as a measure of the hydrophobicity of the core . surprisingly , the ( fgg)8 domain formed a core that was more hydrophobic than that of ( wgg)8 although tryptophan is considered more hydrophobic than phenylalanine . we also observed that decreasing the length of the elp domain from 160 to 40 pentapeptide repeats slightly increases the hydrophobicity and stability of the ( fgg)8 core as a function of elp chain length ( table 3 , supporting information figure s6 ) . despite clear evidence of self - assembled structures by light scattering ( table 2 ) and cryo - tem ( figure 4 ) , the pyrene fluorescence data for a160-y8 indicated that the fluor was in an aqueous environment over a range of concentrations , thus suggesting a lack of self - assembly . cryo - tem experiments were performed to directly visualize the self - assembled structures in their near - native hydrated state . however , due to their relatively low electron density , polypeptide amphiphiles are challenging to image by cryo - tem as they exhibit lower contrast than many synthetic polymer amphiphiles . although the hydrophobic cores have sufficient electron density to be clearly visualized by cryo - tem under these conditions , we were unable to visualize the hydrated elp corona in self - assembled structures or single elp chains ( unimers ) due to their low electron density and high degree of solvation . in contrast to conjugation - driven assembly , which only yields spherical micelles , these asymmetric amphiphiles spontaneously assemble into cylindrical micelles upon achieving sufficient hydrophobicity in the core ( figure 4a f ) . for instance , the a160 amphiphile with a terminal ( ygg)8 domain showed no self - assembly , but as the glycine fraction in the assembly domain was reduced with ( yg)8 and y8 assembly domains , amphiphiles spontaneously self - assembled into cylindrical micelles ( figure 4b c ) . we found that the gly content of the self - assembly domain controls the length of the micelles and hence their aspect ratio . the cryo - tem results were also consistent with results from light scattering . the value of 0.78 for a160-(yg)8 seemingly indicates a spherical morphology ; however , cryo - tem reveals that it is in fact a cylindrical morphology with a low aspect ratio . whereas the composition of the assembly domain played a role in controlling the aspect ratio of the nanoparticles , the length of the elp domain ( 160 , 80 , and 40 pentapeptides ) attached to the ( fgg)8 assembly domain only resulted in minor differences in the aspect ratio ( figure 4d f , table 4 ) . ( a ) constructs such as a160-(ygg)8 that do not self - assemble could not be visualized by cryo - tem because of their high levels of hydration and low densities . ( b , c ) changing the assembly domain from ( yg)8 ( b ) to y8 ( c ) causes a significant decrease in the length of the cylindrical micelles . ( d f ) a160-(fgg)8 ( d ) , a80-(fgg)8 ( e ) , and a40-(fgg)8 ( f ) self - assemble into cylindrical micelles with similar aspect ratios . this apparent aspect ratio is defined only by the portion of the assembly that is visible by cryo - tem and as such differs from the true aspect ratio determined by scattering . vsans ( very small - angle neutron scattering ) experiments were performed at lower q to confirm this size limit ( supporting information figure s7 ) . on the basis of cryo - tem data , we chose a model of isotropically oriented homogeneous cylindrical micelles that also incorporated scattering from individual polypeptide chains at smaller length scales ( see supporting information ) . however , in cryo - tem it is possible that many of the cylindrical micelles are tilted within the vitreous ice layer , leading to a lower observed length . it is also possible that the outer region of the core is too highly solvated to be observed by cryo - tem , thus explaining the larger radius observed by sans . we have observed that amphiphilic block copolymers comprised of a water - soluble polypeptide and an extremely short hydrophobic peptide self - assemble into cylindrical micelles . this observation is highly surprising and contrary to accepted theories of self - assembly of synthetic block copolymers because their extremely high hydrophilic weight fraction would predict either a lack of self - assembly or assembly into star - like micelles . we also observed that self - assembly is highly sensitive to the hydrophobicity of the assembly domain : less hydrophobic residues such as leu and ile do not drive self - assembly , whereas the more hydrophobic tyr , phe , and trp residues spontaneously form nanoparticles . self - assembly is also sensitive to the number of glycine residues between the hydrophobic residues . for instance , the ( ygg)8 domain was not sufficiently hydrophobic to drive self - assembly of a160 elp , whereas both the ( yg)8 and y8 domains drove self - assembly into cylindrical micelles . among the self - assembling constructs , the rg and rh determined by light scattering varied substantially ( from 46.1 to 124.4 nm and from 33.0 to 91.4 nm , respectively ) , while the shape factor ( rg / rh ) remained remarkably constant ( varying only between 1.31 and 1.43 ) for all but one amphiphile . cryo - tem revealed that all constructs other than a160-y8 self - assembled into similar cylindrical morphologies , and a160-y8 self - assembled into cylindrical micelles with a significantly lower aspect ratio . in addition to cryo - tem , sans provided a complementary ensemble measurement that confirmed the cylindrical morphology with core radii of 2123 nm and a length of 170 nm . fluorescence spectroscopy using pyrene as a probe revealed that despite their similar morphologies , the cylindrical micelles formed cores with varying levels of hydrophobicity ( as measured by the characteristic fluorescence behavior of pyrene ) and displayed a range of cacs from 0.4 to 13.0 m . our most unexpected finding is that asymmetric amphiphiles with hydrophilic fractions as high as 97% self - assemble into cylindrical micelles rather than spherical micelles . synthetic block copolymers canonically form spherical micelles when the hydrophilic weight fraction exceeds 60% , cylindrical micelles are favored between 45% and 55% , and vesicles are constrained to the region between 25% and 45% . the free energy of self - assembly and thus the morphology are determined primarily by the stretching of the core - forming block , the repulsion between the corona blocks , and the interfacial tension between the core and the solvent . surprisingly , within the sequence space sampled here the hydrophilic fraction does not seem to play a significant role in determining the morphology , as phenylalanine - based constructs with hydrophilic fractions of 97% ( chain length n = 160 ) , 94% ( n = 80 ) , and 88% ( n = 40 ) all assembled into similar cylindrical nanostructures as seen by cryo - tem and light scattering . the self - assembly of these asymmetric amphiphiles also raises more fundamental questions about how the self - assembly of polypeptide chains differs from that of synthetic polymers . given the aromatic groups present in tyr , phe , and trp side - chains , we believe that stacking of aromatic groups may play an important and as yet unexplored role in amphiphile self - assembly . the fact that the core diameter observed by sans is larger than the stretched length of the hydrophobic assembly domain also indicates that part of the hydrophilic elp chain may be collapsed onto the hydrophobic core . the slight increase in the hydrophobicity of the core as the elp block is shortened from 160 to 80 and 40 with an assembly domain of ( fgg)8 ( as measured by pyrene i1/i3 , table 3 ) is consistent with this hypothesis , as it indicates that the hydrophobic region may consist not only of the ( fgg)8 domain but also of a portion of the elp domain . in addition to their interesting material characteristics , this class of self - assembling amphiphiles should be useful for a number of biomedical applications . for instance , promoting self - assembly via a genetically encoded peptide instead of a chemically conjugated hydrophobe could allow for hydrophilic drugs to be repurposed into therapeutic nanoparticles . in the case of hydrophobic therapeutics , these issues are typically ameliorated by the sequestration of the drug into a protected nanoparticle core . the design of triblock peptide polymers with an elp segment , followed by a drug attachment domain and an assembly domain , could result in self - assembly into stable nanostructures despite the conjugation of a hydrophilic therapeutic within the core . the solution was kept on ice , and 3 m nacl was added to isothermally trigger the phase transition of the elp . dynamic light scattering was used to determine the hydrodynamic radius ( rh ) of the various constructs at 25 c and 25 m amphiphile concentration using a dynapro plate reader ( wyatt technology ; santa barbara , ca ) , following filtration through 0.22 m millex - gv filters ( millipore ; billerica , ma ) . forty microliters of the sample was loaded onto a qnano ( izon ; christchurch , new zealand ) instrument equipped with 50150 nm pores ( a160-(fgg)8 , a160-(fg)8 , a80-(fgg)8 , and a40-(fgg)8 ) or 100300 nm pores ( a160-(yg)8 , a160-f8 , and a160-(wgg)8 ) . pyrene fluorescence displays four peaks ; the intensity of the first ( i1 , 370373 nm ) and third peak ( i3 , 381384 nm ) were recorded . cryo - tem experiments were performed at duke university s shared materials instrumentation facility ( durham , nc ) and at the university of pennsylvania in the penn regional nanotechnology facility ( philadelphia , pa ) . three configurations were used at a fixed wavelength of 0.46 nm ( with full width at half - maximum of 10% on kws-1 and 20% on kws-2 ) , changing the sample - to - detector distance ( 1 , 5 , and 20 m ) . data were reduced in the small - angle approximation using the bersans software program , correcting measured intensities for the transmission , dead - time , detector background ( with b4c as a neutron absorber at the sample position ) , sample background ( either the empty cuvette or the solvent ) and light water ( 1 mm neutron pathway ) for the flat field . very small - angle neutron scattering ( vsans ) data were measured on kws-3 at mlz , at a wavelength of 1.2 nm and a sample - to - detector distance of 9.5 m with a beam aperture of 2 2 mm , the detector being at the focal point of the beam .

a great number of advanced cancer patients are suffering from physical and psychosocial burdens because of cancer cachexia , and these burdens also greatly impact on their family members and relationships between patients and family members.1 , 2 , 3 involuntary weight loss is a main symptom of cancer cachexia , and it is linked to the deterioration of physical function , quality of life , nutritional status , treatment outcomes , and survival in advanced cancer patients.4 , 5 , 6 , 7 , 8 weight loss often follows anorexia and declining food intake , and thus , these are causes of eatingrelated distress for patients and family members.1 , 2 , 3 in a survey of 2074 bereaved family members , 78% remembered the anorexia the patient had experienced , and 23% perceived the patient as very distressed by the anorexia.9 in addition , perspectives on declining food intake often conflict between patients and family members,1 , 10 , 11 and eating habits of patients are influenced by family members.12 , 13 these are also causes of eatingrelated distress for both patients and family members.10 , 11 , 12 , 13 therefore , it is necessary to consider the psychosocial impact of cancer cachexia,1 , 2 , 3 , 10 , 11 , 12 , 13 and currently the consensus that nutritional support is important to treat malnutrition due to cancer cachexia is growing.14 however , the psychosocial impact of cancer cachexia on family members is poorly understood , and their eatingrelated distress and need for nutritional support have not been clarified.1 , 2 , 3 this study was , therefore , designed to investigate the prevalence of eatingrelated distress and need for nutritional support of family members when the advanced cancer patients become unable to take nourishment orally in inpatient hospices , to categorize eatingrelated distress of family members , and to explore the association between their eatingrelated distress and depression . the present study was carried out as part of the japan hospice and palliative care evaluation study 3 , a crosssectional anonymous nationwide survey of bereaved family members of cancer patients to evaluate quality of care , quality of death and dying of the patients , and bereaved family outcomes , including depression and complex grief . the main study included bereaved family members of cancer patients who had died in either of the three palliative care settings ( general ward , inpatient hospice , or home ) , and this study included a subpopulation of the main study : bereaved family members of cancer patients who had died in inpatient hospices . in may 2014 , we mailed a questionnaire to the primary caregiver listed in the hospital medical chart of each patient according to previous surveys in japan.15 , 16 if family members did not want to participate , we requested them to return the questionnaire with the completion and return of the questionnaire were regarded as consent to participate in this study . primary physicians identified potential participants with the following inclusion criteria : ( i ) bereaved adult family members of an adult cancer patient who had died in an inpatient hospice ( one family member was selected for each patient ) ; ( ii ) capable of replying to a selfreport questionnaire ; ( iii ) aware of the diagnosis of malignancy ; and ( iv ) no serious psychological distress recognized by the primary physician . the last criterion was the same as in previous surveys15 , 16 and was adopted on the assumption that primary physicians were able to identify family members suffering from serious psychological distress because they were closely involved in caring for relatives of patients in inpatient hospices . the questionnaire for this study was developed by the authors on the basis of a systematic literature review and discussion among the authors.17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 face validity of the questionnaire was confirmed by a pilot test with five bereaved family members and four physicians . the first question was about the mental status of family members when they were caring for the patient ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( good ) to 4 ( poor ) . questions concerning eatingrelated distress of family members were composed of 19 items ( table 2 ) ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( no ) to 4 ( frequently ) . we then asked whether family members had the need for the following five items , with an answer of yes or no : ( i ) you would like to receive nutritional support for the patient from medical staff members ; ( ii ) you would like to receive sufficient explanations about the reasons for the anorexia and weight loss of the patient ; ( iii ) you would like to be provided with ideas on how to improve the patient 's food ; ( iv ) you would like to receive intervention regarding conflict concerning eating and food between you and the patient ; and ( v ) you would like your eatingrelated distress to be monitored ( table 3 ) . we also requested family members to answer the patient health questionnaire 9 ( phq9 ) , which is a selfadministered questionnaire composed of nine items to screen depression . a phq9 score 10 means that an answerer may suffer from major depression.32 , 33 , 34 primary physicians identified potential participants with the following inclusion criteria : ( i ) bereaved adult family members of an adult cancer patient who had died in an inpatient hospice ( one family member was selected for each patient ) ; ( ii ) capable of replying to a selfreport questionnaire ; ( iii ) aware of the diagnosis of malignancy ; and ( iv ) no serious psychological distress recognized by the primary physician . the last criterion was the same as in previous surveys15 , 16 and was adopted on the assumption that primary physicians were able to identify family members suffering from serious psychological distress because they were closely involved in caring for relatives of patients in inpatient hospices . the questionnaire for this study was developed by the authors on the basis of a systematic literature review and discussion among the authors.17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 face validity of the questionnaire was confirmed by a pilot test with five bereaved family members and four physicians . the first question was about the mental status of family members when they were caring for the patient ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( good ) to 4 ( poor ) . questions concerning eatingrelated distress of family members were composed of 19 items ( table 2 ) ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( no ) to 4 ( frequently ) . we then asked whether family members had the need for the following five items , with an answer of yes or no : ( i ) you would like to receive nutritional support for the patient from medical staff members ; ( ii ) you would like to receive sufficient explanations about the reasons for the anorexia and weight loss of the patient ; ( iii ) you would like to be provided with ideas on how to improve the patient 's food ; ( iv ) you would like to receive intervention regarding conflict concerning eating and food between you and the patient ; and ( v ) you would like your eatingrelated distress to be monitored ( table 3 ) . we also requested family members to answer the patient health questionnaire 9 ( phq9 ) , which is a selfadministered questionnaire composed of nine items to screen depression . a phq9 score 10 means that an answerer may suffer from major depression.32 , 33 , 34 we calculated the proportion of family members with a 95% confidence interval ( ci ) with regard to the questions . we then used explanatory factor analysis , using the principle method with a promax rotation . according to the results of the factor analysis , attributes with factor loadings less than 0.4 ( standardized regression coefficient ) we divided subjects into two categories regarding presence or absence of major depression ( phq9 scores 10 or < 10 ) . multiple logistic regression analysis was performed to identify independent determinants of major depression using demographics , mental status , and factors of eatingrelated distress , which were extracted with factor analysis . in the main study , 175 institutions [ general ward ( 20 ) , inpatient hospice ( 133 ) , and home ( 22 ) ] agreed to participate , and 10 715 family members were included . in this study , 133 inpatient hospices and 925 family members finally , 925 questionnaires were sent to them , and 702 were returned ( response rate , 75.9% ) . among these , 70 refused to participate , and 74 were excluded because of missing data on eatingrelated distress . in addition , 33 and 40 were excluded because of missing data on phq9 and other covariates , such as age , sex , relationship , and mental status , respectively . characteristics of patients were as follows : the mean age standard deviation was 72.7 11.4 years , 55.0% were men , and the mean number of hospital days was 39 53 . upper and lower gastrointestinal tracts ( 26.9% ) were the most common primary cancer site , followed by lung ( 22.6% ) and liver , biliary system , and pancreas ( 18.6% ) . as for bereaved family members , the mean age was 60.3 12.0 years , 33.7% were men , 9.9% and 43.9% had good or moderate mental status , respectively , and spouses made up 44.1% and children 37.5% . characteristics of participants values are mean standard deviation , or n ( % ) . i served what the patient wanted without consideration of calories and nutritional composition was highest ( 75.1% ) , and i tried making many kinds of meals for the patient and i was concerned about planning meals for the patient every day followed ( 63.0% and 59.4% , respectively ) . i felt disregarded when the patient could not eat meals which i made , and i thought that the patient could not eat because of a lack of effort on his / her part were very rare ( 7.8% , 4.7% , and 2.1% , respectively ) . eatingrelated distress of family members the prevalence of need for nutritional support is shown in table 3 . their need for nutritional support for their patients was high ( 72.2% ) , and need for explanations about the reasons for anorexia and weight loss of patients was moderate ( 41.4% ) , while need for intervention regarding conflict between the patient and family members and need for the family member 's eatingrelated distress to be monitored were low ( 27.8% and 20.8% , respectively ) . need for nutritional support of family members in accordance with the aforementioned item reduction procedure , 14 attributes for core domains were selected . the results of the factor analysis for core domains are shown in table 4 . the following four domains were identified : ( factor 1 ) feeling that family members forced the patient to eat to avoid death , ( factor 2 ) feeling that family members made great efforts to help the patient eat , ( factor 3 ) feeling that eating was a cause of conflicts between the patient and family members , and ( factor 4 ) feeling that correct information was insufficient . factor validity of eatingrelated distress of family members : four core domains ( n = 558 ) f # , factors 1 to 4 ; sd , standard deviation ; cronbach 's , cronbach 's alpha coefficients . results of multiple logistic regression analysis performed to identify independent determinants of major depression using demographics , mental status ( good / moderate and fair / poor ) , and the four factors of eatingrelated distress are shown in table 5 . data for factors 1 , 2 , and 4 were classified into three categories ( no , occasionally / sometimes , and frequently ) and data for factor 3 into two categories ( no and occasionally / sometimes / frequently ) after merging due to lack of data . factors of family members related to depression ( n = 485 ) others include childreninlaw , siblings , and parents of the patient . depression was diagnosed when the total score of the patient health questionnaire 9 was 10 points or greater . spouse , fair / poor mental status , factors 1 , and 4 were identified as independent determinants of major depression [ odds ratio ( or ) 3.27 ( 95% ci 1.248.60 ) , p = 0.02 ; or 4.50 ( 95% ci 2.468.25 ) , p < 0.001 ; or 2.51 ( 95% ci 1.165.45 ) , p = 0.02 ; or 2.33 ( 95% ci 1.134.80 ) , p = 0.02 , respectively ] ( table 5 ) . characteristics of patients were as follows : the mean age standard deviation was 72.7 11.4 years , 55.0% were men , and the mean number of hospital days was 39 53 . upper and lower gastrointestinal tracts ( 26.9% ) were the most common primary cancer site , followed by lung ( 22.6% ) and liver , biliary system , and pancreas ( 18.6% ) . as for bereaved family members , the mean age was 60.3 12.0 years , 33.7% were men , 9.9% and 43.9% had good or moderate mental status , respectively , and spouses made up 44.1% and children 37.5% . characteristics of participants values are mean standard deviation , or n ( % ) . i served what the patient wanted without consideration of calories and nutritional composition was highest ( 75.1% ) , and i tried making many kinds of meals for the patient and i was concerned about planning meals for the patient every day followed ( 63.0% and 59.4% , respectively ) . i felt disregarded when the patient could not eat meals which i made , and i thought that the patient could not eat because of a lack of effort on his / her part were very rare ( 7.8% , 4.7% , and 2.1% , respectively ) . their need for nutritional support for their patients was high ( 72.2% ) , and need for explanations about the reasons for anorexia and weight loss of patients was moderate ( 41.4% ) , while need for intervention regarding conflict between the patient and family members and need for the family member 's eatingrelated distress to be monitored were low ( 27.8% and 20.8% , respectively ) . in accordance with the aforementioned item reduction procedure , 14 attributes for core domains were selected . the results of the factor analysis for core domains are shown in table 4 . the following four domains were identified : ( factor 1 ) feeling that family members forced the patient to eat to avoid death , ( factor 2 ) feeling that family members made great efforts to help the patient eat , ( factor 3 ) feeling that eating was a cause of conflicts between the patient and family members , and ( factor 4 ) feeling that correct information was insufficient . factor validity of eatingrelated distress of family members : four core domains ( n = 558 ) f # , factors 1 to 4 ; sd , standard deviation ; cronbach 's , cronbach 's alpha coefficients . results of multiple logistic regression analysis performed to identify independent determinants of major depression using demographics , mental status ( good / moderate and fair / poor ) , and the four factors of eatingrelated distress are shown in table 5 . data for factors 1 , 2 , and 4 were classified into three categories ( no , occasionally / sometimes , and frequently ) and data for factor 3 into two categories ( no and occasionally / sometimes / frequently ) after merging due to lack of data . factors of family members related to depression ( n = 485 ) others include childreninlaw , siblings , and parents of the patient . depression was diagnosed when the total score of the patient health questionnaire 9 was 10 points or greater . spouse , fair / poor mental status , factors 1 , and 4 were identified as independent determinants of major depression [ odds ratio ( or ) 3.27 ( 95% ci 1.248.60 ) , p = 0.02 ; or 4.50 ( 95% ci 2.468.25 ) , p < 0.001 ; or 2.51 ( 95% ci 1.165.45 ) , p = 0.02 ; or 2.33 ( 95% ci 1.134.80 ) , p = 0.02 , respectively ] ( table 5 ) . eatingrelated distress and need for nutritional support of family members of advanced cancer patients have not been clarified . to the best of our knowledge , this study is the first large quantitative survey designed to investigate the prevalence of eatingrelated distress and need for nutritional support of family members , to categorize their eatingrelated distress , and to explore the association between their eatingrelated distress and depression . in this study , nutritional support means that medical staffs with specific nutrition , such as trained physicians , dietitians , pharmacists , and nurses , provide individualized and tailored nutritional support and advice to each patient . eatingrelated distress is emotional strains in advanced cancer patients and their family members caused by anorexia and weight loss of patients . negative impact of cancer cachexia is simply defined as diseaserelated anorexia and weight loss . in studies investigating eatingrelated distress of family members of advanced cancer patients , their responses to declining food intake and weight loss of patients were described as three separate subprocesses : fighting back , letting nature take its course , or waffling , the latter referring to vacillating between the first two patterns.25 , 26 , 27 in this study , the top 5 of the 19 items ( 48.975.1% ) were categorized as fighting back and the sixth item ( 48.5% ) as letting nature take its course. thus , the subprocess of fighting back by family members can cause distress . this study revealed that over 70% of family members needed nutritional support for their patients and over 40% would like to have received sufficient explanations about the reasons for anorexia and weight loss of the patient . this is consistent with a previous study , in which the main causes of psychosocial burdens of advanced cancer patients and family members were lack of knowledge about cancer cachexia , unsuccessful attempts to increase body weight , and expected occurrence of death.2 a study reported eatingrelated distress when the reality of eating differed from the expectations of eating.20 in our previous preliminary study , specific support , such as attention to patient 's distress about eating and explanation of the reasons for anorexia and weight loss , was needed by many patients , and it might alleviate their eatingrelated distress . the study also suggested that hopelessness , fretting , and a shortage of information aggravated their distress.31 in japan , inpatient hospices anorexic cachectic cancer patients are often treated with drugs , for example , corticosteroids , while they scarcely receive specific nutritional support maybe because medical staff members in hospices tend to withhold aggressive care . eatingrelated distress of patients and their family members in japan may be induced against such background . in addition , the identified four domains ( table 4 ) indicated where the focus of intervention for eatingrelated distress of family members should be . this study , as well as previous studies , indicated that medical staff members might contribute to the relief of patients and family members by giving advice on how to avoid futile activities and by explaining why anorexia and weight loss become irreversible.23 , 31 thus , we should explain the mechanism of cancer cachexia as simply as possible , and this may contribute to the relief of patients and family members by paying attention to their distress and by appreciating their efforts to cope . in this study , the experience of conflicts over food was one of the lowest frequency items ( 7.8% ) among the 19 , while the need for intervention in such conflicts was moderate ( 27.8% ) . it seems that overt emotional conflicts over food in families in japan inpatients hospices seldom occur despite family members ' latent distress . in contrast , reid et al.10 suggested that reduced dietary intake frequently became a source of conflict between patients and their family members in inpatients and outpatients units , while the prevalence of conflict was unclear . patients ate not because they wanted food but because they wanted to satisfy their family members , who were encouraging them to eat . family members often experienced feelings of rejection of their affection when food was refused by patients.9 , 10 changes in food preferences and eating habits induced conflict between patients and family members , because they failed to see weight loss as an inevitable consequence of cancer cachexia . while patients often felt dejected and harassed because of this conflict , family members also suffered . contrary to family members ' intention , their approach to the eating problem can become a barrier to food intake.10 , 18 in addition , anorexia is one of the most distressful symptoms in the last week of life in advanced cancer patients , but family members fail to rate it as important.29 a qualitative study suggested that perspectives of patients might not necessarily be shared by their family members and that anorexia of the patients was indeed a source of eatingrelated distress and conflict within the family.35 we can support patients and family members in managing conflict over food . we can draw from our own experiences of patients and family members who have found effective ways of managing conflict . sharing this experiential knowledge can demonstrate that disagreements over food can be temporary and present ideas that enable patients and family members to see new ways of managing their problems.18 our results imply that spouses have a higher risk of depression by loss of their partner . negative impacts of cancer cachexia affect cooking at home , the couple 's daily eating habits , and their spousal relationship.1 when food was rejected by patients , the family members , especially women , responded in two ways . first , they experienced feelings of guilt and fault , because they believed that not offering food might have been viewed as neglect on their part . second , they became angry when patients declined food that they prepared.9 , 10 another study suggested that family members ' eatingrelated distress was more pronounced in women than in men because women expressed their caring through the preparation and serving of food.35 female partners were more concerned about male patients ' weight loss than patients themselves , and patients felt more pressure to eat from partners than they had estimated.23 our results also imply that bereaved family members who feel that they forced the patient to eat to avoid death and those who feel that correct information was insufficient have a higher risk of bereavement depression . although it seems to be difficult to identify family members who may be suffering from depression , this study indicates that a female spouse who is excessively concerned about the patient 's daily diet without sufficient knowledge has a higher risk of depression by loss of her partner . thus , we should give advice on how to avoid futile activities and explain why anorexia and weight loss become irreversible to prevent bereaved family members ' depression . despite the strengths of a large multicenter study and high response rate , this study has several limitations . first , the survey subjects were family members , and care strategies suggested from this study are for family members . the distress of a symptom is unique to the patient and most accurate when described by the patient . family members tend to overrate the intensity of symptoms29 and experience more eatingrelated distress than patients ( 87% versus 36%).36 we do not , however , believe that this is a weakness of this study because family members are a main subject for palliative care . second , the study subjects were limited to family members of patients admitted to inpatient hospices , and nonresponding subjects were not included in the analysis , so the findings can not be generalized to other settings . third , we can not affirm that the patients of responding subjects were suffering from cachexia . however , upper and lower gastrointestinal tracts ( 26.9% ) were the most common primary cancer site , followed by lung ( 22.6% ) and liver , biliary system , and pancreas ( 18.6% ) in this study . fourth , there may have been recall bias because of the retrospective design of the study , including selfassessment of mental state . fifth , the questionnaire for this study had not been validated . however , there is no validated tool to estimate eatingrelated distress of advanced cancer patients and their family members . sixth , several yes / no questions have the potential to be affected by acquiescence bias . no were not high ( table 3 ) . this may mean that influence of acquiescence bias was slight . seventh , we can not compare backgrounds between participating and nonparticipating subjects , and thus there might be a systematic bias . a number of family members of advanced cancer patients admitted to inpatient hospices experienced high levels of eatingrelated distress and had a need for nutritional support for their patients . sufficient explanation about the reasons for anorexia and weight loss of patients ( i.e. mechanism of cancer cachexia ) may contribute to the relief of patients and family members and alleviate their eatingrelated distress . further surveys need to be developed to establish indicators and outcomes of care for eatingrelated distress of advanced cancer patients and family members . the authors declare no potential conflicts of interest with respect to the research , authorship , and publication of this article .

abstractbackgrounda number of advanced cancer patients are suffering from physical and psychosocial burdens because of cancer cachexia , and these burdens also greatly impact on their family members and relationships between patients and family members . it is necessary to consider the psychosocial impact of cancer cachexia on family members of advanced cancer patients.methodsa crosssectional anonymous nationwide survey was conducted involving 925 bereaved family members of cancer patients who had been admitted to 133 inpatient hospices throughout japan.resultsa total of 702 bereaved family members returned the questionnaires ( response rate , 75.9% ) . concerning eatingrelated distress , i served what the patient wanted without consideration of calories and nutritional composition was highest ( 75.1% ) , and i tried making many kinds of meals for the patient and i was concerned about planning meals for the patient every day followed ( 63.0% and 59.4% , respectively ) . the top 5 of the 19 items were categorized as fighting back. need for nutritional support was high ( 72.2% ) , and need for explanations about the reasons for anorexia and weight loss of patients was moderate ( 41.4% ) . explanatory factor analysis of eatingrelated distress identified the following four domains : ( factor 1 ) feeling that family members forced the patient to eat to avoid death , ( factor 2 ) feeling that family members made great efforts to help the patient eat , ( factor 3 ) feeling that eating was a cause of conflicts between the patient and family members , and ( factor 4 ) feeling that correct information was insufficient . results of multiple logistic regression analysis showed that spouse , fair / poor mental status , factors 1 , and 4 were identified as independent determinants of major depression { odds ratio [ or ] 3.27 [ 95% confidence interval ( ci ) 1.248.60 ] , p = 0.02 ; or 4.50 [ 95% ci 2.468.25 ] , p < 0.001 ; or 2.51 [ 95% ci 1.165.45 ] , p = 0.02 ; or 2.33 [ 95% ci 1.134.80 ] , p = 0.02 , respectively}.conclusionsa number of family members of advanced cancer patients experienced high levels of eatingrelated distress and had a need for nutritional support .

Introduction Methods Subjects Questionnaire Statistical analyses Results Characteristics of participants Eatingrelated distress of family members Need for nutritional support of family members Factor analysis Multiple logistic regression analysis Discussion Conclusions Conflict of interest

a great number of advanced cancer patients are suffering from physical and psychosocial burdens because of cancer cachexia , and these burdens also greatly impact on their family members and relationships between patients and family members.1 , 2 , 3 involuntary weight loss is a main symptom of cancer cachexia , and it is linked to the deterioration of physical function , quality of life , nutritional status , treatment outcomes , and survival in advanced cancer patients.4 , 5 , 6 , 7 , 8 weight loss often follows anorexia and declining food intake , and thus , these are causes of eatingrelated distress for patients and family members.1 , 2 , 3 in a survey of 2074 bereaved family members , 78% remembered the anorexia the patient had experienced , and 23% perceived the patient as very distressed by the anorexia.9 in addition , perspectives on declining food intake often conflict between patients and family members,1 , 10 , 11 and eating habits of patients are influenced by family members.12 , 13 these are also causes of eatingrelated distress for both patients and family members.10 , 11 , 12 , 13 therefore , it is necessary to consider the psychosocial impact of cancer cachexia,1 , 2 , 3 , 10 , 11 , 12 , 13 and currently the consensus that nutritional support is important to treat malnutrition due to cancer cachexia is growing.14 however , the psychosocial impact of cancer cachexia on family members is poorly understood , and their eatingrelated distress and need for nutritional support have not been clarified.1 , 2 , 3 this study was , therefore , designed to investigate the prevalence of eatingrelated distress and need for nutritional support of family members when the advanced cancer patients become unable to take nourishment orally in inpatient hospices , to categorize eatingrelated distress of family members , and to explore the association between their eatingrelated distress and depression . the present study was carried out as part of the japan hospice and palliative care evaluation study 3 , a crosssectional anonymous nationwide survey of bereaved family members of cancer patients to evaluate quality of care , quality of death and dying of the patients , and bereaved family outcomes , including depression and complex grief . the main study included bereaved family members of cancer patients who had died in either of the three palliative care settings ( general ward , inpatient hospice , or home ) , and this study included a subpopulation of the main study : bereaved family members of cancer patients who had died in inpatient hospices . primary physicians identified potential participants with the following inclusion criteria : ( i ) bereaved adult family members of an adult cancer patient who had died in an inpatient hospice ( one family member was selected for each patient ) ; ( ii ) capable of replying to a selfreport questionnaire ; ( iii ) aware of the diagnosis of malignancy ; and ( iv ) no serious psychological distress recognized by the primary physician . the first question was about the mental status of family members when they were caring for the patient ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( good ) to 4 ( poor ) . questions concerning eatingrelated distress of family members were composed of 19 items ( table 2 ) ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( no ) to 4 ( frequently ) . we then asked whether family members had the need for the following five items , with an answer of yes or no : ( i ) you would like to receive nutritional support for the patient from medical staff members ; ( ii ) you would like to receive sufficient explanations about the reasons for the anorexia and weight loss of the patient ; ( iii ) you would like to be provided with ideas on how to improve the patient 's food ; ( iv ) you would like to receive intervention regarding conflict concerning eating and food between you and the patient ; and ( v ) you would like your eatingrelated distress to be monitored ( table 3 ) . a phq9 score 10 means that an answerer may suffer from major depression.32 , 33 , 34 primary physicians identified potential participants with the following inclusion criteria : ( i ) bereaved adult family members of an adult cancer patient who had died in an inpatient hospice ( one family member was selected for each patient ) ; ( ii ) capable of replying to a selfreport questionnaire ; ( iii ) aware of the diagnosis of malignancy ; and ( iv ) no serious psychological distress recognized by the primary physician . the last criterion was the same as in previous surveys15 , 16 and was adopted on the assumption that primary physicians were able to identify family members suffering from serious psychological distress because they were closely involved in caring for relatives of patients in inpatient hospices . the first question was about the mental status of family members when they were caring for the patient ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( good ) to 4 ( poor ) . questions concerning eatingrelated distress of family members were composed of 19 items ( table 2 ) ; they were evaluated by the degree of agreement with the following statements on a 4point likerttype scale of 1 ( no ) to 4 ( frequently ) . we then asked whether family members had the need for the following five items , with an answer of yes or no : ( i ) you would like to receive nutritional support for the patient from medical staff members ; ( ii ) you would like to receive sufficient explanations about the reasons for the anorexia and weight loss of the patient ; ( iii ) you would like to be provided with ideas on how to improve the patient 's food ; ( iv ) you would like to receive intervention regarding conflict concerning eating and food between you and the patient ; and ( v ) you would like your eatingrelated distress to be monitored ( table 3 ) . a phq9 score 10 means that an answerer may suffer from major depression.32 , 33 , 34 we calculated the proportion of family members with a 95% confidence interval ( ci ) with regard to the questions . according to the results of the factor analysis , attributes with factor loadings less than 0.4 ( standardized regression coefficient ) we divided subjects into two categories regarding presence or absence of major depression ( phq9 scores 10 or < 10 ) . multiple logistic regression analysis was performed to identify independent determinants of major depression using demographics , mental status , and factors of eatingrelated distress , which were extracted with factor analysis . in this study , 133 inpatient hospices and 925 family members finally , 925 questionnaires were sent to them , and 702 were returned ( response rate , 75.9% ) . in addition , 33 and 40 were excluded because of missing data on phq9 and other covariates , such as age , sex , relationship , and mental status , respectively . as for bereaved family members , the mean age was 60.3 12.0 years , 33.7% were men , 9.9% and 43.9% had good or moderate mental status , respectively , and spouses made up 44.1% and children 37.5% . i served what the patient wanted without consideration of calories and nutritional composition was highest ( 75.1% ) , and i tried making many kinds of meals for the patient and i was concerned about planning meals for the patient every day followed ( 63.0% and 59.4% , respectively ) . i felt disregarded when the patient could not eat meals which i made , and i thought that the patient could not eat because of a lack of effort on his / her part were very rare ( 7.8% , 4.7% , and 2.1% , respectively ) . eatingrelated distress of family members the prevalence of need for nutritional support is shown in table 3 . their need for nutritional support for their patients was high ( 72.2% ) , and need for explanations about the reasons for anorexia and weight loss of patients was moderate ( 41.4% ) , while need for intervention regarding conflict between the patient and family members and need for the family member 's eatingrelated distress to be monitored were low ( 27.8% and 20.8% , respectively ) . need for nutritional support of family members in accordance with the aforementioned item reduction procedure , 14 attributes for core domains were selected . the following four domains were identified : ( factor 1 ) feeling that family members forced the patient to eat to avoid death , ( factor 2 ) feeling that family members made great efforts to help the patient eat , ( factor 3 ) feeling that eating was a cause of conflicts between the patient and family members , and ( factor 4 ) feeling that correct information was insufficient . factor validity of eatingrelated distress of family members : four core domains ( n = 558 ) f # , factors 1 to 4 ; sd , standard deviation ; cronbach 's , cronbach 's alpha coefficients . results of multiple logistic regression analysis performed to identify independent determinants of major depression using demographics , mental status ( good / moderate and fair / poor ) , and the four factors of eatingrelated distress are shown in table 5 . data for factors 1 , 2 , and 4 were classified into three categories ( no , occasionally / sometimes , and frequently ) and data for factor 3 into two categories ( no and occasionally / sometimes / frequently ) after merging due to lack of data . factors of family members related to depression ( n = 485 ) others include childreninlaw , siblings , and parents of the patient . spouse , fair / poor mental status , factors 1 , and 4 were identified as independent determinants of major depression [ odds ratio ( or ) 3.27 ( 95% ci 1.248.60 ) , p = 0.02 ; or 4.50 ( 95% ci 2.468.25 ) , p < 0.001 ; or 2.51 ( 95% ci 1.165.45 ) , p = 0.02 ; or 2.33 ( 95% ci 1.134.80 ) , p = 0.02 , respectively ] ( table 5 ) . as for bereaved family members , the mean age was 60.3 12.0 years , 33.7% were men , 9.9% and 43.9% had good or moderate mental status , respectively , and spouses made up 44.1% and children 37.5% . i served what the patient wanted without consideration of calories and nutritional composition was highest ( 75.1% ) , and i tried making many kinds of meals for the patient and i was concerned about planning meals for the patient every day followed ( 63.0% and 59.4% , respectively ) . i felt disregarded when the patient could not eat meals which i made , and i thought that the patient could not eat because of a lack of effort on his / her part were very rare ( 7.8% , 4.7% , and 2.1% , respectively ) . their need for nutritional support for their patients was high ( 72.2% ) , and need for explanations about the reasons for anorexia and weight loss of patients was moderate ( 41.4% ) , while need for intervention regarding conflict between the patient and family members and need for the family member 's eatingrelated distress to be monitored were low ( 27.8% and 20.8% , respectively ) . the following four domains were identified : ( factor 1 ) feeling that family members forced the patient to eat to avoid death , ( factor 2 ) feeling that family members made great efforts to help the patient eat , ( factor 3 ) feeling that eating was a cause of conflicts between the patient and family members , and ( factor 4 ) feeling that correct information was insufficient . factor validity of eatingrelated distress of family members : four core domains ( n = 558 ) f # , factors 1 to 4 ; sd , standard deviation ; cronbach 's , cronbach 's alpha coefficients . results of multiple logistic regression analysis performed to identify independent determinants of major depression using demographics , mental status ( good / moderate and fair / poor ) , and the four factors of eatingrelated distress are shown in table 5 . data for factors 1 , 2 , and 4 were classified into three categories ( no , occasionally / sometimes , and frequently ) and data for factor 3 into two categories ( no and occasionally / sometimes / frequently ) after merging due to lack of data . factors of family members related to depression ( n = 485 ) others include childreninlaw , siblings , and parents of the patient . spouse , fair / poor mental status , factors 1 , and 4 were identified as independent determinants of major depression [ odds ratio ( or ) 3.27 ( 95% ci 1.248.60 ) , p = 0.02 ; or 4.50 ( 95% ci 2.468.25 ) , p < 0.001 ; or 2.51 ( 95% ci 1.165.45 ) , p = 0.02 ; or 2.33 ( 95% ci 1.134.80 ) , p = 0.02 , respectively ] ( table 5 ) . eatingrelated distress and need for nutritional support of family members of advanced cancer patients have not been clarified . to the best of our knowledge , this study is the first large quantitative survey designed to investigate the prevalence of eatingrelated distress and need for nutritional support of family members , to categorize their eatingrelated distress , and to explore the association between their eatingrelated distress and depression . eatingrelated distress is emotional strains in advanced cancer patients and their family members caused by anorexia and weight loss of patients . negative impact of cancer cachexia is simply defined as diseaserelated anorexia and weight loss . in studies investigating eatingrelated distress of family members of advanced cancer patients , their responses to declining food intake and weight loss of patients were described as three separate subprocesses : fighting back , letting nature take its course , or waffling , the latter referring to vacillating between the first two patterns.25 , 26 , 27 in this study , the top 5 of the 19 items ( 48.975.1% ) were categorized as fighting back and the sixth item ( 48.5% ) as letting nature take its course. this study revealed that over 70% of family members needed nutritional support for their patients and over 40% would like to have received sufficient explanations about the reasons for anorexia and weight loss of the patient . this is consistent with a previous study , in which the main causes of psychosocial burdens of advanced cancer patients and family members were lack of knowledge about cancer cachexia , unsuccessful attempts to increase body weight , and expected occurrence of death.2 a study reported eatingrelated distress when the reality of eating differed from the expectations of eating.20 in our previous preliminary study , specific support , such as attention to patient 's distress about eating and explanation of the reasons for anorexia and weight loss , was needed by many patients , and it might alleviate their eatingrelated distress . the study also suggested that hopelessness , fretting , and a shortage of information aggravated their distress.31 in japan , inpatient hospices anorexic cachectic cancer patients are often treated with drugs , for example , corticosteroids , while they scarcely receive specific nutritional support maybe because medical staff members in hospices tend to withhold aggressive care . eatingrelated distress of patients and their family members in japan may be induced against such background . in addition , the identified four domains ( table 4 ) indicated where the focus of intervention for eatingrelated distress of family members should be . this study , as well as previous studies , indicated that medical staff members might contribute to the relief of patients and family members by giving advice on how to avoid futile activities and by explaining why anorexia and weight loss become irreversible.23 , 31 thus , we should explain the mechanism of cancer cachexia as simply as possible , and this may contribute to the relief of patients and family members by paying attention to their distress and by appreciating their efforts to cope . in this study , the experience of conflicts over food was one of the lowest frequency items ( 7.8% ) among the 19 , while the need for intervention in such conflicts was moderate ( 27.8% ) . patients ate not because they wanted food but because they wanted to satisfy their family members , who were encouraging them to eat . family members often experienced feelings of rejection of their affection when food was refused by patients.9 , 10 changes in food preferences and eating habits induced conflict between patients and family members , because they failed to see weight loss as an inevitable consequence of cancer cachexia . contrary to family members ' intention , their approach to the eating problem can become a barrier to food intake.10 , 18 in addition , anorexia is one of the most distressful symptoms in the last week of life in advanced cancer patients , but family members fail to rate it as important.29 a qualitative study suggested that perspectives of patients might not necessarily be shared by their family members and that anorexia of the patients was indeed a source of eatingrelated distress and conflict within the family.35 we can support patients and family members in managing conflict over food . we can draw from our own experiences of patients and family members who have found effective ways of managing conflict . sharing this experiential knowledge can demonstrate that disagreements over food can be temporary and present ideas that enable patients and family members to see new ways of managing their problems.18 our results imply that spouses have a higher risk of depression by loss of their partner . negative impacts of cancer cachexia affect cooking at home , the couple 's daily eating habits , and their spousal relationship.1 when food was rejected by patients , the family members , especially women , responded in two ways . second , they became angry when patients declined food that they prepared.9 , 10 another study suggested that family members ' eatingrelated distress was more pronounced in women than in men because women expressed their caring through the preparation and serving of food.35 female partners were more concerned about male patients ' weight loss than patients themselves , and patients felt more pressure to eat from partners than they had estimated.23 our results also imply that bereaved family members who feel that they forced the patient to eat to avoid death and those who feel that correct information was insufficient have a higher risk of bereavement depression . although it seems to be difficult to identify family members who may be suffering from depression , this study indicates that a female spouse who is excessively concerned about the patient 's daily diet without sufficient knowledge has a higher risk of depression by loss of her partner . thus , we should give advice on how to avoid futile activities and explain why anorexia and weight loss become irreversible to prevent bereaved family members ' depression . second , the study subjects were limited to family members of patients admitted to inpatient hospices , and nonresponding subjects were not included in the analysis , so the findings can not be generalized to other settings . however , there is no validated tool to estimate eatingrelated distress of advanced cancer patients and their family members . a number of family members of advanced cancer patients admitted to inpatient hospices experienced high levels of eatingrelated distress and had a need for nutritional support for their patients . sufficient explanation about the reasons for anorexia and weight loss of patients ( i.e. mechanism of cancer cachexia ) may contribute to the relief of patients and family members and alleviate their eatingrelated distress . further surveys need to be developed to establish indicators and outcomes of care for eatingrelated distress of advanced cancer patients and family members .

the university of british columbia clinical research ethics board ( certificate nos . h07 - 01707 and h03 - 70046 ) approved the collection of blood , and informed consent was received from nondiabetic control and type 1 diabetic subjects . complete blood - cell counts were performed on fresh blood using a sysmex xe-2100 automated multiparameter blood - cell counter at the children s and women s health centre of british columbia . cells were pretreated with anti - cd16 ( 3g8 ; biolegend ) antibody ( ab ) to block nonspecific binding to fc receptors prior to samples being stained with the indicated markers . abs specific for cd3 ( hit3a ) , cd4 ( rpa - t4 ) , cd8 ( hit8a ) , cd19 ( hib19 ) , cd25 ( 2a3 ) , 2b4 ( 2 - 69 ) , lair-1 ( dx26 ) , nkb1 ( dx9 ) , cd94 ( hp-3d9 ) , cd56 ( b159 ) , cd122 ( mik - b2 ) , and cd132 ( ag184 ) were purchased from bd biosciences . abs recognizing il-15r ( ebiojm7a4 ; ebioscience ) , cd16 ( cb16 , ebioscience ) , nkg2d ( fab139p ; r&d systems ) , major histocompatibility complex class i related chains a and b ( mica / b ) clone 159207 ( r&d systems ) , and mica / b clone 6d4 ( biolegend ) were acquired from the indicated sources . samples were analyzed on a facscalibur flow cytometer using cellquest software ( bd biosciences ) . nk cells were isolated from peripheral blood using a human nk cell enrichment kit ( stemcell technologies ) , and typical isolation resulted in 96% purity , as determined by staining with anti - cd3 and anti - cd56 abs ( data not shown ) . purified nk cells were expanded in rpmi-1640 medium containing 10% ab human serum , 1 mmol / l nonessential amino acids , 5 10 2-me , 1000 units / ml human ril-2 ( bd biosciences ) , and 50 units / ml ril-15 ( ebioscience ) . nk cells were expanded in vitro for 57 days prior to their use in cytotoxicity and enzyme - linked immunosorbent spot ( elispot ) assays . target cell lines k562 , raji , and daudi were acquired from the american type culture collection . one million target cells were labeled by incubating cells with 100 ci cr for 90 min at 37c , washing them three times with pbs , and seeding them at 10 cells / well in round - bottom 96-well plates . various numbers of effectors were added to each well , and plates were centrifuged at 500 rpm for 2 min and incubated at 37c . after 4 h incubation , 100-l volumes of supernatant were collected and the amount of cr released was measured using a counter . for nkg2d stimulation , nk cells were treated with 10 g / ml anti - nkg2d ( mab139 ; r&d systems ) abs for 20 min at 37c . after stimulation , cells were washed twice with complete rpmi medium prior to their use as effectors in cytotoxic t - lymphocyte ( ctl ) assays . the preparation of nk cell effectors was carried out identically as that performed for the ctl assays . elispots were performed in 96-well flat - bottom maip s4510 plates ( millipore ) using a human ifn- elispot kit from mabtech . immunospot plates were coated with 15 g / ml capture anti - human ifn- mabs ( clone 1-d1 k ) by overnight incubation at 4c . a total of 5,000 of the indicated target cells were mixed with 50,000 or 25,000 lymphokine - activated killer ( lak ) cells ( 10:1 or 5:1 effector : target ratios ) and cultured for 24 h. captured cytokines were detected with biotinylated anti ifn- mabs ( clone 7-b6 - 1 ) , and spots were developed using streptavidin - alkaline phosphatase and counted with bioreader-4000 ( bio - sys ) . il-2/il-15cultured nk cells ( 10 10 cells / ml ) were stimulated with 10 g / ml of anti - nkg2d ab ( mab139 ; r&d systems ) for 15 min at 37c . cells were lysed in ice - cold lysis buffer ( 20 mmol / l tris - hcl , ph 8.2 ; 100 mmol / l nacl ; and 10 mmol / l edta ) containing a protease inhibitor cocktail ( sigma ) . nkg2d was immunoprecipitated using anti - nkg2d abs ( 1d11 ; ebioscience ) and a combination of protein g - sepharose and anti - mouse igg - agarose ( santa cruz biotechnology ) . cell lysates and immunoprecipitates were analyzed by blotting with either anti - pi3k ( 06 - 496 ; upstate biotechnology ) or anti - nkg2d ( 1d11 ; ebioscience ) abs . anti - mouse igg abs coupled to horseradish peroxidase ( biorad ) and electrochemiluminescence ( pierce biotechnology ) were used to detect membrane - bound anti - pi3k and anti - nkg2d abs . nk cells , expanded in complete medium containing 1,000 units / ml il-2 and il-15 ( bd biosciences ) for 10 days , were serum starved for 4 h. for nkg2d - signaling studies , nk cells ( 10 cells / ml ) were incubated with 10 g / ml of anti - nkg2d mabs ( r&d systems ) for 10 min on ice , washed twice with pbs , and incubated for 1 min or 5 min in 37c warmed pbs containing affinity - purified rabbit anti - mouse igg f(ab')2 ( jackson immunoresearch laboratories ) . the fabrication and processing of lysate arrays has previously been discussed in detail ( 30 ) . 9271 ) , and p(s473)-akt ( no . 4058 ; mabs ) primary abs from cell signaling technology . the processed slides were scanned using a genepix 4000a microarray scanner ( molecular devices ) and analyzed with genepix pro 6.0 software ( molecular devices ) . for each sample printed in triplicate , the background - subtracted median fluorescence intensities were averaged and the intensity fold - change compared with the unstimulated sample calculated as a ratio of background - subtracted median fluorescence intensities for each time point versus the background - subtracted median fluorescence intensities of the unstimulated sample . the log base 2 values of these ratios were depicted in heatmap format using tigr multiexperiment viewer software , and data were expressed as the means sd ( 31 ) . a student t test was used to calculate statistical significance where indicated , and a single - factor anova was used for multigroup comparison . prism software ( graphpad software ) was used to create graphs and provided assistance with statistical tests . the university of british columbia clinical research ethics board ( certificate nos . h07 - 01707 and h03 - 70046 ) approved the collection of blood , and informed consent was received from nondiabetic control and type 1 diabetic subjects . complete blood - cell counts were performed on fresh blood using a sysmex xe-2100 automated multiparameter blood - cell counter at the children s and women s health centre of british columbia . cells were pretreated with anti - cd16 ( 3g8 ; biolegend ) antibody ( ab ) to block nonspecific binding to fc receptors prior to samples being stained with the indicated markers . abs specific for cd3 ( hit3a ) , cd4 ( rpa - t4 ) , cd8 ( hit8a ) , cd19 ( hib19 ) , cd25 ( 2a3 ) , 2b4 ( 2 - 69 ) , lair-1 ( dx26 ) , nkb1 ( dx9 ) , cd94 ( hp-3d9 ) , cd56 ( b159 ) , cd122 ( mik - b2 ) , and cd132 ( ag184 ) were purchased from bd biosciences . abs recognizing il-15r ( ebiojm7a4 ; ebioscience ) , cd16 ( cb16 , ebioscience ) , nkg2d ( fab139p ; r&d systems ) , major histocompatibility complex class i related chains a and b ( mica / b ) clone 159207 ( r&d systems ) , and mica / b clone 6d4 ( biolegend ) were acquired from the indicated sources . samples were analyzed on a facscalibur flow cytometer using cellquest software ( bd biosciences ) . nk cells were isolated from peripheral blood using a human nk cell enrichment kit ( stemcell technologies ) , and typical isolation resulted in 96% purity , as determined by staining with anti - cd3 and anti - cd56 abs ( data not shown ) . purified nk cells were expanded in rpmi-1640 medium containing 10% ab human serum , 1 mmol / l nonessential amino acids , 5 10 2-me , 1000 units / ml human ril-2 ( bd biosciences ) , and 50 units / ml ril-15 ( ebioscience ) . nk cells were expanded in vitro for 57 days prior to their use in cytotoxicity and enzyme - linked immunosorbent spot ( elispot ) assays . target cell lines k562 , raji , and daudi were acquired from the american type culture collection . one million target cells were labeled by incubating cells with 100 ci cr for 90 min at 37c , washing them three times with pbs , and seeding them at 10 cells / well in round - bottom 96-well plates . various numbers of effectors were added to each well , and plates were centrifuged at 500 rpm for 2 min and incubated at 37c . after 4 h incubation , 100-l volumes of supernatant were collected and the amount of cr released was measured using a counter . for nkg2d stimulation , nk cells were treated with 10 g / ml anti - nkg2d ( mab139 ; r&d systems ) abs for 20 min at 37c . after stimulation , cells were washed twice with complete rpmi medium prior to their use as effectors in cytotoxic t - lymphocyte ( ctl ) assays . the preparation of nk cell effectors was carried out identically as that performed for the ctl assays . elispots were performed in 96-well flat - bottom maip s4510 plates ( millipore ) using a human ifn- elispot kit from mabtech . immunospot plates were coated with 15 g / ml capture anti - human ifn- mabs ( clone 1-d1 k ) by overnight incubation at 4c . a total of 5,000 of the indicated target cells were mixed with 50,000 or 25,000 lymphokine - activated killer ( lak ) cells ( 10:1 or 5:1 effector : target ratios ) and cultured for 24 h. captured cytokines were detected with biotinylated anti ifn- mabs ( clone 7-b6 - 1 ) , and spots were developed using streptavidin - alkaline phosphatase and counted with bioreader-4000 ( bio - sys ) . il-2/il-15cultured nk cells ( 10 10 cells / ml ) were stimulated with 10 g / ml of anti - nkg2d ab ( mab139 ; r&d systems ) for 15 min at 37c . cells were lysed in ice - cold lysis buffer ( 20 mmol / l tris - hcl , ph 8.2 ; 100 mmol / l nacl ; and 10 mmol / l edta ) containing a protease inhibitor cocktail ( sigma ) . nkg2d was immunoprecipitated using anti - nkg2d abs ( 1d11 ; ebioscience ) and a combination of protein g - sepharose and anti - mouse igg - agarose ( santa cruz biotechnology ) . cell lysates and immunoprecipitates were analyzed by blotting with either anti - pi3k ( 06 - 496 ; upstate biotechnology ) or anti - nkg2d ( 1d11 ; ebioscience ) abs . anti - mouse igg abs coupled to horseradish peroxidase ( biorad ) and electrochemiluminescence ( pierce biotechnology ) were used to detect membrane - bound anti - pi3k and anti - nkg2d abs . nk cells , expanded in complete medium containing 1,000 units / ml il-2 and il-15 ( bd biosciences ) for 10 days , were serum starved for 4 h. for nkg2d - signaling studies , nk cells ( 10 cells / ml ) were incubated with 10 g / ml of anti - nkg2d mabs ( r&d systems ) for 10 min on ice , washed twice with pbs , and incubated for 1 min or 5 min in 37c warmed pbs containing affinity - purified rabbit anti - mouse igg f(ab')2 ( jackson immunoresearch laboratories ) . the fabrication and processing of lysate arrays has previously been discussed in detail ( 30 ) . 9271 ) , and p(s473)-akt ( no . 4058 ; mabs ) primary abs from cell signaling technology . the processed slides were scanned using a genepix 4000a microarray scanner ( molecular devices ) and analyzed with genepix pro 6.0 software ( molecular devices ) . for each sample printed in triplicate , the background - subtracted median fluorescence intensities were averaged and the intensity fold - change compared with the unstimulated sample calculated as a ratio of background - subtracted median fluorescence intensities for each time point versus the background - subtracted median fluorescence intensities of the unstimulated sample . the log base 2 values of these ratios were depicted in heatmap format using tigr multiexperiment viewer software , and data were expressed as the means sd ( 31 ) . a student t test was used to calculate statistical significance where indicated , and a single - factor anova was used for multigroup comparison . prism software ( graphpad software ) was used to create graphs and provided assistance with statistical tests . to address whether numerical or functional nk cell defects are present in human type 1 diabetic subjects , we analyzed peripheral blood mononuclear cells ( pbmcs ) from subjects with established type 1 diabetes ( > 0.5 years and < 2 years ; mean age 9.3 4.5 years ; mean type 1 diabetes duration 1.4 0.5 years ) and age - matched nondiabetic control subjects ( mean age 10.7 4.0 years ) using great care to follow standardized and consistent processing of blood samples and experimental conditions ( table 1 ) . we rationalized that if nk cell dysfunction was an intrinsic property of the type 1 diabetes immune system , longstanding measurable defects would still be present in subjects after establishment of disease . we also limited our subjects to those whose onset of diabetes was no greater than 2 years in order to minimize the potential effects of chronic hyperglycemia on lymphocyte number and function . frequencies of nk ( cd3cd56 ) , nkt ( cd3cd56 ) , cd4 t ( cd3cd56cd4 ) , cd8 t ( cd3cd56cd8 ) , and b - cell ( cd3cd19 ) subsets among pbmcs were assessed using standard flow cytometric techniques ( fig . 1a and b ) . in contrast to the similar proportions of cd4 t- , cd8 t- , and b - cells in the peripheral blood of type 1 diabetic subjects relative to control subjects , the nk cell fraction in type 1 diabetic subjects was markedly reduced ( ~37% ) relative to nondiabetic age - matched control subjects ( control subjects : 6.58 2.93% vs. type 1 diabetic subjects : 4.18 1.66% ; p < 0.0005 ) . to ascertain whether type 1 diabetic subjects exhibit a decrease in absolute nk cell numbers , complete blood - cell counts were performed on fresh blood samples from type 1 diabetic and nondiabetic subjects ( fig . 1c ) . total lymphocyte numbers were found to be modestly reduced in type 1 diabetic subjects relative to control subjects , although these numbers both fell within the normal range for age at our institution ( type 1 diabetic subjects : 2.21 0.74 10/l , n = 11 vs. control subjects : 2.96 0.74 10/l , n = 10 ; p < 0.02 ) , and absolute nk cell numbers per blood volume were decreased approximately twofold in type 1 diabetic subjects relative to control subjects ( type 1 diabetic subjects : 0.92 0.37 10/l vs. control subjects : 1.94 0.86 10/l ; p < 0.0001 ) . characteristics of the type 1 diabetic subjects and nondiabetic control groups from british columbia 's children 's hospital are presented data are means sd . all type 1 diabetic subjects were being treated with insulin and did not show evidence of other autoimmune diseases . age - matched subjects with no autoimmune or metabolic diseases were used as nondiabetic control subjects . nk cells from pbmcs of type 1 diabetic ( t1d ) subjects are present at reduced cell frequencies and numbers . a : flow cytometric analyses of nondiabetic control ( ctl ) and type 1 diabetic pbmcs using abs against cd3 and cd56 markers . b : the frequencies of nk cells ( ctl : n = 36 ; t1d : n = 22 ) and other lymphocyte subsets ( ctl : n = 11 ; t1d : n = 14 ) among pbmcs obtained from type 1 diabetic subjects ( ) and age - matched , nondiabetic control subjects ( ) were determined by flow cytometry : nk cells ( cd3cd56 ) , b - cells ( cd3cd19 ) , t - cells ( cd3cd19 ) , cd4 t - cells ( cd4cd19 ) , cd8 t - cells ( cd8cd19 ) , and nk t - cells ( cd3cd56 ) . complete blood - cell counts were used to determine the nk cell numbers present per liter of blood . * * * p < 0.0005 . the critical roles of the cytokines il-2 and il-15 in nk cell homeostasis ( 32,33 ) led us to hypothesize that a lack of responsiveness by type 1 diabetic nk cells to il-2 and il-15 could underlie their decreased representation . to address this question , purified nk cells from type 1 diabetic and age - matched control subjects were labeled with the mitotic tracker carboxyfluorescein succinimidyl ester ( cfse ) , as described previously ( 34 ) , and cultured in vitro either in media alone or with addition of il-2 and il-15 ( fig . 2a ) . after 1 week , measurements of cellular proliferation indicated that very few type 1 diabetic nk cells had proliferated . in contrast , significant numbers of control nk cells had undergone one or more cell divisions . the vast majority of nk cells , whether type 1 diabetic or control derived , failed to proliferate in the absence of exogenous cytokines , demonstrating that cell division was dependent upon cytokine stimulation ( data not shown ) . to determine whether the lack of proliferation by type 1 diabetic nk cells was associated with a decreased cellular recovery , equivalent numbers of control and type 1 diabetic nk cells were placed into culture with il-2 and il-15 ( fig . one week later , cell counts of cultures revealed that the yield from wells containing type 1 diabetic nk cells was decreased twofold relative to the control group . these findings indicate that reduced frequencies of nk cells in type 1 diabetic subjects are correlated with poor responsiveness to il-2 and il-15 . type 1 diabetic nk cells are poorly responsive to il-2/il-15 stimulation . a : one million nk cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 4 ) were cultured for 1 week with ril-2 and ril-15 and were subsequently counted . b : purified nk cells from the peripheral blood of type 1 diabetic subjects ( , n = 8) and age - matched control subjects ( , n = 4 ) were labeled with cfse and cultured for 1 week with ril-2 and ril-15 . representative ( histograms ) and cumulative data ( bar graphs ) are shown for the cell - division history of cultured nk cell populations . c : expression of il-2r/il-15r ( cd122 ) and the common- chain receptor ( cd132 ) on nk cells was determined directly ex vivo by flow cytometry . error bars represent the sd . to address whether poor il-2/il-15 responsiveness by type 1 diabetic nk cells is a result of insufficient cytokine receptor expression , we compared levels of il-2 and il-15 receptor subunits ( fig . 2c ) . flow cytometric analyses revealed that type 1 diabetic nk cells expressed modestly reduced levels , as determined by comparison of mean fluorescence intensity ( mfi ) values , of il-2r/il-15r ( cd122 ) and il-2r/il-15r ( cd132 or common- chain ) relative to control ( cd122 : type 1 diabetic = 65.3 5.8 vs. control = 75.2 12.5 ; cd132 : type 1 diabetic = 26.1 5.9 vs. control = 29.8 2.7 ) . cd122 and cd132 interact with cd25 to form the high - affinity il-2 receptor , whereas these two subunits are thought to bind il-15 through trans - presentation by il-15r chain on an accessory cell ( 35 ) . regardless of the nk cell origin , we were unable to detect significant expression of either of the unique subunits of these two cytokine receptors , il-2r ( cd25 ) and il-15r ( data not shown ) . these results indicate that the hyporesponsiveness of type 1 diabetic nk cells to il-2/il-15 stimulation is not a result of a lack of cytokine receptor expression . to investigate the surface phenotype of type 1 diabetic nk cells and potential causes of their dysfunction , we analyzed the expression of different nk cell markers on cells directly ex vivo and after in vitro activation with il-2/il-15 ( fig . type 1 diabetic nk cells were found to express normal levels of 2b4 , cd94 , lair , and nkb-1 directly ex vivo , as judged by percent - positive and mfi values ( fig . type 1 diabetic and control nk cells induced the expression of the c - type lectin cd94 and extinguished the signaling lymphocyte activation molecule family receptor , 2b4 . next , we assessed levels of nkg2d ligands on the surface of control and type 1 diabetic nk cells because previous experiments in diabetic nod mice attributed their altered expression to nk cell dysfunction ( 27 ) . resting nk cells have been reported to express a mica and micb message ( http://biogps.gnf.org ) and mica protein ( 36 ) . using a specific monoclonal ab anti - mica / b ab ( clone 6d4 ) for detection , we also detected mica / b expression on control and type 1 diabetic nk cells directly ex vivo ( fig . however , upon activation in vitro , mica / b levels on control nk cells were almost completely lost , whereas type 1 diabetic nk cells maintained strong mica / b expression ( control = 4.8 0.3 mfi ; type 1 diabetic = 30.3 7.6 mfi ; 6.3-fold change in mfi ) . experiments performed with monoclonal anti - mica ab corroborated these conclusions ( ab clone 159227 ; data not shown ) . despite retaining high mica / b levels , activated type 1 diabetic nk cells expressed nkg2d levels that were comparable to control nk cells ( fig . together , these experiments reveal that type 1 diabetic nk cells exhibit dysregulated mica / b but normal cd94 and 2b4 expression upon stimulation with il-2/il-15 . type 1 diabetic nk cells fail to downregulate the nkg2d ligands mica / b upon activation . a : surface marker analyses were performed on type 1 diabetic ( t1d ; n = 10 ) and age - matched control ( ctl ; n = 10 ) nk cells either directly ex vivo ( nk ) or after 1 week of in vitro activation with il-2/il-15 ( lak ) . nk cells were pretreated with anti - cd16 ab to block nonspecific fcr binding and were subsequently stained with abs recognizing mica / b , nkg2d , 2b4 , cd94 , lair , nkb-1 , nkp46 m or cd16 , electronically gated on cd56cd3 cells and the percent positive for the indicated marker determined . b : representative histograms illustrate staining with anti - nkg2d ( igg1 ) or anti - mica / b ( igg2a ) abs , both directly conjugated with phycoerythrin ( pe ) , on freshly isolated ( nk ) and 1-week - activated nk cells ( lak ) from the peripheral blood of type 1 diabetic and age - matched control subjects . shaded histograms represent staining with isotype - control abs ( igg1 or igg2a ) bound to pe and include relevant antibodies from other channels to account for fluorescence spillover . c : cumulative data comparing nkg2d and mica / b expression , as net mfi values ( mfi of specific ab - stained cells minus mfi of isotype control ab - stained cells ) , on type 1 diabetic ( n = 10 ) and age - matched control ( n = 10 ) nk cells directly ex vivo and 1 week after activation with il-2/il-15 . * p < 0.05 ; * * * p < 0.0001 . diabetic and control nk cells were expanded with il-2 to generate laks and were assessed for their ability to lyse either hla - negative , nk cell sensitive k562 , or nk cell resistant lak - sensitive raji targets using standard cr - release assays ( fig . type 1 diabetic lak cells were found to be at least twofold less efficient killers of k562 cells on a per - cell basis than control laks ( type 1 diabetic lak = 70.4 3.0% kill at 10:1 effector : target [ e : t ] ratio kill vs. control lak = 80.4 2.6 kill at 5:1 e : t ratio ) . in addition , a similar deficit in type 1 diabetic lak cytotoxicity also was observed against raji targets ( type 1 diabetic lak = 78.6 1.8% kill at 10:1 e : t ratio kill vs. control lak = 80.5 5.7 kill at 5:1 e : t ratio ) . next , we assessed the ability of control and type 1 diabetic lak cells to produce ifn- upon exposure to target cells ( fig . 4b ) . control or type 1 diabetic lak cells were incubated with either k562 or raji cells for 24 h and ifn- cellular secretion enumerated by elispot assays . similar to the cytotoxicity results , type 1 diabetic lak cells demonstrated a twofold - decreased capacity to produce ifn- when stimulated with k562 targets ( type 1 diabetic lak = 220 13 spots at 10:1 e : t ratio vs. control lak = 210 29 spots at 5:1 e : t ratio ) . likewise , type 1 diabetic lak cells also displayed marked reductions in ifn- secretion relative to control subjects when treated with either 10:1 ( 220 12 vs. 320 18 ) or 5:1 ( 140 10 vs. 190 4 ) ratios of raji stimulators . together , these findings demonstrate that lak cells derived from type 1 diabetic subjects display reduced effector function compared with those derived from nondiabetic control subjects . type 1 diabetic lak cells exhibit reduced cytotoxicity , ifn- secretion , and nkg2d function . lak cells were generated by treating purified nk cells with ril-2/ril-15 and were tested for effector function . a : the cytotoxicity of lak cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 14 ) was assessed using standard chromium - release assays with either k562 or raji cell lines as targets and indicated numbers of e : t ratios . b : the capacity of type 1 diabetic ( n = 8) and age - matched control ( n = 14 ) lak cells to produce ifn- following stimulation with k562 or raji cells was measured with elispot assays , using the indicated e : t ratios . c : type 1 diabetic ( n = 6 ) and control lak cells ( n = 6 ) were cultured with cr - labeled daudi target cells at a 5:1 e : t ratio , and cytolytic activity was assessed at the end of 4 h. data are presented in both dot graph ( , control ig ; , nkg2d ) and bar graph ( , control ; , type 1 diabetic ) format . d : type 1 diabetic ( n = 6 ) and control ( n = 6 ) lak cells were treated with daudi stimulators , and ifn- production was measured by an elispot assay . data are presented in the same fashion as in c ( dot graph and bar graph format ) . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.0005 . previous work in nod mice has suggested that the expression of nkg2d ligands on activated nk cells affects nkg2d signaling and results in decreased nkg2d - dependent cytotoxicity and cytokine production ( 27 ) . because activated type 1 diabetic nk cells possess unusually high levels of nkg2d ligands , we sought to examine whether these cells also exhibited defects in nkg2d function ( fig . type 1 diabetic and control lak cells were treated with either anti - nkg2d abs or control murine abs for 20 min , washed , and subsequently incubated with cr - labeled daudi targets , a cell line known both to express nkg2d ligands and to be sensitive to nkg2d - mediated killing ( 37,38 ) . stimulation of control lak cells with anti - nkg2d abs resulted in markedly improved killing of targets versus control murine abs ( 70.1 2.8% vs. 88.4 1.7% ; 26.1% increase ; p < 0.0005 ) , whereas type 1 diabetic lak cells were unaffected by treatment ( 54.5 5.1% vs. 59.0 7.4% ; 8.2% increase ; p = 0.39 ) . using elispot assays , we also assessed the effect of anti - nkg2d ab treatment on the ability of nondiabetic control and type 1 diabetic lak cells to secrete ifn- after incubation with daudi stimulators ( fig . 4d ) . as with the cytotoxicity results , anti - nkg2d ab stimulation had a more profound and significant effect on ifn- production by control lak cells ( 183 19 vs. 241 19 spots ; 31.7% increase ; p = 0.031 ) . in comparison , type 1 diabetic lak cells treated with anti - nkg2d abs displayed an insignificant rise ( 96 8 vs. 116 12 ; 20.8% increase ; p = 0.096 ) . these results suggest that a defect in the nkg2d - dependent activation pathway of type 1 diabetic nk cells may be responsible for their diminished effector functions . nkg2d - mediated effector functions are triggered through its association with the transmembrane adaptor molecule dap10 ( 26,39 ) . coupling of nkg2d to dap10 leads to formation of a multimolecular signaling complex and the activation of multiple downstream signaling cascades , including the pi3k - akt pathway ( summarized in fig . 5a ) , which is critically involved in effector function , cell growth , and cell survival ( 39,40 ) . to investigate whether nkg2d signaling is altered in type 1 diabetic subjects , we first measured pi3k association with nkg2d - dap10 complexes in control and type 1 diabetic lak cells after treatment with either anti - nkg2d abs or control murine abs ( fig . nkg2d - dap10 complexes were pulled down by immunoprecipitation and probed with either anti - nkg2d or anti - p85 subunit of pi3k abs . strikingly , nkg2d stimulation resulted in the efficient association of pi3k with nkg2d - dap10 complexes in lak cells from three nondiabetic control subjects but not from type 1 diabetic subjects . to measure the activation status of pi3k and the downstream - acting serine / threonine kinase akt , we next used reverse - phase protein lysate microarrays to measure their phosphorylation with phospho-(p)specific abs , as previously described ( 30 ) . nk cells expanded from six type 1 diabetic subjects , and six nondiabetic control subjects were serum - starved for 4 h then stimulated with anti - nkg2d abs over a time course of 5 min and their lysates probed with two p - akt(s473) , one p - akt(t308) , and one p - pi3k p85(y458)-specific abs ( fig . the use of two separate ab clones , both recognizing p - akt(s473 ) , allowed us to assess the internal reproducibility of the assay . robust phosphorylation of pi3k and akt was detected in all six control samples over the sampled times . by contrast , five of six type 1 diabetic samples showed no evidence of stimulation - induced phosphorylation and three of six in this group exhibited stimulation - induced dephosphorylation . the cumulative mean phosphorylation by type 1 diabetic nk cells was significantly decreased relative to control samples at both 1 and 5 min after anti - nkg2d stimulation ( fig . , these findings suggest that impaired effector functions by type 1 diabetic lak cells may be a consequence of aberrant signaling through the nkg2d receptor . type 1 diabetic lak cells exhibit defective nkg2d signaling . a : dap10 phosphorylation at its yinm motif ( blue box ) results in the activation of the pi3k pathway . b : type 1 diabetic ( t1d ; n = 3 ; c052 , c053 , and c068 ) and control ( ctl ; n = 3 ; d068 , d069 , and d088 ) lak cells were stimulated with either anti - nkg2d ab or murine igg for 15 min . nkg2d was pulled down with anti - nkg2d abs and blotted with either anti - nkg2d or anti - pi3k abs . c : densitometric measurements are presented on nkg2d and pi3k band intensities and ratios of cumulative means sd . * p < 0.05 . d : purified nk cells from nondiabetic control subjects ( c ; n = 6 ) and type 1 diabetic subjects ( d ; n = 6 ) were stimulated with anti - nkg2d abs for a time course . reverse - phase protein lysate microarrays were used to detect phosphorylation of p85 pi3k and akt . results are expressed as log base 2 mfi ratio values and presented as heatmaps of phosphorylation changes over time , with yellow reflecting an increase , blue reflecting a decrease compared with baseline time zero , and black representing no change . e : cumulative data from control and type 1 diabetic samples in d. * p < 0.05 ; * * p < 0.01 ; * * * p < 0.005 . to address whether numerical or functional nk cell defects are present in human type 1 diabetic subjects , we analyzed peripheral blood mononuclear cells ( pbmcs ) from subjects with established type 1 diabetes ( > 0.5 years and < 2 years ; mean age 9.3 4.5 years ; mean type 1 diabetes duration 1.4 0.5 years ) and age - matched nondiabetic control subjects ( mean age 10.7 4.0 years ) using great care to follow standardized and consistent processing of blood samples and experimental conditions ( table 1 ) . we rationalized that if nk cell dysfunction was an intrinsic property of the type 1 diabetes immune system , longstanding measurable defects would still be present in subjects after establishment of disease . we also limited our subjects to those whose onset of diabetes was no greater than 2 years in order to minimize the potential effects of chronic hyperglycemia on lymphocyte number and function . frequencies of nk ( cd3cd56 ) , nkt ( cd3cd56 ) , cd4 t ( cd3cd56cd4 ) , cd8 t ( cd3cd56cd8 ) , and b - cell ( cd3cd19 ) subsets among pbmcs were assessed using standard flow cytometric techniques ( fig . 1a and b ) . in contrast to the similar proportions of cd4 t- , cd8 t- , and b - cells in the peripheral blood of type 1 diabetic subjects relative to control subjects , the nk cell fraction in type 1 diabetic subjects was markedly reduced ( ~37% ) relative to nondiabetic age - matched control subjects ( control subjects : 6.58 2.93% vs. type 1 diabetic subjects : 4.18 1.66% ; p < 0.0005 ) . to ascertain whether type 1 diabetic subjects exhibit a decrease in absolute nk cell numbers , complete blood - cell counts were performed on fresh blood samples from type 1 diabetic and nondiabetic subjects ( fig . 1c ) . total lymphocyte numbers were found to be modestly reduced in type 1 diabetic subjects relative to control subjects , although these numbers both fell within the normal range for age at our institution ( type 1 diabetic subjects : 2.21 0.74 10/l , n = 11 vs. control subjects : 2.96 0.74 10/l , n = 10 ; p < 0.02 ) , and absolute nk cell numbers per blood volume were decreased approximately twofold in type 1 diabetic subjects relative to control subjects ( type 1 diabetic subjects : 0.92 0.37 10/l vs. control subjects : 1.94 0.86 10/l ; p < 0.0001 ) . characteristics of the type 1 diabetic subjects and nondiabetic control groups from british columbia 's children 's hospital are presented data are means sd . all type 1 diabetic subjects were being treated with insulin and did not show evidence of other autoimmune diseases . age - matched subjects with no autoimmune or metabolic diseases were used as nondiabetic control subjects . nk cells from pbmcs of type 1 diabetic ( t1d ) subjects are present at reduced cell frequencies and numbers . a : flow cytometric analyses of nondiabetic control ( ctl ) and type 1 diabetic pbmcs using abs against cd3 and cd56 markers . b : the frequencies of nk cells ( ctl : n = 36 ; t1d : n = 22 ) and other lymphocyte subsets ( ctl : n = 11 ; t1d : n = 14 ) among pbmcs obtained from type 1 diabetic subjects ( ) and age - matched , nondiabetic control subjects ( ) were determined by flow cytometry : nk cells ( cd3cd56 ) , b - cells ( cd3cd19 ) , t - cells ( cd3cd19 ) , cd4 t - cells ( cd4cd19 ) , cd8 t - cells ( cd8cd19 ) , and nk t - cells ( cd3cd56 ) . complete blood - cell counts were used to determine the nk cell numbers present per liter of blood . * * * p < 0.0005 . the critical roles of the cytokines il-2 and il-15 in nk cell homeostasis ( 32,33 ) led us to hypothesize that a lack of responsiveness by type 1 diabetic nk cells to il-2 and il-15 could underlie their decreased representation . to address this question , purified nk cells from type 1 diabetic and age - matched control subjects were labeled with the mitotic tracker carboxyfluorescein succinimidyl ester ( cfse ) , as described previously ( 34 ) , and cultured in vitro either in media alone or with addition of il-2 and il-15 ( fig . 2a ) . after 1 week , measurements of cellular proliferation indicated that very few type 1 diabetic nk cells had proliferated . in contrast , significant numbers of control nk cells had undergone one or more cell divisions . the vast majority of nk cells , whether type 1 diabetic or control derived , failed to proliferate in the absence of exogenous cytokines , demonstrating that cell division was dependent upon cytokine stimulation ( data not shown ) . to determine whether the lack of proliferation by type 1 diabetic nk cells was associated with a decreased cellular recovery , equivalent numbers of control and type 1 diabetic nk cells were placed into culture with il-2 and il-15 ( fig . one week later , cell counts of cultures revealed that the yield from wells containing type 1 diabetic nk cells was decreased twofold relative to the control group . these findings indicate that reduced frequencies of nk cells in type 1 diabetic subjects are correlated with poor responsiveness to il-2 and il-15 . type 1 diabetic nk cells are poorly responsive to il-2/il-15 stimulation . a : one million nk cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 4 ) were cultured for 1 week with ril-2 and ril-15 and were subsequently counted . b : purified nk cells from the peripheral blood of type 1 diabetic subjects ( , n = 8) and age - matched control subjects ( , n = 4 ) were labeled with cfse and cultured for 1 week with ril-2 and ril-15 . representative ( histograms ) and cumulative data ( bar graphs ) are shown for the cell - division history of cultured nk cell populations . c : expression of il-2r/il-15r ( cd122 ) and the common- chain receptor ( cd132 ) on nk cells was determined directly ex vivo by flow cytometry . error bars represent the sd . to address whether poor il-2/il-15 responsiveness by type 1 diabetic nk cells is a result of insufficient cytokine receptor expression , we compared levels of il-2 and il-15 receptor subunits ( fig . 2c ) . flow cytometric analyses revealed that type 1 diabetic nk cells expressed modestly reduced levels , as determined by comparison of mean fluorescence intensity ( mfi ) values , of il-2r/il-15r ( cd122 ) and il-2r/il-15r ( cd132 or common- chain ) relative to control ( cd122 : type 1 diabetic = 65.3 5.8 vs. control = 75.2 12.5 ; cd132 : type 1 diabetic = 26.1 5.9 vs. control = 29.8 2.7 ) . cd122 and cd132 interact with cd25 to form the high - affinity il-2 receptor , whereas these two subunits are thought to bind il-15 through trans - presentation by il-15r chain on an accessory cell ( 35 ) . regardless of the nk cell origin , we were unable to detect significant expression of either of the unique subunits of these two cytokine receptors , il-2r ( cd25 ) and il-15r ( data not shown ) . these results indicate that the hyporesponsiveness of type 1 diabetic nk cells to il-2/il-15 stimulation is not a result of a lack of cytokine receptor expression . to investigate the surface phenotype of type 1 diabetic nk cells and potential causes of their dysfunction , we analyzed the expression of different nk cell markers on cells directly ex vivo and after in vitro activation with il-2/il-15 ( fig . type 1 diabetic nk cells were found to express normal levels of 2b4 , cd94 , lair , and nkb-1 directly ex vivo , as judged by percent - positive and mfi values ( fig . type 1 diabetic and control nk cells induced the expression of the c - type lectin cd94 and extinguished the signaling lymphocyte activation molecule family receptor , 2b4 . next , we assessed levels of nkg2d ligands on the surface of control and type 1 diabetic nk cells because previous experiments in diabetic nod mice attributed their altered expression to nk cell dysfunction ( 27 ) . resting nk cells have been reported to express a mica and micb message ( http://biogps.gnf.org ) and mica protein ( 36 ) . using a specific monoclonal ab anti - mica / b ab ( clone 6d4 ) for detection , we also detected mica / b expression on control and type 1 diabetic nk cells directly ex vivo ( fig . however , upon activation in vitro , mica / b levels on control nk cells were almost completely lost , whereas type 1 diabetic nk cells maintained strong mica / b expression ( control = 4.8 0.3 mfi ; type 1 diabetic = 30.3 7.6 mfi ; 6.3-fold change in mfi ) . experiments performed with monoclonal anti - mica ab corroborated these conclusions ( ab clone 159227 ; data not shown ) . despite retaining high mica / b levels , activated type 1 diabetic nk cells expressed nkg2d levels that were comparable to control nk cells ( fig . together , these experiments reveal that type 1 diabetic nk cells exhibit dysregulated mica / b but normal cd94 and 2b4 expression upon stimulation with il-2/il-15 . type 1 diabetic nk cells fail to downregulate the nkg2d ligands mica / b upon activation . a : surface marker analyses were performed on type 1 diabetic ( t1d ; n = 10 ) and age - matched control ( ctl ; n = 10 ) nk cells either directly ex vivo ( nk ) or after 1 week of in vitro activation with il-2/il-15 ( lak ) . nk cells were pretreated with anti - cd16 ab to block nonspecific fcr binding and were subsequently stained with abs recognizing mica / b , nkg2d , 2b4 , cd94 , lair , nkb-1 , nkp46 m or cd16 , electronically gated on cd56cd3 cells and the percent positive for the indicated marker determined . b : representative histograms illustrate staining with anti - nkg2d ( igg1 ) or anti - mica / b ( igg2a ) abs , both directly conjugated with phycoerythrin ( pe ) , on freshly isolated ( nk ) and 1-week - activated nk cells ( lak ) from the peripheral blood of type 1 diabetic and age - matched control subjects . shaded histograms represent staining with isotype - control abs ( igg1 or igg2a ) bound to pe and include relevant antibodies from other channels to account for fluorescence spillover . c : cumulative data comparing nkg2d and mica / b expression , as net mfi values ( mfi of specific ab - stained cells minus mfi of isotype control ab - stained cells ) , on type 1 diabetic ( n = 10 ) and age - matched control ( n = 10 ) nk cells directly ex vivo and 1 week after activation with il-2/il-15 . * p < 0.05 ; * * * to evaluate their effector function , purified type 1 diabetic and control nk cells were expanded with il-2 to generate laks and were assessed for their ability to lyse either hla - negative , nk cell sensitive k562 , or nk cell resistant lak - sensitive raji targets using standard cr - release assays ( fig . type 1 diabetic lak cells were found to be at least twofold less efficient killers of k562 cells on a per - cell basis than control laks ( type 1 diabetic lak = 70.4 3.0% kill at 10:1 effector : target [ e : t ] ratio kill vs. control lak = 80.4 2.6 kill at 5:1 e : t ratio ) . in addition , a similar deficit in type 1 diabetic lak cytotoxicity also was observed against raji targets ( type 1 diabetic lak = 78.6 1.8% kill at 10:1 e : t ratio kill vs. control lak = 80.5 5.7 kill at 5:1 e : t ratio ) . next , we assessed the ability of control and type 1 diabetic lak cells to produce ifn- upon exposure to target cells ( fig . 4b ) . control or type 1 diabetic lak cells were incubated with either k562 or raji cells for 24 h and ifn- cellular secretion enumerated by elispot assays . similar to the cytotoxicity results , type 1 diabetic lak cells demonstrated a twofold - decreased capacity to produce ifn- when stimulated with k562 targets ( type 1 diabetic lak = 220 13 spots at 10:1 e : t ratio vs. control lak = 210 29 spots at 5:1 e : t ratio ) . likewise , type 1 diabetic lak cells also displayed marked reductions in ifn- secretion relative to control subjects when treated with either 10:1 ( 220 12 vs. 320 18 ) or 5:1 ( 140 10 vs. 190 4 ) ratios of raji stimulators . together , these findings demonstrate that lak cells derived from type 1 diabetic subjects display reduced effector function compared with those derived from nondiabetic control subjects . type 1 diabetic lak cells exhibit reduced cytotoxicity , ifn- secretion , and nkg2d function . lak cells were generated by treating purified nk cells with ril-2/ril-15 and were tested for effector function . a : the cytotoxicity of lak cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 14 ) was assessed using standard chromium - release assays with either k562 or raji cell lines as targets and indicated numbers of e : t ratios . b : the capacity of type 1 diabetic ( n = 8) and age - matched control ( n = 14 ) lak cells to produce ifn- following stimulation with k562 or raji cells was measured with elispot assays , using the indicated e : t ratios . c : type 1 diabetic ( n = 6 ) and control lak cells ( n = 6 ) were cultured with cr - labeled daudi target cells at a 5:1 e : t ratio , and cytolytic activity was assessed at the end of 4 h. data are presented in both dot graph ( , control ig ; , nkg2d ) and bar graph ( , control ; , type 1 diabetic ) format . d : type 1 diabetic ( n = 6 ) and control ( n = 6 ) lak cells were treated with daudi stimulators , and ifn- production was measured by an elispot assay . data are presented in the same fashion as in c ( dot graph and bar graph format ) . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.0005 . previous work in nod mice has suggested that the expression of nkg2d ligands on activated nk cells affects nkg2d signaling and results in decreased nkg2d - dependent cytotoxicity and cytokine production ( 27 ) . because activated type 1 diabetic nk cells possess unusually high levels of nkg2d ligands , we sought to examine whether these cells also exhibited defects in nkg2d function ( fig . type 1 diabetic and control lak cells were treated with either anti - nkg2d abs or control murine abs for 20 min , washed , and subsequently incubated with cr - labeled daudi targets , a cell line known both to express nkg2d ligands and to be sensitive to nkg2d - mediated killing ( 37,38 ) . stimulation of control lak cells with anti - nkg2d abs resulted in markedly improved killing of targets versus control murine abs ( 70.1 2.8% vs. 88.4 1.7% ; 26.1% increase ; p < 0.0005 ) , whereas type 1 diabetic lak cells were unaffected by treatment ( 54.5 5.1% vs. 59.0 7.4% ; 8.2% increase ; p = 0.39 ) . using elispot assays , we also assessed the effect of anti - nkg2d ab treatment on the ability of nondiabetic control and type 1 diabetic lak cells to secrete ifn- after incubation with daudi stimulators ( fig . , anti - nkg2d ab stimulation had a more profound and significant effect on ifn- production by control lak cells ( 183 19 vs. 241 19 spots ; 31.7% increase ; p = 0.031 ) . in comparison , type 1 diabetic lak cells treated with anti - nkg2d abs displayed an insignificant rise ( 96 8 vs. 116 12 ; 20.8% increase ; p = 0.096 ) . these results suggest that a defect in the nkg2d - dependent activation pathway of type 1 diabetic nk cells may be responsible for their diminished effector functions . nkg2d - mediated effector functions are triggered through its association with the transmembrane adaptor molecule dap10 ( 26,39 ) . coupling of nkg2d to dap10 leads to formation of a multimolecular signaling complex and the activation of multiple downstream signaling cascades , including the pi3k - akt pathway ( summarized in fig . 5a ) , which is critically involved in effector function , cell growth , and cell survival ( 39,40 ) . to investigate whether nkg2d signaling is altered in type 1 diabetic subjects , we first measured pi3k association with nkg2d - dap10 complexes in control and type 1 diabetic lak cells after treatment with either anti - nkg2d abs or control murine abs ( fig . nkg2d - dap10 complexes were pulled down by immunoprecipitation and probed with either anti - nkg2d or anti - p85 subunit of pi3k abs . strikingly , nkg2d stimulation resulted in the efficient association of pi3k with nkg2d - dap10 complexes in lak cells from three nondiabetic control subjects but not from type 1 diabetic subjects . to measure the activation status of pi3k and the downstream - acting serine / threonine kinase akt , we next used reverse - phase protein lysate microarrays to measure their phosphorylation with phospho-(p)specific abs , as previously described ( 30 ) . nk cells expanded from six type 1 diabetic subjects , and six nondiabetic control subjects were serum - starved for 4 h then stimulated with anti - nkg2d abs over a time course of 5 min and their lysates probed with two p - akt(s473) , one p - akt(t308) , and one p - pi3k p85(y458)-specific abs ( fig . the use of two separate ab clones , both recognizing p - akt(s473 ) , allowed us to assess the internal reproducibility of the assay . robust phosphorylation of pi3k and akt was detected in all six control samples over the sampled times . by contrast , five of six type 1 diabetic samples showed no evidence of stimulation - induced phosphorylation and three of six in this group exhibited stimulation - induced dephosphorylation . the cumulative mean phosphorylation by type 1 diabetic nk cells was significantly decreased relative to control samples at both 1 and 5 min after anti - nkg2d stimulation ( fig . , these findings suggest that impaired effector functions by type 1 diabetic lak cells may be a consequence of aberrant signaling through the nkg2d receptor . type 1 diabetic lak cells exhibit defective nkg2d signaling . a : dap10 phosphorylation at its yinm motif ( blue box ) results in the activation of the pi3k pathway . b : type 1 diabetic ( t1d ; n = 3 ; c052 , c053 , and c068 ) and control ( ctl ; n = 3 ; d068 , d069 , and d088 ) lak cells were stimulated with either anti - nkg2d ab or murine igg for 15 min . nkg2d was pulled down with anti - nkg2d abs and blotted with either anti - nkg2d or anti - pi3k abs . c : densitometric measurements are presented on nkg2d and pi3k band intensities and ratios of cumulative means sd . * d : purified nk cells from nondiabetic control subjects ( c ; n = 6 ) and type 1 diabetic subjects ( d ; n = 6 ) were stimulated with anti - nkg2d abs for a time course . reverse - phase protein lysate microarrays were used to detect phosphorylation of p85 pi3k and akt . results are expressed as log base 2 mfi ratio values and presented as heatmaps of phosphorylation changes over time , with yellow reflecting an increase , blue reflecting a decrease compared with baseline time zero , and black representing no change . e : cumulative data from control and type 1 diabetic samples in d. * p < 0.05 ; * * p < 0.01 ; * * * p < 0.005 . our analysis of pbmcs from type 1 diabetic subjects revealed that nk cell frequency ( cd3cd56 ) was decreased ~37% relative to age - matched nondiabetic control subjects ( fig . ( 41 ) also have reported that nk cell frequencies were reduced in type 1 diabetic subjects , although in their study , the reduced frequencies were present in recent - onset ( < 1 month ) but not in long - standing ( > 1 year ; mean 10 years postdiagnosis ) type 1 diabetic subjects . our observation that decreased nk cell frequencies in pbmcs from type 1 diabetic subjects were associated with impaired responsiveness to il-2/il-15 stimulation suggests that cell - intrinsic mechanisms may be responsible for their reduced frequencies ( fig . ( 42 ) have proposed that murine nk cell homeostasis and nkg2d function are coregulated through the coupling of nkg2d and il-15 receptors , suggesting that a common pathway may be responsible for defects in both cytokine responsiveness and nkg2d function exhibited by type 1 diabetic nk cells . consistent with these findings , nkg2d - deficient mice possess perturbations to nk cell numbers , nk cell apoptosis , and nk cell proliferation , implying that nkg2d plays a critical role in the regulation of nk cell homeostasis ( 43 ) . the decreased responsiveness to il-2/il-15 led us to compare markers of nk cell activation and differentiation between type 1 diabetic and nondiabetic control nk cells directly ex vivo and after cytokine stimulation ( fig . 3 ) . of the nk cell markers assessed , the only difference seen between type 1 diabetic and control nk cells was in the failure of type 1 diabetic nk cell to downmodulate expression of the nkg2d ligands mica / b . however , despite aberrant maintenance of mica / b expression on activated type 1 diabetic nk cells , we did not see signs of nkg2d receptor downmodulation , a pi3k - dependent phenomenon seen in nod nk cells ( 27 ) . the finding that surface levels of nkg2d on type 1 diabetic nk cells continued to match closely those of nondiabetic control nk cells suggested that impaired nkg2d function by type 1 diabetic nk cells was a consequence of downstream ( intracellular ) signaling rather than insufficient receptor expression ( fig . consistent with this interpretation and with the diminished nkg2d - mediated effector function observed ( fig . 4c and d ) , type 1 diabetic nk cells were found to possess an intracellular signal transduction defect proximal to the nkg2d receptor affecting the pi3k - akt pathway ( fig . additional examination of nkg2d signal transduction in type 1 diabetic nk cells , including the grb2/vav1 pathway , is being pursued . nk cells share an increasing number of traits with the adaptive immune system , including the formation of self - tolerance and the generation of long - lived memory cells , despite their exclusive use of germline - encoded antigen receptors ( 44,45 ) . continuous exposure of nk cells to ligands recognizing their activating receptors has been shown to result in nk cell tolerance and , therefore , argues that regulatory mechanisms exist to limit their autoimmune potential ( 46,47 ) . moreover , these experiments suggest that the expression of nkg2d ligands on activated type 1 diabetic nk cells could result in their chronic stimulation through the nkg2d receptor , inducing nk cell hyporesponsiveness . notably , ectopic expression of the murine nkg2d ligand rae-1 in the epithelium of mice has been shown to result in nkg2d downregulation and defective nk cell cytotoxicity ( 48 ) . our findings of dysregulated nkg2d ligand expression on type 1 diabetic nk cells are reminiscent of a previous report ( 27 ) describing the expression of nkg2d ligands on activated nk cells from diabetes - prone nod , but not diabetes - resistant c57bl/6 , mice . in nod mice , it has been postulated that the expression of nkg2d ligands by activated nk cells results in chronic nkg2d stimulation , nkg2d downmodulation through pi3k - dependent ligand - induced internalization , and , eventually , desensitization ( 27 ) . as a consequence of the aforementioned study , as well as our own , we hypothesize that chronic exposure to nkg2d ligands , either on the same cell ( cis ) or on an adjacent nk cell ( trans ) , may result in prolonged signaling and eventually lead to nk cell dysfunction . given their propensity to produce ifn- and kill other cells , nk cells may influence the development of autoimmune diseases through direct tissue destruction or indirectly via the regulation of adaptive immune responses or the modification of antigen - presenting cells ( 49 ) . examples of nk cells playing a causative role in disease exist ( 5 ) ; for instance , nk cells have also been suggested to mediate a protective function in subjects with multiple sclerosis and their depletion in rodent models of experimental autoimmune encephalomyelitis exacerbates autoimmunity ( 50,51 ) . in addition , low nk cell activity has been observed in other autoimmune settings , including systemic lupus erythematosus ( sle ) subjects and the lpr murine model of sle . adoptive transfer of nk1.1 cells into lpr mice has been found to slow down the lupus - like disease process ( 5254 ) . with respect to type 1 diabetes , we have previously shown that enhancement of nk cell function through cfa treatment , resulting in improved nkg2d receptor levels and decreased nkg2d ligand expression , reduces autoreactive ctl numbers and protects nod mice from disease ( 28,29 ) . the data above indicate that nk cells in type 1 diabetic subjects are defective in number , signaling , and function and suggest that augmentation of nk cell function may prove valuable as an immune - modifying therapy for type 1 diabetes or other autoimmune diseases .

objectivenatural killer ( nk ) cells from nod mice have numeric and functional abnormalities , and restoration of nk cell function prevents autoimmune diabetes in nod mice . however , little is known about the number and function of nk cells in humans affected by type 1 diabetes . therefore , we evaluated the phenotype and function of nk cells in a large cohort of type 1 diabetic children.research design and methodsperipheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years . nk cells were characterized by flow cytometry , enzyme - linked immunosorbent spot assays , and cytotoxicity assays . signaling through the activating nk cell receptor , nkg2d , was assessed by immunoblotting and reverse - phase phosphoprotein lysate microarray.resultsnk cells from type 1 diabetic subjects were present at reduced cell numbers compared with age - matched , nondiabetic control subjects and had diminished responses to the cytokines interleukin ( il)-2 and il-15 . analysis before and after il-2 stimulation revealed that unlike nk cells from nondiabetic control subjects , nk cells from type 1 diabetic subjects failed to downregulate the nkg2d ligands , major histocompatibility complex class i related chains a and b , upon activation . moreover , type 1 diabetic nk cells also exhibited decreased nkg2d - dependent cytotoxicity and interferon- secretion . finally , type 1 diabetic nk cells showed clear defects in nkg2d - mediated activation of the phosphoinositide 3-kinase akt pathway.conclusionsthese results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the nkg2d receptor and suggest that nk cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes .

RESEARCH DESIGN AND METHODS Subject recruitment, sample collection, and complete blood-cell counts. Antibodies and flow cytometry. Purification and in vitro culture of human NK cells. Cytotoxicity assays. ELISpot assays. Cell-signaling studies. Lysate preparation, microarray production, data acquisition, and array analyses. Statistical analyses. RESULTS NK cells from type 1 diabetic subjects are present at reduced frequencies and respond poorly to IL-2 and IL-15. Activated type 1 diabetic NK cells fail to downregulate the NKG2D ligands, MICA/B. Type 1 diabetic LAK cells exhibit reduced cytotoxicity, IFN- secretion, and NKG2D function. Type 1 diabetic LAK cells exhibit defective NKG2D signaling. DISCUSSION

abs recognizing il-15r ( ebiojm7a4 ; ebioscience ) , cd16 ( cb16 , ebioscience ) , nkg2d ( fab139p ; r&d systems ) , major histocompatibility complex class i related chains a and b ( mica / b ) clone 159207 ( r&d systems ) , and mica / b clone 6d4 ( biolegend ) were acquired from the indicated sources . nk cells were expanded in vitro for 57 days prior to their use in cytotoxicity and enzyme - linked immunosorbent spot ( elispot ) assays . abs recognizing il-15r ( ebiojm7a4 ; ebioscience ) , cd16 ( cb16 , ebioscience ) , nkg2d ( fab139p ; r&d systems ) , major histocompatibility complex class i related chains a and b ( mica / b ) clone 159207 ( r&d systems ) , and mica / b clone 6d4 ( biolegend ) were acquired from the indicated sources . to address whether numerical or functional nk cell defects are present in human type 1 diabetic subjects , we analyzed peripheral blood mononuclear cells ( pbmcs ) from subjects with established type 1 diabetes ( > 0.5 years and < 2 years ; mean age 9.3 4.5 years ; mean type 1 diabetes duration 1.4 0.5 years ) and age - matched nondiabetic control subjects ( mean age 10.7 4.0 years ) using great care to follow standardized and consistent processing of blood samples and experimental conditions ( table 1 ) . in contrast to the similar proportions of cd4 t- , cd8 t- , and b - cells in the peripheral blood of type 1 diabetic subjects relative to control subjects , the nk cell fraction in type 1 diabetic subjects was markedly reduced ( ~37% ) relative to nondiabetic age - matched control subjects ( control subjects : 6.58 2.93% vs. type 1 diabetic subjects : 4.18 1.66% ; p < 0.0005 ) . total lymphocyte numbers were found to be modestly reduced in type 1 diabetic subjects relative to control subjects , although these numbers both fell within the normal range for age at our institution ( type 1 diabetic subjects : 2.21 0.74 10/l , n = 11 vs. control subjects : 2.96 0.74 10/l , n = 10 ; p < 0.02 ) , and absolute nk cell numbers per blood volume were decreased approximately twofold in type 1 diabetic subjects relative to control subjects ( type 1 diabetic subjects : 0.92 0.37 10/l vs. control subjects : 1.94 0.86 10/l ; p < 0.0001 ) . nk cells from pbmcs of type 1 diabetic ( t1d ) subjects are present at reduced cell frequencies and numbers . b : the frequencies of nk cells ( ctl : n = 36 ; t1d : n = 22 ) and other lymphocyte subsets ( ctl : n = 11 ; t1d : n = 14 ) among pbmcs obtained from type 1 diabetic subjects ( ) and age - matched , nondiabetic control subjects ( ) were determined by flow cytometry : nk cells ( cd3cd56 ) , b - cells ( cd3cd19 ) , t - cells ( cd3cd19 ) , cd4 t - cells ( cd4cd19 ) , cd8 t - cells ( cd8cd19 ) , and nk t - cells ( cd3cd56 ) . the critical roles of the cytokines il-2 and il-15 in nk cell homeostasis ( 32,33 ) led us to hypothesize that a lack of responsiveness by type 1 diabetic nk cells to il-2 and il-15 could underlie their decreased representation . to address this question , purified nk cells from type 1 diabetic and age - matched control subjects were labeled with the mitotic tracker carboxyfluorescein succinimidyl ester ( cfse ) , as described previously ( 34 ) , and cultured in vitro either in media alone or with addition of il-2 and il-15 ( fig . to determine whether the lack of proliferation by type 1 diabetic nk cells was associated with a decreased cellular recovery , equivalent numbers of control and type 1 diabetic nk cells were placed into culture with il-2 and il-15 ( fig . these findings indicate that reduced frequencies of nk cells in type 1 diabetic subjects are correlated with poor responsiveness to il-2 and il-15 . a : one million nk cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 4 ) were cultured for 1 week with ril-2 and ril-15 and were subsequently counted . b : purified nk cells from the peripheral blood of type 1 diabetic subjects ( , n = 8) and age - matched control subjects ( , n = 4 ) were labeled with cfse and cultured for 1 week with ril-2 and ril-15 . to investigate the surface phenotype of type 1 diabetic nk cells and potential causes of their dysfunction , we analyzed the expression of different nk cell markers on cells directly ex vivo and after in vitro activation with il-2/il-15 ( fig . next , we assessed levels of nkg2d ligands on the surface of control and type 1 diabetic nk cells because previous experiments in diabetic nod mice attributed their altered expression to nk cell dysfunction ( 27 ) . however , upon activation in vitro , mica / b levels on control nk cells were almost completely lost , whereas type 1 diabetic nk cells maintained strong mica / b expression ( control = 4.8 0.3 mfi ; type 1 diabetic = 30.3 7.6 mfi ; 6.3-fold change in mfi ) . type 1 diabetic nk cells fail to downregulate the nkg2d ligands mica / b upon activation . a : surface marker analyses were performed on type 1 diabetic ( t1d ; n = 10 ) and age - matched control ( ctl ; n = 10 ) nk cells either directly ex vivo ( nk ) or after 1 week of in vitro activation with il-2/il-15 ( lak ) . b : representative histograms illustrate staining with anti - nkg2d ( igg1 ) or anti - mica / b ( igg2a ) abs , both directly conjugated with phycoerythrin ( pe ) , on freshly isolated ( nk ) and 1-week - activated nk cells ( lak ) from the peripheral blood of type 1 diabetic and age - matched control subjects . together , these findings demonstrate that lak cells derived from type 1 diabetic subjects display reduced effector function compared with those derived from nondiabetic control subjects . a : the cytotoxicity of lak cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 14 ) was assessed using standard chromium - release assays with either k562 or raji cell lines as targets and indicated numbers of e : t ratios . previous work in nod mice has suggested that the expression of nkg2d ligands on activated nk cells affects nkg2d signaling and results in decreased nkg2d - dependent cytotoxicity and cytokine production ( 27 ) . because activated type 1 diabetic nk cells possess unusually high levels of nkg2d ligands , we sought to examine whether these cells also exhibited defects in nkg2d function ( fig . these results suggest that a defect in the nkg2d - dependent activation pathway of type 1 diabetic nk cells may be responsible for their diminished effector functions . strikingly , nkg2d stimulation resulted in the efficient association of pi3k with nkg2d - dap10 complexes in lak cells from three nondiabetic control subjects but not from type 1 diabetic subjects . nk cells expanded from six type 1 diabetic subjects , and six nondiabetic control subjects were serum - starved for 4 h then stimulated with anti - nkg2d abs over a time course of 5 min and their lysates probed with two p - akt(s473) , one p - akt(t308) , and one p - pi3k p85(y458)-specific abs ( fig . , these findings suggest that impaired effector functions by type 1 diabetic lak cells may be a consequence of aberrant signaling through the nkg2d receptor . d : purified nk cells from nondiabetic control subjects ( c ; n = 6 ) and type 1 diabetic subjects ( d ; n = 6 ) were stimulated with anti - nkg2d abs for a time course . to address whether numerical or functional nk cell defects are present in human type 1 diabetic subjects , we analyzed peripheral blood mononuclear cells ( pbmcs ) from subjects with established type 1 diabetes ( > 0.5 years and < 2 years ; mean age 9.3 4.5 years ; mean type 1 diabetes duration 1.4 0.5 years ) and age - matched nondiabetic control subjects ( mean age 10.7 4.0 years ) using great care to follow standardized and consistent processing of blood samples and experimental conditions ( table 1 ) . in contrast to the similar proportions of cd4 t- , cd8 t- , and b - cells in the peripheral blood of type 1 diabetic subjects relative to control subjects , the nk cell fraction in type 1 diabetic subjects was markedly reduced ( ~37% ) relative to nondiabetic age - matched control subjects ( control subjects : 6.58 2.93% vs. type 1 diabetic subjects : 4.18 1.66% ; p < 0.0005 ) . total lymphocyte numbers were found to be modestly reduced in type 1 diabetic subjects relative to control subjects , although these numbers both fell within the normal range for age at our institution ( type 1 diabetic subjects : 2.21 0.74 10/l , n = 11 vs. control subjects : 2.96 0.74 10/l , n = 10 ; p < 0.02 ) , and absolute nk cell numbers per blood volume were decreased approximately twofold in type 1 diabetic subjects relative to control subjects ( type 1 diabetic subjects : 0.92 0.37 10/l vs. control subjects : 1.94 0.86 10/l ; p < 0.0001 ) . nk cells from pbmcs of type 1 diabetic ( t1d ) subjects are present at reduced cell frequencies and numbers . b : the frequencies of nk cells ( ctl : n = 36 ; t1d : n = 22 ) and other lymphocyte subsets ( ctl : n = 11 ; t1d : n = 14 ) among pbmcs obtained from type 1 diabetic subjects ( ) and age - matched , nondiabetic control subjects ( ) were determined by flow cytometry : nk cells ( cd3cd56 ) , b - cells ( cd3cd19 ) , t - cells ( cd3cd19 ) , cd4 t - cells ( cd4cd19 ) , cd8 t - cells ( cd8cd19 ) , and nk t - cells ( cd3cd56 ) . the critical roles of the cytokines il-2 and il-15 in nk cell homeostasis ( 32,33 ) led us to hypothesize that a lack of responsiveness by type 1 diabetic nk cells to il-2 and il-15 could underlie their decreased representation . to address this question , purified nk cells from type 1 diabetic and age - matched control subjects were labeled with the mitotic tracker carboxyfluorescein succinimidyl ester ( cfse ) , as described previously ( 34 ) , and cultured in vitro either in media alone or with addition of il-2 and il-15 ( fig . to determine whether the lack of proliferation by type 1 diabetic nk cells was associated with a decreased cellular recovery , equivalent numbers of control and type 1 diabetic nk cells were placed into culture with il-2 and il-15 ( fig . these findings indicate that reduced frequencies of nk cells in type 1 diabetic subjects are correlated with poor responsiveness to il-2 and il-15 . a : one million nk cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 4 ) were cultured for 1 week with ril-2 and ril-15 and were subsequently counted . b : purified nk cells from the peripheral blood of type 1 diabetic subjects ( , n = 8) and age - matched control subjects ( , n = 4 ) were labeled with cfse and cultured for 1 week with ril-2 and ril-15 . to investigate the surface phenotype of type 1 diabetic nk cells and potential causes of their dysfunction , we analyzed the expression of different nk cell markers on cells directly ex vivo and after in vitro activation with il-2/il-15 ( fig . next , we assessed levels of nkg2d ligands on the surface of control and type 1 diabetic nk cells because previous experiments in diabetic nod mice attributed their altered expression to nk cell dysfunction ( 27 ) . however , upon activation in vitro , mica / b levels on control nk cells were almost completely lost , whereas type 1 diabetic nk cells maintained strong mica / b expression ( control = 4.8 0.3 mfi ; type 1 diabetic = 30.3 7.6 mfi ; 6.3-fold change in mfi ) . type 1 diabetic nk cells fail to downregulate the nkg2d ligands mica / b upon activation . b : representative histograms illustrate staining with anti - nkg2d ( igg1 ) or anti - mica / b ( igg2a ) abs , both directly conjugated with phycoerythrin ( pe ) , on freshly isolated ( nk ) and 1-week - activated nk cells ( lak ) from the peripheral blood of type 1 diabetic and age - matched control subjects . together , these findings demonstrate that lak cells derived from type 1 diabetic subjects display reduced effector function compared with those derived from nondiabetic control subjects . a : the cytotoxicity of lak cells from type 1 diabetic subjects ( t1d ; n = 8) and age - matched control subjects ( ctl ; n = 14 ) was assessed using standard chromium - release assays with either k562 or raji cell lines as targets and indicated numbers of e : t ratios . previous work in nod mice has suggested that the expression of nkg2d ligands on activated nk cells affects nkg2d signaling and results in decreased nkg2d - dependent cytotoxicity and cytokine production ( 27 ) . because activated type 1 diabetic nk cells possess unusually high levels of nkg2d ligands , we sought to examine whether these cells also exhibited defects in nkg2d function ( fig . type 1 diabetic and control lak cells were treated with either anti - nkg2d abs or control murine abs for 20 min , washed , and subsequently incubated with cr - labeled daudi targets , a cell line known both to express nkg2d ligands and to be sensitive to nkg2d - mediated killing ( 37,38 ) . these results suggest that a defect in the nkg2d - dependent activation pathway of type 1 diabetic nk cells may be responsible for their diminished effector functions . strikingly , nkg2d stimulation resulted in the efficient association of pi3k with nkg2d - dap10 complexes in lak cells from three nondiabetic control subjects but not from type 1 diabetic subjects . nk cells expanded from six type 1 diabetic subjects , and six nondiabetic control subjects were serum - starved for 4 h then stimulated with anti - nkg2d abs over a time course of 5 min and their lysates probed with two p - akt(s473) , one p - akt(t308) , and one p - pi3k p85(y458)-specific abs ( fig . , these findings suggest that impaired effector functions by type 1 diabetic lak cells may be a consequence of aberrant signaling through the nkg2d receptor . * d : purified nk cells from nondiabetic control subjects ( c ; n = 6 ) and type 1 diabetic subjects ( d ; n = 6 ) were stimulated with anti - nkg2d abs for a time course . our analysis of pbmcs from type 1 diabetic subjects revealed that nk cell frequency ( cd3cd56 ) was decreased ~37% relative to age - matched nondiabetic control subjects ( fig . ( 41 ) also have reported that nk cell frequencies were reduced in type 1 diabetic subjects , although in their study , the reduced frequencies were present in recent - onset ( < 1 month ) but not in long - standing ( > 1 year ; mean 10 years postdiagnosis ) type 1 diabetic subjects . ( 42 ) have proposed that murine nk cell homeostasis and nkg2d function are coregulated through the coupling of nkg2d and il-15 receptors , suggesting that a common pathway may be responsible for defects in both cytokine responsiveness and nkg2d function exhibited by type 1 diabetic nk cells . consistent with these findings , nkg2d - deficient mice possess perturbations to nk cell numbers , nk cell apoptosis , and nk cell proliferation , implying that nkg2d plays a critical role in the regulation of nk cell homeostasis ( 43 ) . the decreased responsiveness to il-2/il-15 led us to compare markers of nk cell activation and differentiation between type 1 diabetic and nondiabetic control nk cells directly ex vivo and after cytokine stimulation ( fig . of the nk cell markers assessed , the only difference seen between type 1 diabetic and control nk cells was in the failure of type 1 diabetic nk cell to downmodulate expression of the nkg2d ligands mica / b . however , despite aberrant maintenance of mica / b expression on activated type 1 diabetic nk cells , we did not see signs of nkg2d receptor downmodulation , a pi3k - dependent phenomenon seen in nod nk cells ( 27 ) . 4c and d ) , type 1 diabetic nk cells were found to possess an intracellular signal transduction defect proximal to the nkg2d receptor affecting the pi3k - akt pathway ( fig . moreover , these experiments suggest that the expression of nkg2d ligands on activated type 1 diabetic nk cells could result in their chronic stimulation through the nkg2d receptor , inducing nk cell hyporesponsiveness . in nod mice , it has been postulated that the expression of nkg2d ligands by activated nk cells results in chronic nkg2d stimulation , nkg2d downmodulation through pi3k - dependent ligand - induced internalization , and , eventually , desensitization ( 27 ) . with respect to type 1 diabetes , we have previously shown that enhancement of nk cell function through cfa treatment , resulting in improved nkg2d receptor levels and decreased nkg2d ligand expression , reduces autoreactive ctl numbers and protects nod mice from disease ( 28,29 ) . the data above indicate that nk cells in type 1 diabetic subjects are defective in number , signaling , and function and suggest that augmentation of nk cell function may prove valuable as an immune - modifying therapy for type 1 diabetes or other autoimmune diseases .

alzheimer 's disease ( ad ) is a devastating late - onset neurodegenerative condition that affects many regions of the human brain . although the most obvious disease symptoms involve the inability to form and store new memories , the neurological and psychiatric description of an individual with ad includes a wide range of symptoms such as depression , apathy , episodic behavioral outbursts , deteriorating executive functioning , and others . the biological substrates of these symptoms are only partially understood , but imaging and neuropathological studies have revealed important facets of their diverse and distributed nature . there is a clear loss of volume and pathologically visible degeneration in the brain 's memory centers , which include the entorhinal cortex , hippocampus , and basal forebrain nucleus . but there are also functional and structural abnormalities found in the locus coeruleus , dorsal raphe , cingulate gyrus , amygdala and prefrontal cortex as well as other cortical and subcortical regions [ 13 ] . amyloid plaques and neurofibrillary tangles are the widely accepted biochemical signatures of ad , used to confirm the clinical diagnosis upon final neuropathological examination . these plaques and tangles are found in conjunction with significant and progressive neurodegeneration affecting both synapses and cell bodies . while the appearance of the abnormal deposits is disease specific , their anatomical locations in human ad mark only a subset of the brain regions that are identified as undergoing significant atrophy during the progress of the disease . recent work from our laboratory and many others has explored the use of abnormal neuronal cell cycle processes as an additional pathological marker of disease [ 411 ] . the timing and location of neuronal cell death in ad has been intimately associated with the unscheduled appearance of events related to mitotic cell division . both cell cycle - related proteins and evidence of dna replication have been found in neurons that are considered at risk for death . it is hypothesized that , although the neurons are able to initiate a true cell cycle and replicate most if not all of their genome , they are incapable of completing the process and are believed to die . using immunohistochemical analysis , cell cycle events ( cces ) have been identified in subcortical brain regions of individuals with ad as well as those with mild cognitive impairment ( mci considered by many to be the clinical precursor of ad ) . in age - matched controls and in ad brain regions where neurons are not susceptible to death , cell cycle - related protein expression is significantly lower . this has led to the hypothesis that cell cycle events represent the first step of a process that leads to neuronal cell death in ad . significantly , these unexpected attempts by neurons to reenter a cell cycle provide one of the few homologies observed between mouse models of ad and the pathogenesis of the human condition . a number of different ad models have been created , most of which rely on transgenes encoding the gene for -amyloid precursor protein ( app ) , presenilin-1 ( psen1 ) or both . produced in a number of different laboratories , however , none produces the neurofibrillary tangles of ad or the severe behavioral changes that mark the end stages of the human disease . other researchers have developed models of human tauopathies based on transgenes expressing disease causing variants of the microtubule - associated protein tau [ 13 , 14 ] . paradoxically , despite the fact that familial ad has not been significantly associated with tau ( mapt ) mutations in humans , the tauopathy mouse models have been reasonably successful in reproducing many of the pathological characteristics of ad . with age , the brains of these animals display both tangles of hyperphosphorylated tau and progressive neurodegeneration in some cases . thus , mapt models reproduce the tangles and degeneration but not the plaques , while the app / ps1 models reproduce the alzheimer 's plaques but not the associated tangles or neurodegeneration . from the standpoint of the plaques and tangles , therefore , the app / ps1 mice are the better genocopies of ad while the mapt mice are somewhat better phenocopies . we have elected to focus on the pattern of neurodegeneration in app transgenic mice in order to expand the characterization of this group of ad models , and we have used cces as outcome measures . previously , where they have been studied in depth , the appearance of cces in many human disease models show an age - dependent increase in prevalence that often closely mimics the pattern of neuronal cell death in the human disease . for example , there is a significant correlation between the regional pattern of cell loss in human ataxia - telangiectasia and its mouse model . the same is true for amyotrophic lateral sclerosis . for human ad , the temporal and spatial appearance of cces in the r1.40 ad mouse model [ 1820 ] accurately recapitulate the anatomical progression of cell death in the human . in the current study , we expand the use of cell cycle markers as a benchmark of neuronal distress in the mouse . our goal was to determine the phenotypic variability among the various mouse models of ad and to learn whether the similarities and differences observed are informative as to their relative fidelity to the human disease . we were particularly interested in exploring the involvement of the subcortical areas affected in ad since these regions typically receive less attention yet are likely to contribute significantly to the symptoms of ad . we describe a pattern of selective neuronal vulnerability similar to human ad which is recapitulated in some , but not all of the five . five transgenic mouse models of familial ad were used in the current studies . in each model , amyloid plaques induced by app develop at different ages . most animals ( r1.40 b6.129-tg(appsw)40btla / j , app / ps1 b6.cg-tg(appswe,psen1de9)85dbo/j , app8.9 b6.129s2-tg(app)8.9btla / j and wild type ) were housed at rutgers university animal center . brain tissues from the tg2576 and tg6799 mouse models were a generous gift from dr . three animals from the r1.40 , tg2576 , and tg6799 lines were examined for this study . two each of the 8.9 , app / ps1 and wild - type strains were used . animals were anesthetized with avertin ( 0.02 cc / g body weight ) and perfused through the heart with 0.1 m phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m pbs solution . the brain was immediately removed from the skull and transferred to 4% paraformaldehyde at 4c overnight . the brains were then cryoprotected by sinking in 30% sucrose in 0.1 m pbs at 4c overnight . after cutting along the midline cryostat sections were cut at 10 microns and air - dried on superfrost / plus glass slides overnight . for hematoxylin staining , they were exposed to hematoxylin for 4560 sec then washed with double distilled water until clear . a rabbit monoclonal antibody ( abcam , cambridge , uk ) to proliferating cell nuclear antigen ( pcna ) was diluted 1 : 3000 in 10% goat serum / pbs blocking buffer before use . a rabbit polyclonal cyclin a antibody ( abcam , cambridge , uk ) was used at a working dilution of 1 : 500 . the beta amyloid , 116 ( 6e10 ) mouse monoclonal antibody ( covance , princeton , nj ) , was used at a working dilution of 1 : 3000 . the anti - phf - tau antibody clone at8 mouse monoclonal antibody ( thermo scientific , rockoff , il ) was used at a working dilution of 1 : 1000 . to perform fluorescent immunohistochemistry , sections were first rinsed twice in pbs , followed by pretreatment in antigen unmask solution ( vector laboratories , burlingame , ca ; working dilution 1 : 100 ) for 4 - 5 min at 95c . after the slides had cooled in buffer for 1020 min at room temperature , they were rinsed twice in double distilled h2o . for dab staining , slides were subjected to an additional pretreatment step : 0.3% hydrogen peroxide in double distilled water for 30 min to remove endogenous peroxidase activity . subsequently , all sections were washed with pbs and incubated for 1 h at room temperature in 10% goat serum and 0.25% tween-20 in pbs to block nonspecific binding . all primary antibodies , diluted in pbs containing 0.25% tween-20 and 10% goat serum , were applied to sections and then incubated overnight at 4c . after rinsing in three washes of pbs , they were incubated for 1 h with a secondary antibody , which was conjugated with fluorescent alexa dyes ( dilution , 1 : 500 ) . antifade with dapi was applied before sealing the sections under a glass coverslip . for dab staining , secondary antibody ( 1 : 500 dilution ) was applied for 1 h at room temperature , washed three times in pbs , and incubated in vectastain abc elite reagent ( vector laboratories , burlingame , ca ) for thirty minutes . after three more pbs washes , sections were dehydrated through double distilled water , graded ethanol , and washed twice in xylene . control sections were subjected to the same staining procedure , except that primary antibody was omitted . positive controls were obtained using cerebellar cortex of 15-day - old wild - type mice . to analyze the immunocytochemical data in a more quantitative fashion , we developed a rating scale to rate the cell cycle , at-8 , and 6e10 markers . the values assigned to the rating scale were 0 ( no staining or very little staining of cell cycle events ) , 1 ( a few staining of cell cycle events 515% ) , 2 ( low staining of cell cycle events , 1530% ) , 3 ( moderate staining of cell cycle events 3050% ) , and 4 ( > 50% cell cycle events ) . the r1.40 yac transgenic line contains a 650-kb yeast artificial chromosome ( yac ) with the entire 400-kb huapp gene modified with the swedish mutation ( k670n / m671l ) . r1.40 mice exhibit a preferential deposition of a 1 - 42 , which results in the appearance of amyloid deposits in parietal cortex beginning at 13.514 months . in addition , the r1.40 cortex displays extensive neuritic abnormalities as evidenced by staining with app , ubiquitin , neurofilament , and hyperphosphorylated tau antibodies [ 21 , 22 ] . an app695 human cdna transgene was used , with the swedish ( k670n / m671l ) double mutation , under the regulation of the hamster prp promoter . tg6799 is also known as the 5xtg transgenic mouse ; it carries a single human app cdna with the swedish k670n / m671l double mutation as well as the florida i716v mutation , and the london v717i mutation . the transgene includes a cdna sequence encoding the human app gene with the swedish mutation as well as a psen1 cdna transgene carrying the e9 mutation ( the sequence for exon 9 of ps1 is deleted ) . these were microinjected together resulting in the insertion of both app and psen1 transgenes at a sinigle locus . the app8.9 line has a yac genomic transgene similar to r1.40 , but the transgene encodes a wild - type human app gene instead of the swedish mutation found in r1.40 . the levels of app transgene expression have been found to be similar to both normal levels of app expression in humans as well as to the endogenous murine app gene . although no plaque pathology has been described , the app8.9 mouse should recapitulate the app dosage imbalance found in down syndrome [ 26 , 27 ] . we used both dab and fluorescent immunostaining of proliferating cell nuclear antigen ( pcna ) to study the regional variations of cces in dorsal raphe , hippocampus , cerebellum , pons , amygdala , and locus coeruleus . pcna is known to play an essential role in positioning the dna polymerase in replication and repair of dna . our results were confirmed using cyclin a , the activating subunit for several of the cdks ( cyclin - dependent kinases ) , which served as a second cell cycle marker . as the cyclin a results were comparable to those with pcna , only the pcna data are shown . the at8 and 6e10 antibodies were used to identify neurofibrillary tangles and plaques , respectively . immunostaining for tyrosine hydroxylase ( th ) combined with their anatomical location was used to identify locus coeruleus neurons . tryptophan hydroxylase ( tph ) immunoreactivity plus anatomical location was used to identify the neurons of the dorsal raphe . the results from the locus coeruleus are illustrated in figure 1 . in 3 of the 5 models app / ps1 , r1.40 and app8.9 ( figure 1(b))greater than 50% of these brainstem noradrenergic neurons were found to have immunocytochemical evidence of cell cycle activity . no cces were detected in the wild - type locus coeruleus ( figure 1(c ) ) . despite the absence of cell cycle activity , tg6799 scored the highest for phospho - tau staining , with greater than 50% of the neurons staining positive . tg6799 was also the only model to show 6e10 staining in this region ( figure 1(d ) ) ; none of the other four ad models displayed any a plaque deposition in the brainstem . a graphical summary of the cces and associated amyloid and tau staining patterns is shown in figure 1(d ) . most of the tryptophan hydroxylase immunoreactive neurons analyzed were from the region indicated by the box in figure 2(a ) . of the five models , r1.40 had the highest cce rating ( 4 ) , with greater than 50% of the neurons scoring positive for cell cycle staining ( figure 2(b ) ) . app8.9 had the second highest level of pcna staining with moderate number of neurons positively stained . age - matched wild - type mice showed virtually no cell cycle staining in this area ( figure 2(c ) ) . similar to our finding in the locus coeruleus , at8 staining was variable and the dorsal raphe of the tg6799 mouse model showed the highest levels at8 staining . across all genotypes , no correlation was observed between the cce score and the presence or absence of at8 or 6e10 immunoreactivity . our analysis of the amygdala was performed in sagittal sections , which makes the reliable identification of the specific subnuclei more difficult . to address this we divided the structure into three subregions as illustrated by the black boxes in figure 3(a ) . the anterior region contained predominantly the anterior cortical amygdaloid nucleus , with small contributions of the nuclear ansae lenticularis and the medial and cortical amygdaloid nuclei ; the middle region contained predominantly the posterolateral cortical , with lesser amounts of the anterior - lateral and central amygdaloid nuclei ; the posterior region contained predominantly the posteromedial cortical amygdaloid nucleus with small contributions from the medial basal amygdaloid nucleus . using this scheme , regional differences were found in the involvement of neuronal cell cycle events in the ad models . for all five models , the anterior portion of the amygdala demonstrated lower levels of cell cycle staining , while the medial and posterior portions showed higher levels of staining . of the 5 transgenic models , r1.40 showed the highest level of immunoreactivity , with greater than 50% of the neurons in the posterior and medial portions of the amygdala staining positive for cell cycle events . surprisingly , the wild - type mice showed modest levels of cces in the anterior and medial portions , but no cces in the posterior portions . tg2576 and app8.9 showed little staining or no staining in any of the three regions . in two models , r1.40 and app / ps1 , at8 was distinctively higher in the posterior and medial regions . amyloid deposits in the amygdala , as revealed by 6e10 staining , were observed only in the anterior and medial regions of app / ps1 mice . all other regions of the cns were substantially negative for cell cycle protein expression , as expected . the one exception to this was a small region of the ventral brainstem . in the pons and the more dorsal nucleus reticulari tegmentis pontis ( nrtp ) , wild - type animals showed moderate levels of cces , with similar levels in the app8.9 and tg2576 app / ps1 and tg6799 . the only exception to this pattern was found in the r1.40 model , which had low levels of cces in this region . the r1.40 showed high pontine levels of hyperphosphorylated tau , but low levels of 6e10-positive beta amyloid deposits . tg6799 exhibited a very high level of 6e10 staining at greater than 50% , the highest score of any of the models . in addition to these results , there were observations of cell cycle staining that were more unexpected . for example , the deep nuclei of the cerebellum showed strong levels of cces in almost all the models ( figure 4 ) . while at8 and 6e10 staining was observed only in tg6799 mice , four out of five transgenic models showed moderate cce staining ; r1.40 showed greater than 50% of the neurons positive for cce staining . a more detailed analysis of the involvement of the cerebellar deep nuclei in the pathogenesis of ad is the subject of a newly published report . cces have been reported in this region for two mouse models of ad , the pdapp mouse and the r1.40 model . in the current study , we did not choose to explore the pdapp mice , but were able to replicate our findings in the r1.40 ( figure 5(b ) ) . curiously , none of the other models we examined had evidence of cell cycle activity in this region . we observed no significant at8 or 6e10 staining in the dentate gyrus or in the pyramidal cells of any of the mouse models ( figure 5(d ) ) . transgenic mouse models have long been used to study the molecular mechanisms of disease . to be considered useful , such models must recapitulate the human disease in as many ways as possible , and by this criterion the mouse models of ad have been at least partially successful . no wild - type mouse has been reported to naturally develop amyloid plaques , neurofibrillary tangles , or an alzheimer's - like loss of neurons in any brain region . by contrast , mouse lines expressing app and/or psen1 transgenes with ad - related mutations display an age - related appearance of -amyloid plaques and hyperphosphorylated tau . behavioral and neurophysiological abnormalities have also been observed in some transgenic models , as well as neurophysiological defects , inflammation , and occasionally a decrease in the numbers of ca1 pyramidal neurons . impressively , the yac r.140 model shows a regional pattern of cerebral and vascular amyloid deposits along with reactive astrocytes and microglia that is consistent with the pattern of these events in human ad . in the end , however , the reproduction of the human disease has been incomplete in all of these lines . no neurofibrillary tangles are found ; neurodegeneration is limited even in the best models ; and the behavioral changes are mild compared to those observed in humans with mid- to late - stage alzheimer 's disease . also , because most successful app - expressing mouse lines require highly elevated levels of transgene expression to form plaques , the use of plaque deposition as the major outcome measure for evaluating the effectiveness of the models represents somewhat of a self - fulfilling prophecy . this raises concerns that the effectiveness of the current app - based ad mouse models might be compromised . one of these concerns that we attempt to address here is the poor reproduction in the mouse of the neuronal cell death found in ad . in human ad , there is a massive degeneration of neurons and this is observed in a pattern with pronounced temporal and regional variability . while the reasons for the discordance between mouse and human phenotypes are unknown , if cces are used in place of actual neuronal cell loss as an index of neurodegeneration , our previous studies of the r1.40 model suggest a remarkably faithful replication of the progression of neuronal cell death in human ad . what remains unknown is why , if cces are correlated with neuronal cell death , the subsequent death of neurons is not immediate . indeed , studies in both mouse and human suggest that cell cycling and cell death can be months apart [ 19 , 34 ] . to the appearance of cell cycle events , we add the correlation with more traditional neuropathological indicators of alzheimer 's disease . our findings show that -amyloid deposits appear in several subcortical regions not just in the r1.40 mouse , but in most models . -amyloid deposition appears in some but not all of the structures studied and some but not all of the models stain with the at8 phospho - tau antibody . based on the models we examined , it would appear that amyloid deposits and tau expression levels showed region- and model - specificity and thus cautions should apply . in this study , we have expanded the range of biological responses in the 5 different ad mouse models to include cell cycle events ( cces ) as direct cell - autonomous indices of neuronal distress . since cces have been observed in both the human ad brain and in the analogous regions of certain ad mouse models , characterizing cce expression patterns provides a logical and independent outcome measure for the study of neuronal death process in human ad . it is significant , therefore , that the results reported here demonstrate clear differences among the mouse models examined in the pattern of cce expression . we find a high level of cces in the brain stem of all 5 models , including the pons , locus coeruleus , dorsal raphe , and deep nuclei of the cerebellum . the consistent appearance of cces in these more caudal regions of the cns despite differing transgene properties and -amyloid and tau pathologies is significant . it implies that the existence of neuronal stress in metencephalic and myelencephalic regions in familial ad may represent a central feature of disease pathogenesis . immunocytochemical and biochemical techniques revealed that cells produced from a locus coeruleus - derived cell line , but not hippocampal and cortical neurons , exhibited beta - amyloid accumulation and concentrate -secretase at process terminals . in the same study , it was shown that intracellular a plaques can become extracellular when neurites degenerate , which leads to additional accumulation and extracellular aggregation . in a different domain , the locus coeruleus has been proposed to play a role in brain inflammatory processes , such as those seen in ad [ 36 , 37 ] . more recently , braak and tredici , using tau phosphorylation as an index , also report very early disease pathology in the locus coeruleus . the pattern of cces in the amygdala suggests internal variations in ad pathology in this structure such as is seen in the cortex . in several ad models , the posterior and central amygdaloid nuclei showed stronger levels of cce expression when compared to more anterior amygdaloid neurons , suggesting a common impact of app transgene expression in this region . this variation is not apparent in the 6e10 or at8 staining suggesting that specific neuroanatomical phenotypes may be uncovered when cce markers are used . our findings in the ad mouse models are consistent with the well - documented involvement of the structure in human ad pathology [ 3941 ] . these studies have shown that significant numbers of neurons in the amygdala die during the early stages of ad . although there is less information available on the regional variability within the amygdala , attempts have been made to use degeneration in this structure to detect the onset of ad [ 34 , 42 ] . the r1.40 model showed the strongest staining for cces in most subcortical regions and was the only model in the current study to show significant cce expression in the hippocampal region . the absence of cce expression in the 4 other ad models we studied is noteworthy , albeit without explanation . nearly all of the models we studied have been shown to have deficits in behavioral tasks that are known to involve the hippocampus . this discordance between function and pyramidal cell body neuropathology suggests several hypotheses , none of which are mutually exclusive . perhaps the behavioral changes are due to synaptic loss or atrophy , but the cell body , as seen through the appearance of cces or neuronal cell death , remains largely unaffected . a related hypothesis is that the transgene - dependent excess of a at the synapse is the cause of the behavioral and physiological changes . a , especially the lower molecular weight form , is recognized as having a neuromodulatory function . finally , as the ages of the animals we examined were mostly one year or less , it is also possible that the disease process in the hippocampus was not sufficiently advanced at the time of perfusion . all of these alternatives are consistent with the proposal that alzheimer 's begins as a synaptic disease . this would appear to be the situation in hippocampus where slices , isolated in vitro , show impaired ltp ; whether this accounts for all of the behavioral changes or whether some might be due to the aberrant cell cycle activity is unknown at this time . although no single mouse model provides a complete recapitulation of human ad , based on the regions we examined , the yac r1.40 would appear to be the most reliable model , especially when using cces as an outcome measure . one possible explanation for r1.40 's strong fidelity as a model is the close reproduction of the pattern of transgenic app expression to that found in human . this in turn is most likely due to the method used to insert the mutant gene [ 21 , 22 ] . with the exception of app8.9 , the r1.40 mouse model carries the entire human app gene , including all introns and all 3 and 5 regulatory elements within 3050 kb of the coding sequence . this allows for a more faithful temporal and spatial expression pattern , possibly contributing to a more faithful reproduction of the human disease . we have shown here that cce markers are a reasonable way of studying ad mouse model fidelity to human ad . since no transgenic mouse model is able to perfectly capture the complexities of the human ad pathology , using several phenotypic markers to study the effects of transgene insertions is well advised . distinguishing the role of species differences and the effects of transgenes in ad pathogenesis through rigorous characterization of mouse models and ad will be important to uncovering the mechanisms of ad pathogenesis and lead to the more rapid identification of useful therapeutic targets .

ectopic cell cycle events ( cces ) in postmitotic neurons link the neurodegenerative process in human alzheimer 's disease ( ad ) with the brain phenotype of transgenic mouse models with known familial ad genes . most reports on the mouse models use the appearance of brain amyloid pathology as a key outcome measure . in the current paper , we focus on the induction of neurodegeneration using cces as markers for impending neuronal loss . we compare 5 mouse models of familial ad for the appearance of cces in subcortical regions deep cerebellar nuclei , amygdala , locus coeruleus , hippocampus , and dorsal raphe . we find that the models differ in their cce involvement as well as in the appearance of phosphorylated tau and amyloid deposition , suggesting that each model represents a different disease phenotype . comparison with the pattern of neuron death in human ad suggests that each may represent a distinctly different disease model when used in preclinical trials .

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

alzheimer 's disease ( ad ) is a devastating late - onset neurodegenerative condition that affects many regions of the human brain . although the most obvious disease symptoms involve the inability to form and store new memories , the neurological and psychiatric description of an individual with ad includes a wide range of symptoms such as depression , apathy , episodic behavioral outbursts , deteriorating executive functioning , and others . there is a clear loss of volume and pathologically visible degeneration in the brain 's memory centers , which include the entorhinal cortex , hippocampus , and basal forebrain nucleus . but there are also functional and structural abnormalities found in the locus coeruleus , dorsal raphe , cingulate gyrus , amygdala and prefrontal cortex as well as other cortical and subcortical regions [ 13 ] . while the appearance of the abnormal deposits is disease specific , their anatomical locations in human ad mark only a subset of the brain regions that are identified as undergoing significant atrophy during the progress of the disease . recent work from our laboratory and many others has explored the use of abnormal neuronal cell cycle processes as an additional pathological marker of disease [ 411 ] . the timing and location of neuronal cell death in ad has been intimately associated with the unscheduled appearance of events related to mitotic cell division . both cell cycle - related proteins and evidence of dna replication have been found in neurons that are considered at risk for death . it is hypothesized that , although the neurons are able to initiate a true cell cycle and replicate most if not all of their genome , they are incapable of completing the process and are believed to die . using immunohistochemical analysis , cell cycle events ( cces ) have been identified in subcortical brain regions of individuals with ad as well as those with mild cognitive impairment ( mci considered by many to be the clinical precursor of ad ) . in age - matched controls and in ad brain regions where neurons are not susceptible to death , cell cycle - related protein expression is significantly lower . this has led to the hypothesis that cell cycle events represent the first step of a process that leads to neuronal cell death in ad . significantly , these unexpected attempts by neurons to reenter a cell cycle provide one of the few homologies observed between mouse models of ad and the pathogenesis of the human condition . other researchers have developed models of human tauopathies based on transgenes expressing disease causing variants of the microtubule - associated protein tau [ 13 , 14 ] . paradoxically , despite the fact that familial ad has not been significantly associated with tau ( mapt ) mutations in humans , the tauopathy mouse models have been reasonably successful in reproducing many of the pathological characteristics of ad . with age , the brains of these animals display both tangles of hyperphosphorylated tau and progressive neurodegeneration in some cases . thus , mapt models reproduce the tangles and degeneration but not the plaques , while the app / ps1 models reproduce the alzheimer 's plaques but not the associated tangles or neurodegeneration . we have elected to focus on the pattern of neurodegeneration in app transgenic mice in order to expand the characterization of this group of ad models , and we have used cces as outcome measures . previously , where they have been studied in depth , the appearance of cces in many human disease models show an age - dependent increase in prevalence that often closely mimics the pattern of neuronal cell death in the human disease . for example , there is a significant correlation between the regional pattern of cell loss in human ataxia - telangiectasia and its mouse model . for human ad , the temporal and spatial appearance of cces in the r1.40 ad mouse model [ 1820 ] accurately recapitulate the anatomical progression of cell death in the human . in the current study , we expand the use of cell cycle markers as a benchmark of neuronal distress in the mouse . our goal was to determine the phenotypic variability among the various mouse models of ad and to learn whether the similarities and differences observed are informative as to their relative fidelity to the human disease . we describe a pattern of selective neuronal vulnerability similar to human ad which is recapitulated in some , but not all of the five . five transgenic mouse models of familial ad were used in the current studies . in each model , amyloid plaques induced by app develop at different ages . brain tissues from the tg2576 and tg6799 mouse models were a generous gift from dr . three animals from the r1.40 , tg2576 , and tg6799 lines were examined for this study . the brain was immediately removed from the skull and transferred to 4% paraformaldehyde at 4c overnight . for dab staining , secondary antibody ( 1 : 500 dilution ) was applied for 1 h at room temperature , washed three times in pbs , and incubated in vectastain abc elite reagent ( vector laboratories , burlingame , ca ) for thirty minutes . after three more pbs washes , sections were dehydrated through double distilled water , graded ethanol , and washed twice in xylene . to analyze the immunocytochemical data in a more quantitative fashion , we developed a rating scale to rate the cell cycle , at-8 , and 6e10 markers . the values assigned to the rating scale were 0 ( no staining or very little staining of cell cycle events ) , 1 ( a few staining of cell cycle events 515% ) , 2 ( low staining of cell cycle events , 1530% ) , 3 ( moderate staining of cell cycle events 3050% ) , and 4 ( > 50% cell cycle events ) . the r1.40 yac transgenic line contains a 650-kb yeast artificial chromosome ( yac ) with the entire 400-kb huapp gene modified with the swedish mutation ( k670n / m671l ) . r1.40 mice exhibit a preferential deposition of a 1 - 42 , which results in the appearance of amyloid deposits in parietal cortex beginning at 13.514 months . in addition , the r1.40 cortex displays extensive neuritic abnormalities as evidenced by staining with app , ubiquitin , neurofilament , and hyperphosphorylated tau antibodies [ 21 , 22 ] . an app695 human cdna transgene was used , with the swedish ( k670n / m671l ) double mutation , under the regulation of the hamster prp promoter . tg6799 is also known as the 5xtg transgenic mouse ; it carries a single human app cdna with the swedish k670n / m671l double mutation as well as the florida i716v mutation , and the london v717i mutation . the transgene includes a cdna sequence encoding the human app gene with the swedish mutation as well as a psen1 cdna transgene carrying the e9 mutation ( the sequence for exon 9 of ps1 is deleted ) . these were microinjected together resulting in the insertion of both app and psen1 transgenes at a sinigle locus . the levels of app transgene expression have been found to be similar to both normal levels of app expression in humans as well as to the endogenous murine app gene . we used both dab and fluorescent immunostaining of proliferating cell nuclear antigen ( pcna ) to study the regional variations of cces in dorsal raphe , hippocampus , cerebellum , pons , amygdala , and locus coeruleus . our results were confirmed using cyclin a , the activating subunit for several of the cdks ( cyclin - dependent kinases ) , which served as a second cell cycle marker . immunostaining for tyrosine hydroxylase ( th ) combined with their anatomical location was used to identify locus coeruleus neurons . tryptophan hydroxylase ( tph ) immunoreactivity plus anatomical location was used to identify the neurons of the dorsal raphe . in 3 of the 5 models app / ps1 , r1.40 and app8.9 ( figure 1(b))greater than 50% of these brainstem noradrenergic neurons were found to have immunocytochemical evidence of cell cycle activity . no cces were detected in the wild - type locus coeruleus ( figure 1(c ) ) . despite the absence of cell cycle activity , tg6799 scored the highest for phospho - tau staining , with greater than 50% of the neurons staining positive . tg6799 was also the only model to show 6e10 staining in this region ( figure 1(d ) ) ; none of the other four ad models displayed any a plaque deposition in the brainstem . of the five models , r1.40 had the highest cce rating ( 4 ) , with greater than 50% of the neurons scoring positive for cell cycle staining ( figure 2(b ) ) . age - matched wild - type mice showed virtually no cell cycle staining in this area ( figure 2(c ) ) . similar to our finding in the locus coeruleus , at8 staining was variable and the dorsal raphe of the tg6799 mouse model showed the highest levels at8 staining . using this scheme , regional differences were found in the involvement of neuronal cell cycle events in the ad models . for all five models , the anterior portion of the amygdala demonstrated lower levels of cell cycle staining , while the medial and posterior portions showed higher levels of staining . of the 5 transgenic models , r1.40 showed the highest level of immunoreactivity , with greater than 50% of the neurons in the posterior and medial portions of the amygdala staining positive for cell cycle events . surprisingly , the wild - type mice showed modest levels of cces in the anterior and medial portions , but no cces in the posterior portions . in two models , r1.40 and app / ps1 , at8 was distinctively higher in the posterior and medial regions . amyloid deposits in the amygdala , as revealed by 6e10 staining , were observed only in the anterior and medial regions of app / ps1 mice . all other regions of the cns were substantially negative for cell cycle protein expression , as expected . in the pons and the more dorsal nucleus reticulari tegmentis pontis ( nrtp ) , wild - type animals showed moderate levels of cces , with similar levels in the app8.9 and tg2576 app / ps1 and tg6799 . the only exception to this pattern was found in the r1.40 model , which had low levels of cces in this region . tg6799 exhibited a very high level of 6e10 staining at greater than 50% , the highest score of any of the models . in addition to these results , there were observations of cell cycle staining that were more unexpected . for example , the deep nuclei of the cerebellum showed strong levels of cces in almost all the models ( figure 4 ) . a more detailed analysis of the involvement of the cerebellar deep nuclei in the pathogenesis of ad is the subject of a newly published report . cces have been reported in this region for two mouse models of ad , the pdapp mouse and the r1.40 model . in the current study , we did not choose to explore the pdapp mice , but were able to replicate our findings in the r1.40 ( figure 5(b ) ) . curiously , none of the other models we examined had evidence of cell cycle activity in this region . we observed no significant at8 or 6e10 staining in the dentate gyrus or in the pyramidal cells of any of the mouse models ( figure 5(d ) ) . transgenic mouse models have long been used to study the molecular mechanisms of disease . to be considered useful , such models must recapitulate the human disease in as many ways as possible , and by this criterion the mouse models of ad have been at least partially successful . by contrast , mouse lines expressing app and/or psen1 transgenes with ad - related mutations display an age - related appearance of -amyloid plaques and hyperphosphorylated tau . behavioral and neurophysiological abnormalities have also been observed in some transgenic models , as well as neurophysiological defects , inflammation , and occasionally a decrease in the numbers of ca1 pyramidal neurons . impressively , the yac r.140 model shows a regional pattern of cerebral and vascular amyloid deposits along with reactive astrocytes and microglia that is consistent with the pattern of these events in human ad . in the end , however , the reproduction of the human disease has been incomplete in all of these lines . no neurofibrillary tangles are found ; neurodegeneration is limited even in the best models ; and the behavioral changes are mild compared to those observed in humans with mid- to late - stage alzheimer 's disease . also , because most successful app - expressing mouse lines require highly elevated levels of transgene expression to form plaques , the use of plaque deposition as the major outcome measure for evaluating the effectiveness of the models represents somewhat of a self - fulfilling prophecy . this raises concerns that the effectiveness of the current app - based ad mouse models might be compromised . one of these concerns that we attempt to address here is the poor reproduction in the mouse of the neuronal cell death found in ad . in human ad , there is a massive degeneration of neurons and this is observed in a pattern with pronounced temporal and regional variability . while the reasons for the discordance between mouse and human phenotypes are unknown , if cces are used in place of actual neuronal cell loss as an index of neurodegeneration , our previous studies of the r1.40 model suggest a remarkably faithful replication of the progression of neuronal cell death in human ad . to the appearance of cell cycle events , we add the correlation with more traditional neuropathological indicators of alzheimer 's disease . our findings show that -amyloid deposits appear in several subcortical regions not just in the r1.40 mouse , but in most models . -amyloid deposition appears in some but not all of the structures studied and some but not all of the models stain with the at8 phospho - tau antibody . based on the models we examined , it would appear that amyloid deposits and tau expression levels showed region- and model - specificity and thus cautions should apply . in this study , we have expanded the range of biological responses in the 5 different ad mouse models to include cell cycle events ( cces ) as direct cell - autonomous indices of neuronal distress . since cces have been observed in both the human ad brain and in the analogous regions of certain ad mouse models , characterizing cce expression patterns provides a logical and independent outcome measure for the study of neuronal death process in human ad . it is significant , therefore , that the results reported here demonstrate clear differences among the mouse models examined in the pattern of cce expression . we find a high level of cces in the brain stem of all 5 models , including the pons , locus coeruleus , dorsal raphe , and deep nuclei of the cerebellum . the consistent appearance of cces in these more caudal regions of the cns despite differing transgene properties and -amyloid and tau pathologies is significant . it implies that the existence of neuronal stress in metencephalic and myelencephalic regions in familial ad may represent a central feature of disease pathogenesis . immunocytochemical and biochemical techniques revealed that cells produced from a locus coeruleus - derived cell line , but not hippocampal and cortical neurons , exhibited beta - amyloid accumulation and concentrate -secretase at process terminals . in the same study , it was shown that intracellular a plaques can become extracellular when neurites degenerate , which leads to additional accumulation and extracellular aggregation . in a different domain , the locus coeruleus has been proposed to play a role in brain inflammatory processes , such as those seen in ad [ 36 , 37 ] . more recently , braak and tredici , using tau phosphorylation as an index , also report very early disease pathology in the locus coeruleus . the pattern of cces in the amygdala suggests internal variations in ad pathology in this structure such as is seen in the cortex . in several ad models , the posterior and central amygdaloid nuclei showed stronger levels of cce expression when compared to more anterior amygdaloid neurons , suggesting a common impact of app transgene expression in this region . this variation is not apparent in the 6e10 or at8 staining suggesting that specific neuroanatomical phenotypes may be uncovered when cce markers are used . our findings in the ad mouse models are consistent with the well - documented involvement of the structure in human ad pathology [ 3941 ] . these studies have shown that significant numbers of neurons in the amygdala die during the early stages of ad . although there is less information available on the regional variability within the amygdala , attempts have been made to use degeneration in this structure to detect the onset of ad [ 34 , 42 ] . the r1.40 model showed the strongest staining for cces in most subcortical regions and was the only model in the current study to show significant cce expression in the hippocampal region . the absence of cce expression in the 4 other ad models we studied is noteworthy , albeit without explanation . nearly all of the models we studied have been shown to have deficits in behavioral tasks that are known to involve the hippocampus . perhaps the behavioral changes are due to synaptic loss or atrophy , but the cell body , as seen through the appearance of cces or neuronal cell death , remains largely unaffected . a related hypothesis is that the transgene - dependent excess of a at the synapse is the cause of the behavioral and physiological changes . finally , as the ages of the animals we examined were mostly one year or less , it is also possible that the disease process in the hippocampus was not sufficiently advanced at the time of perfusion . all of these alternatives are consistent with the proposal that alzheimer 's begins as a synaptic disease . this would appear to be the situation in hippocampus where slices , isolated in vitro , show impaired ltp ; whether this accounts for all of the behavioral changes or whether some might be due to the aberrant cell cycle activity is unknown at this time . although no single mouse model provides a complete recapitulation of human ad , based on the regions we examined , the yac r1.40 would appear to be the most reliable model , especially when using cces as an outcome measure . one possible explanation for r1.40 's strong fidelity as a model is the close reproduction of the pattern of transgenic app expression to that found in human . with the exception of app8.9 , the r1.40 mouse model carries the entire human app gene , including all introns and all 3 and 5 regulatory elements within 3050 kb of the coding sequence . we have shown here that cce markers are a reasonable way of studying ad mouse model fidelity to human ad . since no transgenic mouse model is able to perfectly capture the complexities of the human ad pathology , using several phenotypic markers to study the effects of transgene insertions is well advised . distinguishing the role of species differences and the effects of transgenes in ad pathogenesis through rigorous characterization of mouse models and ad will be important to uncovering the mechanisms of ad pathogenesis and lead to the more rapid identification of useful therapeutic targets .

adenosine triphosphate ( atp ) is probably one of the oldest signalling molecules appeared during evolution of living organisms . albeit it is basically impossible to gather experimental proofs , it would not be surprising that atp was used as a signalling molecules at the same time as it started to be exploited as an intracellular energy source . there are several reports of the activity of extracellular atp in primitive organisms such as bacteria , algae and slime moulds . evidence for a signalling role of extracellular nucleotides is compelling in lower invertebrates and overwhelming in mammals . atp , and its degradation product adenosine , are well recognized neurotransmitters in both the central and peripheral nervous system , but their signalling role in other systems is not yet fully acknowledged . in this respect , it is ironic that identification of atp as the high energy intermediate in muscle and first description of extracellular atp and adenosine effects on heart beat occurred in the same year , 1929 , but while the role of atp as an intracellular high energy intermediate was plainly accepted , it took almost a century to acknowledge its participation in extracellular signalling . students of inflammation and innate immunity have always been busy investigating the elementary signals released by distressed cells that alert the immune system of an impending danger . nowadays , it is a solid observation that detection of a foreign microorganism is not by itself sufficient to start inflammation , but recognition of a damage is also needed . in fact , recognition of damage is even more important than detection of a foreign agent , as clearly shown by the occurrence of sterile inflammation . cells of innate immunity recognize pamps ( pathogen associated molecular patterns ) released by invading microorganisms , but they also need to detect damps ( damage associated molecular patterns ) in order to be fully activated . all damps share the unique feature of being virtually absent in the extracellular environment in healthy conditions , while on the contrary are released quickly upon cell damage . since their extracellular concentration is normally close to nil , even a small leakage of these compounds generates a large signal that can be quickly detected by nearby immune cells . several intracellular molecules have been so far listed in the damp family : high mobility group box 1 ( hmgb1 ) protein , heat - shock proteins , nucleosomes , dna and atp . several features make atp an ideal damp , probably better than the other molecules so far proposed to this role . first of all its huge outward - directed transmembrane gradient ( cytosol concentration in the millimolar range , extracellular concentration in the low nanomolar range ) makes it very easy to rapidly generate a large extracellular signal following even minor cellular insults ; secondly , atp is highly diffusible through the aqueous extracellular environment thanks to its charges ( 2 or 4 negative charges depending on the ph and extracellular cation concentration ) ; thirdly , atp binds with varying affinity to a large family of specific cognate receptors , a feature that confers to atp - based signalling a unique plasticity ; fourthly , the atp signal can be quickly terminated by ubiquitous ecto - atpases , thus fulfilling a basic requirement of every bona fide messenger molecule , i.e. rapid inactivation when the message is no more needed . in the initial phases of inflammation damp signalling is exquisitely needed to activate dendritic cells ( dcs ) and therefore to modulate the ensuing immune response , thus it is anticipated that an additional requirement of atp in order to be considered a damp is that this nucleotide acts on the dcs to potentiate antigen presentation and direct the evolution of the immune response . several reports now confirm the key immunoregulatory role of extracellular atp proposed in early studies [ 912 ] . as any extracellular , membrane - impermeant , , adenosine is sensed by the other members of the purinergic receptor family named p1 receptors . p2 receptors are further subdivided into p2y ( g protein coupled ) and p2x ( intrinsic ion channels ) . four p1 receptor subtypes ( a1 , a2a , a2b and a3 ) , eight p2y ( p2y1,2,4,6,11,12,13,14 ) and seven p2x ( p2x1 - 7 ) subtypes are known . basically all p1 and p2 receptor subtypes are expressed by immune cells , in a cell type- and differentiation - dependent fashion . as it is often the case in the signalling field the fundamental question of the detection and measurement of the putative mediator ( atp ) in the extracellular environment has remained unanswered for a long time due to lack of suitable tools to measure the atp concentration in the interstitial fluid in vivo . measuring atp in the extracellular environment avoiding at the same time any possible cell damage that might by itself trigger atp release is a daunting task , something comparable to eisenberg uncertainty principle , if parva licet componere magnis . it is in fact a common observation that even minor cell perturbations , such as for example rinsing cell monolayers with culture medium , trigger large atp release , thus we can imagine what happens any time investigators stick an electrode into a tissue to measure adenosine or atp release . this intrinsic difficulty in atp measurement has been tackled ( and hopefully solved ) by our group with the engineering of a modified luciferase targeted to and stably expressed on the cell plasma membrane ( catalytic site outside ) . we anticipated that such a probe , named pmeluc ( plasma membrane luciferase ) would allow measurement of atp in the pericellular milieu . this prediction has been fulfilled in numerous experimental settings [ 1719 ] , thus making pmeluc the probe of choice to measure extracellular atp in vivo . the convergent indication stemming from the application of pmeluc to extracellular atp measurement has been that while concentration of this nucleotide in healthy tissues is negligible , and in any case below detection level , at sites of inflammation or within tumor microenvironment the concentration can be as high as hundreds micromolar . this concentration is sufficient to activate even the low affinity p2x7 receptor and to generate large amounts of the anti - inflammatory agent adenosine . the obvious question then arises as to how this large extracellular atp concentration is generated and exploited by inflammatory cells to orient themselves through the inflamed tissue , reach and destroy the causative agent , and finally repair the tissue . for a long time it has been assumed that the only pathway for cellular atp release was plasma membrane damage or overt cell injury . we now know that all cells are capable of non - lytic atp release and that this autocrine / paracrine purinergic stimulation has a key role in the pathophysiology of immune cells . several mechanisms are responsible for atp release : exocytosis of atp - containing granules , plasma membrane carriers , large conductance channels such as connexins and pannexins , and p2 receptors themselves such as p2x7 . it is not clear which atp release mechanism predominates in immune cells , but it is likely that multiple release pathways are involved , possibly also depending on the activating stimulus and the given pathophysiological condition . some atp release pathways are of particular interest because they might trigger an autoregenerative atp release loop resulting in spreading and amplification of the atp signal . this is the case of atp release through p2x7 , as released atp might feed back onto the p2x7 receptor itself to keep it in an open state and thus allow further atp release . release of atp at the inflammatory site generates an atp concentration gradient that might be exploited by inflammatory cells for chemotaxis . there is evidence that chemotactic peptides trigger atp release from the leading edge of neutrophils and that atp activates p2y2 receptors that in turn potentiate the chemotactic signal . activation of neutrophils movement through the chemotactic gradient seems to be due to adenosine accumulation at the leading edge of the chemotacting neutrophil and the following a3 receptor stimulation . more recent studies suggest that atp is not directly involved in inflammatory cell chemotaxis , but rather is a prerequisite for the production of chemotactic factors at the site of inflammation . it is proposed that atp activates stromal or resident inflammatory cells ( very likely tissue macrophages ) to generate an inflammatory microenvironment that produces the chemotactic gradient for the neutrophils . other investigators also see atp not as true chemotactic signal but rather as a signal of cell distress that in turn a ) induces the release of chemotactic factors from nearby cells and b ) modulates activation of receptors for chemotactic factors . however , evidence obtained by other investigators suggest that atp ( and adp ) might function as a true chemotactic signal and that pannexins might be involved in the generation of the atp gradient generated by apoptosing cells . among other nucleotides , utp has been shown to have a strong chemotactic activity against neutrophils and mast cells . receptors involved are the g - protein coupled p2yrs , mainly p2y2 , p2y6 , p2y12 and p2y13 . thus , inflammatory cells respond to a graded nucleotide concentration by moving towards the gradient core . once inflammatory cells have reached the center of the inflammatory site , it is likely that the elevated atp levels function as a dendritic cells ( dcs ) offer an interesting example of immune cell responses to a nucleotide gradient . in fact , only immature dcs chemotact to nucleotides , while mature dcs , despite they express p2 receptors to the same level as immature dcs , are fully non - responsive . this suggests that , once dcs have localized and captured the ag and migrated to the lymph nodes , there is no need to remain sensitive to attraction generated by cell - released danger signals , in fact mature dcs should lose nucleotide - dependent chemotactic activity in order to migrate from the atp - rich inflammatory site to the lymph nodes . it is well known that after ag capture dc start a complex differentiation process that leads to maturation and therefore to full ability to present ag to lymphocyte and start an immune response . depending on the type and amount of cytokine released and the co - stimulatory molecules expressed , dc will drive nave cd t lymphocyte differentiation towards a th1 , th2 , th17 or treg phenotype . such a differentiation process is strongly affected by the inflammatory microenvironment and by the atp concentration . it has been shown by several groups that incubation of dcs in the presence of micromolar atp concentrations drives dc maturation towards a th2 phenotype , and more recently that autocrine atp release modulates the differentiation of treg cells . the atp - rich microenvironment affects other key immune cell functions such as phagocytosis , phagosome - lysosome fusion and release of cytotoxic mediators . long ago , high extracellular atp levels were shown to inhibit phagocytosis in mouse macrophages . the inhibitory effect of atp on particle ingestion was confirmed in human monocytes and shown to be p2x7-dependent . on the other hand , extracellular atp has further distinct effects downhill to phagocytosis as atp - mediated p2x7 stimulation is a strong inducer of phagosome - lysosome fusion and subsequent killing of ingested microorganisms . the microbicidal activity due to p2x7 stimulation had been originally assigned to its pro - apoptotic effect , however subsequent experiments suggest that facilitation of phagosome - lysosome fusion has itself a potent microbicidal activity by exposing the ingested microrgansim to lysosomal content . this view is further supported by the ability of extracellular atp to cause the release of reactive oxygen species ( ros ) via the mitochondria or nadph oxidase activation . an essential aspect of the immunomodulatory activity of atp and p2 receptors is the cytokine - releasing activity . this was first described for il-1b , but has been later showed for several other crucial cytokines [ 4144 ] . secretion of other cytokines , e.g. il-12 , on the other hand is inhibited by extracellular atp . it must be stressed that atp effects are very much dose- and receptor subtype- dependent , as while at low doses atp has mainly an immunosuppressive , tolerogenic , activity ( presumably acting at p2y1 and/or p2y11 receptors , at high doses the effect is mainly pro - inflammatory , very likely acting at p2x7 . therefore , low atp doses preferentially activate immunosuppressive or tolerogenic pathways , while high doses trigger pro - inflammatory pathways . atp exerts a potent modulatory activity also on chemokine secretion , inducing release of ccl22 and decreasing lps - induced secretion of cxcl10 and ccl5 . the net effect of this modulation of cytokine and chemokine release in dcs is on one hand to reduce th1 cell differentiation and recruitment and on the other to drive th2 cell differentiation . paradoxically , immunosuppressive pathways may also be activated via the classical pro - inflammatory p2x7 receptor , as reported by robson and co - workers . these authors showed that mice deficient of cd39 , the main ecto - atp / adpase , are protected against con - a - induced hepatonecrosis because failure to degrade atp causes an accumulation of this nucleotide to a level sufficient to trigger p2x7-mediated apoptosis of nkt cells , the main effector of con - a induced liver injury . atp might also down - modulate the immune response by up - regulating thrombospondin and indoleamine 2,3-dioxigenase , factors known to inhibit t cell proliferation and stimulate tgf- release . the shift in dc responses ( from immunosuppresion to immunostimulation ) in presence of increasing concentrations of extracellular atp is consistent with the view that while graded exposure to damps may preferentially cause adaptation to the new condition rather than an overt defensive response , a brisk and large increase in damps is more likely to cause inflammation . identification of p2 receptors mediating the pro - inflammatory effects of extracellular nucleotides ( mainly atp ) is a crucial issue in purinergic signalling as this might lead to the development of novel anti - inflammatory drugs . in vitro and in vivo data point to p2y2 and p2x7 as the main p2 subtypes mediating the pro - inflammatory effects of atp . p2y2 is most probably responsible for recruitment of neutrophils , dendritic cells , eosinophils and macrophages at inflammatory sites , but it may also participate in release of pro - inflammatory factors such as for example elastase il-33 or mcp-1/ccl2 . among p2 receptors , p2x7 has received special attention since its participation to inflammation is more extensive and more thoroughly characterized . in the hope to develop novel drugs for the treatment of inflammation more information have been gathered on the involvement of this receptor in the immune response than for the other p2 receptors . p2x7 belongs to the p2x receptor family of atp - gated cation channels , but differs from the other p2x receptors in its c - terminus which is about 200 amino acid longer . brief activation of p2x7 with extracellular atp in its tetra - anionic form , atp , opens cation - specific ion channels . prolonged ligation of p2x7 results in the formation of non - selective membrane pores , permeable to molecules of molecular mass up to 900 da , as shown experimentally by the uptake of fluorescent dyes . depending on the cell type , p2x7 stimulation triggers opening of non - selective pores which allows cationic and anionic dyes uptake . formation of the non - selective pore is dependent on the cytoplasmic c - terminal domain of p2x7 . prolonged p2x7 activation can lead to membrane blebbing and cell death by lysis / necrosis or apoptosis , depending on the cell type . the cytotoxic effect dependent on p2x7 ( originally named p2z ) activation was first described in mouse lymphocytes and tentatively thought to be implicated in t - cell dependent cytotoxicity , but this hypothesis never received strong experimental support , thus it is fair to say that physiological significance of cell death mediated by the ligation of p2x7 by atp remains unclear . however , in the light of the recent demonstration that atp may reach several hundred micromolar levels in the interstitial fluid , the cytotoxic effect mediated by p2x7 should be re - considered as it is not unlikely that part of the cell injurious effects of inflammation are indeed explained by the cytotoxic activity of atp via the p2x7 receptor . in this respect , it also needs to be stressed that albeit two other p2 receptors have been implicated in cytotoxicity , p2x2 and p2x4 , p2x7 expression is both necessary and sufficient to support atp - mediated cytotoxicity in all cell models so far investigated . more recent evidence suggest that tonic , low level , activation of p2x7 may in some conditions trigger growth or promote survival via a combined effect on endoplasmic reticulum ( er ) and mitochondria ca content , mitochondrial potential and oxidative phosphorylation , and nfatc1 activation . growth - promoting effects of p2x7 are relevant in t lymphocyte proliferation where a functional p2x7 receptor is needed to start mitogenic activation and to support growth . it is not known if p2x7 is also implicated in proliferation in other immune cells . further complexity in the participation of p2x7 in the regulation of immune cell functions is added by the recent discovery that a shorter p2x7 natural splice variant ( named p2x7b ) lacking the c - terminal region is highly expressed in lymphocytes . this short isoform possesses most properties of the longer isoform except for the non - selective pore formation . in a cell model ( hek293 cells ) transfected with both receptors , the longer p2x7 isoform ( p2x7a ) and p2x7 b can assemble on the cell plasma membrane forming a heterotrimeric receptor with distinct functional properties . surprisingly , the shorter p2x7 isoform , rather than acting as a dominant negative , stabilizes plasma membrane expression of p2x7 and potentiates its responses . thus , p2x7 may have different properties depending upon the ratio of shorter vs. longer isoforms in the heterotrimer . it is unknown whether native p2x7a / b heterotrimers exist and if so what their function might be , but it is intriguing that mitogenic stimulation of t lymphocytes causes an increase in p2x7a , but most notably in p2x7b transcription . stimulaton of t cell receptors on t lymphocytes triggers the release of intracellular atp which stimulates cell surface p2x7 leading to ca influx , nfat activation and il-2 synthesis . there is growing evidence that p2x7 participates in cd t lymphocytes differentiation in multiple and as yet only partially understood ways . one one hand p2x7 appears to be constitutively expressed by treg cells , but on the other p2x7 stimulation inhibits treg responses and skews their differentiation towards a th17 phenotype . a role for p2x7 has also been shown in macrophage differentiation where this receptor modulates cell fusion in the typical process of multinucleated giant cell formation occurring in granuloma , or during osteoclast differentiation in the bone . these studies highlighted an additional function of p2x7 in purinergic signalling : a pathway for atp release . in fact , until recently search for the elusive pathways mediating non - lytic atp release had mainly concentrated on vesicle - mediated release or plasma membrane channels belonging to the connexin / pannexin family . now , we know that p2x7 can be a pathway for atp release thus generating its own ligand . experiments in osteoclasts further point to the existence on the plasma membrane of a structured complex of molecules involved in atp signalling ( atp signalosome ? ) comprising atp - generating systems ( p2x7 and other plasma membrane conduits ) , p2 receptors , ecto - atpases and p1 receptors . it is increasingly clear that the multiple facets of purinergic signalling and its profound pathophysiological implications can only be understood in the context of the atp signalosome . it has been shown by several investigators that p2x7 stimulation leads to the activation of various caspases or metalloproteases . a paradigm of this activity is the proteolytic processing and release of il-1 and il-18 by microglia and macrophages . the first signal via toll - like receptors drives pro - il-1 and pro - il-18 expression and accumulation in the cytosol . the second signal via p2x7 triggers the proteolytic cleavage of leaderless pro - il-1 and pro - il-18 and the release of the mature cytokines . in fact , p2x7 is a potent activator of the nlrp3 inflammasome , which is a large multimeric protein platform composed of nlrp3 , the adaptator asc and procaspase 1 . oligomerisation of procaspase 1 leads to its proteolytic activation and the production of active caspase 1 which is involved in the proteolytic processing of pro - interleukins into their active form . p2x7 activation also triggers the proteolytic cleavage of plasma membrane proteins such as l - selectin , cd23 , tnf , cd27 , matrix metalloproteinase-9 and interleukin-6 receptor [ 5357 ] . have shown that p2x7 activation induces the proteolytic cleavage and shedding of the soluble fragment of the amyloid precursor protein ( sapp ) from neuroblastoma cells , in the absence of adam9 , 10 and 17 . the p2x7-dependent protease involved in the generation of sapp fragment was not identified but several pharmacological inhibitors suggested that a metalloprotease(s ) is involved in this processing pathway . macrophages from p2x7 ko mice are unable to release mature il-1 and il-18 after lps stimulation followed by p2x7 activation . therefore , the potential role of p2x7 in systemic or organ - specific auto - immune diseases has been tested in mice deficient for the p2x7 gene . in a monoclonal anti - collagen induced arthritis , less severe cartilage destructions and synovial inflammation as well as decrease in collagen cleavage products were found in p2x7-deficient mice suggesting that the lack of p2x7 leads to a decrease in pro - inflammatory cytokines such as il-1 known to be involved in arthritis severity . in humans , portales - cervantes et al . have analysed the role of p2x7 in systemic lupus erythematosus ( sle ) and rheumatoid arthritis ( ra ) . they studied p2x7 expression and function and two genetic polymorphisms ( 1513 a / c and 762 t / c ) in 101 sle and 122 ra patients compared to healthy controls . they did not find differences in the frequency of the genetic polymorphisms in patients and controls . in contrast , al - shukaili et al . found that the presence of rheumatoid factor and anti - mcv autoantibody was significantly associated with the 1513 a / c polymorphism , in ra patients . in ra patients , portales - cervantes et al . found a significant increase in il-1 released from atp - stimulated monocytes as compared to those of healthy controls . in contrast , atp - triggered monocytes from sle patients showed a significant decrease in il-1 production as compared to controls . thus , the observations made in ra patients are compatible with those made in the mouse model of arthritis even though the defects found in humans are moderate . overall , p2x7 seems to be involved in arthritis severity while its role in sle remains elusive . the potential involvement of p2x7 in sle relies on genomic studies which identified 14 lupus susceptibility loci among which the human sle locus sleb4 at 12q24 which includes the gene encoding p2x7 . since the incidence and gravity of arthritis induced by anti - collagen antibodies is reduced in p2x7 ko mice , two different groups used this mouse strain to study the role of p2x7 in experimental autoimmune encephalomyelitis ( eae ) , an inflammatory demyelinating disease of the cns induced in susceptible species and strains of mice by immunisation with cns myelin , or myelin proteins . interestingly , chen and brosnan found that p2x7 deficient animals develop more severe eae than wt animals . indeed , a significant increase in the number of lesions in the cns was found in p2x7 ko as compared to wt animals . importantly , using bone marrow chimeras , chen and brosnan showed that bone marrow derived cells from p2x7 ko mice increased the severity of the disease in irradiated wt mice . furthermore , the number of apoptotic cells in brain and spinal cord was higher in wt cns than in p2x7 ko animals . overall , these data suggest that the decrease in apoptosis of lymphocytes in the cns plays a major role in the worsening of eae in p2x7 ko . in contrast to this study , sharp et al . found , in a different p2x7 ko mouse strain , that the incidence of mog - induced eae is reduced when compared to wt animals . the discrepancy between these studies might due to the usage of two different p2x7 ko lines : pfizer vs glaxosmithkline ( gsk ) p2x7 ko animals . indeed , it was recently suggested that the gsk - p2x7 ko mice express a functional p2x7 in t lymphocytes but not in macrophages and dendritic cells . have identified a p2x7 ( k ) isoform which is expressed in the gsk but not in the pfizer p2x7 ko . this p2x7 ( k ) variant was shown to have an 8-fold higher ligand sensitivity and to transduce signals more efficiently than the commonly expressed p2x7 ( a ) isoform . thus , these results showing that the gsk p2x7 ko mice might express a functional isoform of p2x7 in t lymphocytes suggest that in these ko animals the decrease in the eae incidence might be due to the presence of p2x7 ( k ) on t lymphocytes , which down modulates autoimmune responses . it is well established that cell populations different from lymphocytes express p2x7 in the cns . in particular , microglial cells also express p2x7 which , after activation , triggers the production , among several other inflammatory mediators , of 2-arachidonoylglycerol ( 2-ag ) an endocannabinoid that activates neuronal cb1 receptors . in addition , activation of cb2 receptors expressed by microglial cells and lymphocytes decreases the production and release of pro - inflammatory cytokines and free radicals . have also determined that stimulation of p2x7 during eae leads to increased endocannabinoid production and reduces cellular destructions . they found that the brain amounts of endocannabinoid were not augmented in wt mice suffering of eae and showing axonal damages as compared to control animals . these observations show that cellular destruction occurring during eae does not lead to increased production of neuroprotective endocannabinoids as found in various neuropathologies . in addition , in animals undergoing eae , the production of endocannabinoid ( 2-ag ) was significantly decreased in the cns of p2x7 ko animals as compared to wt mice witting . this reduction in 2-ag levels found in p2x7 ko mice correlated well with increased tissue destructions found in these animals compared to wt mice . it is worth noticing that in addition to microglial cells , astrocytes express p2x7 and are able to produce 2-ag albeit in much lower amounts as compared to microglia . however , being the astrocyte population larger than microglia , one can hypothesize that astrocyte production of 2-ag significantly contributes to neuroprotection . thus , expression of p2x7 by microglial cells and astrocytes should protect from eae even though endocannabinoid production is partially inhibited by eae indepently of p2x7 . oligodendrocytes which are the target of autoimmune attacks in eae were shown to express functional p2x7 and triggering of this receptor lead to oligodendrocyte death in vitro and in vivo . interestingly , treatment of mice with pharmacological inhibitors of p2x7 inhibited chronic eae by decreasing demyelination . the analyses of optic nerves from ms patients and healthy controls revealed that p2x7 mrna and protein were significantly increased in patients compared to controls . thus , p2x7 stimulation may increase tissue damage in the cns of ms patients as it does in mice suffering from eae . altogether , these results suggest that p2x7 on oligodendrocytes aggravate eae while its presence on aggressive t lymphocytes , microglial cells and astrocytes protects from this organ - specific autoimmune disease . in four mouse models of inflammatory bowel disease gulbransen inhibition of p2x7 with its pharmacological inhibitor o - atp prevented myenteric neuronal death but did not block the pathological signs associated with colitis such as macroscopic damages and weight losses . in addition , neuronal death was shown to be caspase - dependent and blocked by the pan - caspase inhibitor z - vad . the authors suggested that enteric neuronal death was dependent on pannexin-1 ( panx1 ) because it was inhibited by the selective peptide inhibitor panx and the pharmacological inhibitor probenicid . the authors suggested that panx-1 activation might be needed to activate the inflammasome and determined which components of this complex were required for neuronal death . using two ko mouse strains importantly , they found that blocking panx1 , caspases or the asc pathway inhibited neuronal death but did not improve weight loss and macroscopic damage . overall , these results may be therapeutically relevant because panx1 inhibition could protect enteric neurons during colitis and secure innervation of colonic muscle . however , in this study the direct involvement of p2x7 and panx1 in enteric neuronal death was mainly shown with the use of pharmacological inhibitors , p2x7 and/or panx1 ko animals should have been used to strengthen their conclusions . it is known that cona induced hepatitis is mediated by nkt cells because their elimination is protective . it has previously been shown that cell surface proteins from mouse t lymphocytes treated with nad are adp - ribosylated by the cell surface enzyme adp - ribosyl transferase 2 ( art2 ) . adp - ribosylation of p2x7 induces its activation leading to ca influx , non selective pore formation and cell death . among liver mononuclear cells , nkt and t lymphocytes but not nk cells express adp - ribosyltranferase activity and are adp - ribosylated on p2x7 provided that nad is added . mice treated by nad 2 h before cona injection are protected from hepatitis because liver nkt cells are functionally inactivated . importantly , when nad was injected into mice 3 h after cona , hepatitis severity was increased and 40% of the treated mice died . the involvement of p2x7 in this response is cleary established because administration of nad into cona - stimulated wt mice induces important liver destruction while in p2x7 ko mice liver damage was strongly reduced . these experiments clearly show that p2x7 mediates opposite effects in cona - induced autoimmune hepatitis . inhibitory signals are delivered to naive nkt cells protecting from liver injury while stimulatory ones are given to activated nkt lymphocytes exacerbating autoimmune hepatitis . recently , the role of p2x7 in the development of type 1 diabetes in non obese diabetic ( nod ) mice was evaluated by yi - guang chen et al . . they compared the incidence of type 1 diabetes in nod mice of three genotypes : p2x7 wt , p2x7 , p2x7 ko . however , the same authors previously established that type 1 diabetes is accelerated in nod mice lacking cd38 . cd38 is an ectoenzyme which hydrolyses nad and thus down modulates adp - ribosylation of p2x7 by art2 on t lymphocytes . hence , the lack of cd38 leads to a decrease in p2x7 dependent nad - induced cell death . in nod.cd38 ko mice , the acceleration of type i diabetes was attributed to nad - induced cell death of cd invariant nkt cells and foxp3 regulatory t lymphocytes . cd38 ko mice , they found that the lack of p2x7 abolished accelerated development of diabetes . interestingly , yi - guang chen et al . demonstrated that the numbers of cd invariant nkt cells and foxp3 regulatory t lymphocytes were restored in nod double ko mice to the levels found in nod mice . altogether , these results demonstrate that adp - ribosylation of p2x7 triggers nad - induced cell death of subpopulations of lymphocytes involved in the down modulation of type 1 diabetes in nod mice . the evaluation of p2x7 role in autoimmune diseases is complicated by its presence on various sub - populations of lymphocytes , on antigen presenting cells and macrophages and on cells which are the target of the autoimmune attack . in addition , the biological function of p2x7 on these cells varies , its stimulation can lead to cell death , cell proliferation , secretion of pro - inflammatory cytokines , production and release of neuroprotective endocannabinoids . thus , the production and use of p2x7 conditional ko animals is required to improve and clarify our knowledge on the role of p2x7 in autoimmune diseases . although major progress has been achieved over the past ten years in the field of purinergic signalling several exciting areas of research remain to be investigated . the biochemical characterization of p2x7 heterotrimers with distinct isoform composition should bring important information on the function of p2x7 on different subpopulations of t lymphocytes . this should stimulate proteomic analyses to identify the protein partners of these receptors and bolster efforts to characterize the biochemical pathways stimulated by the mitogenic p2x7 vs those triggering cell death . up to now , the p2x7 ko mouse lines available have been produced by methods in which the neomycin cassette has not been removed from the inactivated gene . thus , the impact of this cassette on the p2x4 gene , closely linked to p2x7 , has not been evaluated . in addition , the removal of p2x7 from all cells in which it is naturally expressed prevents detailed analyses of its function on defined sub - populations of cells . the production of conditional ko mice in which one can control where and/or when p2x7 is expressed would be of major interest to delineate its role in physiological or pathological conditions . the development of more selective pharmacological agonists and antagonists of p2x receptors is required to analyse biochemical purinergic pathways more precisely . in addition , some of these drugs might be useful as therapeutic agents to block or stimulate p2x receptors in various pathophysiological conditions .

immune cells express receptors for extracellular nucleotides named p2 receptors . p2 receptors transduce signals delivered by nucleotides present in the extracellular environment . accruing evidence shows that purinergic signalling has a profound effect on multiple immune cell responses such as t lymphocyte proliferation , chemotaxis , cytokine release , phagocytosis , ag presentation and cytotoxicity . this makes p2 receptors an attractive target for the therapy of immuno - mediated disease and cancer .

Nucleotides as extracellular messengers ATP as a DAMP Extracellular ATP: an immunomodulatory factor P2 receptors (P2X7) as drug targets P2X7-dependent activation of proteolytic pathways P2X7 and autoimmune diseases Conclusion

there are several reports of the activity of extracellular atp in primitive organisms such as bacteria , algae and slime moulds . evidence for a signalling role of extracellular nucleotides is compelling in lower invertebrates and overwhelming in mammals . in this respect , it is ironic that identification of atp as the high energy intermediate in muscle and first description of extracellular atp and adenosine effects on heart beat occurred in the same year , 1929 , but while the role of atp as an intracellular high energy intermediate was plainly accepted , it took almost a century to acknowledge its participation in extracellular signalling . all damps share the unique feature of being virtually absent in the extracellular environment in healthy conditions , while on the contrary are released quickly upon cell damage . since their extracellular concentration is normally close to nil , even a small leakage of these compounds generates a large signal that can be quickly detected by nearby immune cells . several intracellular molecules have been so far listed in the damp family : high mobility group box 1 ( hmgb1 ) protein , heat - shock proteins , nucleosomes , dna and atp . first of all its huge outward - directed transmembrane gradient ( cytosol concentration in the millimolar range , extracellular concentration in the low nanomolar range ) makes it very easy to rapidly generate a large extracellular signal following even minor cellular insults ; secondly , atp is highly diffusible through the aqueous extracellular environment thanks to its charges ( 2 or 4 negative charges depending on the ph and extracellular cation concentration ) ; thirdly , atp binds with varying affinity to a large family of specific cognate receptors , a feature that confers to atp - based signalling a unique plasticity ; fourthly , the atp signal can be quickly terminated by ubiquitous ecto - atpases , thus fulfilling a basic requirement of every bona fide messenger molecule , i.e. in the initial phases of inflammation damp signalling is exquisitely needed to activate dendritic cells ( dcs ) and therefore to modulate the ensuing immune response , thus it is anticipated that an additional requirement of atp in order to be considered a damp is that this nucleotide acts on the dcs to potentiate antigen presentation and direct the evolution of the immune response . several reports now confirm the key immunoregulatory role of extracellular atp proposed in early studies [ 912 ] . p2 receptors are further subdivided into p2y ( g protein coupled ) and p2x ( intrinsic ion channels ) . basically all p1 and p2 receptor subtypes are expressed by immune cells , in a cell type- and differentiation - dependent fashion . as it is often the case in the signalling field the fundamental question of the detection and measurement of the putative mediator ( atp ) in the extracellular environment has remained unanswered for a long time due to lack of suitable tools to measure the atp concentration in the interstitial fluid in vivo . measuring atp in the extracellular environment avoiding at the same time any possible cell damage that might by itself trigger atp release is a daunting task , something comparable to eisenberg uncertainty principle , if parva licet componere magnis . it is in fact a common observation that even minor cell perturbations , such as for example rinsing cell monolayers with culture medium , trigger large atp release , thus we can imagine what happens any time investigators stick an electrode into a tissue to measure adenosine or atp release . we anticipated that such a probe , named pmeluc ( plasma membrane luciferase ) would allow measurement of atp in the pericellular milieu . we now know that all cells are capable of non - lytic atp release and that this autocrine / paracrine purinergic stimulation has a key role in the pathophysiology of immune cells . several mechanisms are responsible for atp release : exocytosis of atp - containing granules , plasma membrane carriers , large conductance channels such as connexins and pannexins , and p2 receptors themselves such as p2x7 . it is not clear which atp release mechanism predominates in immune cells , but it is likely that multiple release pathways are involved , possibly also depending on the activating stimulus and the given pathophysiological condition . this is the case of atp release through p2x7 , as released atp might feed back onto the p2x7 receptor itself to keep it in an open state and thus allow further atp release . more recent studies suggest that atp is not directly involved in inflammatory cell chemotaxis , but rather is a prerequisite for the production of chemotactic factors at the site of inflammation . it is proposed that atp activates stromal or resident inflammatory cells ( very likely tissue macrophages ) to generate an inflammatory microenvironment that produces the chemotactic gradient for the neutrophils . other investigators also see atp not as true chemotactic signal but rather as a signal of cell distress that in turn a ) induces the release of chemotactic factors from nearby cells and b ) modulates activation of receptors for chemotactic factors . however , evidence obtained by other investigators suggest that atp ( and adp ) might function as a true chemotactic signal and that pannexins might be involved in the generation of the atp gradient generated by apoptosing cells . among other nucleotides , utp has been shown to have a strong chemotactic activity against neutrophils and mast cells . once inflammatory cells have reached the center of the inflammatory site , it is likely that the elevated atp levels function as a dendritic cells ( dcs ) offer an interesting example of immune cell responses to a nucleotide gradient . in fact , only immature dcs chemotact to nucleotides , while mature dcs , despite they express p2 receptors to the same level as immature dcs , are fully non - responsive . this suggests that , once dcs have localized and captured the ag and migrated to the lymph nodes , there is no need to remain sensitive to attraction generated by cell - released danger signals , in fact mature dcs should lose nucleotide - dependent chemotactic activity in order to migrate from the atp - rich inflammatory site to the lymph nodes . depending on the type and amount of cytokine released and the co - stimulatory molecules expressed , dc will drive nave cd t lymphocyte differentiation towards a th1 , th2 , th17 or treg phenotype . it has been shown by several groups that incubation of dcs in the presence of micromolar atp concentrations drives dc maturation towards a th2 phenotype , and more recently that autocrine atp release modulates the differentiation of treg cells . the atp - rich microenvironment affects other key immune cell functions such as phagocytosis , phagosome - lysosome fusion and release of cytotoxic mediators . on the other hand , extracellular atp has further distinct effects downhill to phagocytosis as atp - mediated p2x7 stimulation is a strong inducer of phagosome - lysosome fusion and subsequent killing of ingested microorganisms . this view is further supported by the ability of extracellular atp to cause the release of reactive oxygen species ( ros ) via the mitochondria or nadph oxidase activation . an essential aspect of the immunomodulatory activity of atp and p2 receptors is the cytokine - releasing activity . secretion of other cytokines , e.g. atp exerts a potent modulatory activity also on chemokine secretion , inducing release of ccl22 and decreasing lps - induced secretion of cxcl10 and ccl5 . atp might also down - modulate the immune response by up - regulating thrombospondin and indoleamine 2,3-dioxigenase , factors known to inhibit t cell proliferation and stimulate tgf- release . identification of p2 receptors mediating the pro - inflammatory effects of extracellular nucleotides ( mainly atp ) is a crucial issue in purinergic signalling as this might lead to the development of novel anti - inflammatory drugs . p2y2 is most probably responsible for recruitment of neutrophils , dendritic cells , eosinophils and macrophages at inflammatory sites , but it may also participate in release of pro - inflammatory factors such as for example elastase il-33 or mcp-1/ccl2 . among p2 receptors , p2x7 has received special attention since its participation to inflammation is more extensive and more thoroughly characterized . in the hope to develop novel drugs for the treatment of inflammation more information have been gathered on the involvement of this receptor in the immune response than for the other p2 receptors . brief activation of p2x7 with extracellular atp in its tetra - anionic form , atp , opens cation - specific ion channels . prolonged ligation of p2x7 results in the formation of non - selective membrane pores , permeable to molecules of molecular mass up to 900 da , as shown experimentally by the uptake of fluorescent dyes . the cytotoxic effect dependent on p2x7 ( originally named p2z ) activation was first described in mouse lymphocytes and tentatively thought to be implicated in t - cell dependent cytotoxicity , but this hypothesis never received strong experimental support , thus it is fair to say that physiological significance of cell death mediated by the ligation of p2x7 by atp remains unclear . however , in the light of the recent demonstration that atp may reach several hundred micromolar levels in the interstitial fluid , the cytotoxic effect mediated by p2x7 should be re - considered as it is not unlikely that part of the cell injurious effects of inflammation are indeed explained by the cytotoxic activity of atp via the p2x7 receptor . in this respect , it also needs to be stressed that albeit two other p2 receptors have been implicated in cytotoxicity , p2x2 and p2x4 , p2x7 expression is both necessary and sufficient to support atp - mediated cytotoxicity in all cell models so far investigated . more recent evidence suggest that tonic , low level , activation of p2x7 may in some conditions trigger growth or promote survival via a combined effect on endoplasmic reticulum ( er ) and mitochondria ca content , mitochondrial potential and oxidative phosphorylation , and nfatc1 activation . growth - promoting effects of p2x7 are relevant in t lymphocyte proliferation where a functional p2x7 receptor is needed to start mitogenic activation and to support growth . it is not known if p2x7 is also implicated in proliferation in other immune cells . further complexity in the participation of p2x7 in the regulation of immune cell functions is added by the recent discovery that a shorter p2x7 natural splice variant ( named p2x7b ) lacking the c - terminal region is highly expressed in lymphocytes . this short isoform possesses most properties of the longer isoform except for the non - selective pore formation . thus , p2x7 may have different properties depending upon the ratio of shorter vs. longer isoforms in the heterotrimer . it is unknown whether native p2x7a / b heterotrimers exist and if so what their function might be , but it is intriguing that mitogenic stimulation of t lymphocytes causes an increase in p2x7a , but most notably in p2x7b transcription . there is growing evidence that p2x7 participates in cd t lymphocytes differentiation in multiple and as yet only partially understood ways . a role for p2x7 has also been shown in macrophage differentiation where this receptor modulates cell fusion in the typical process of multinucleated giant cell formation occurring in granuloma , or during osteoclast differentiation in the bone . these studies highlighted an additional function of p2x7 in purinergic signalling : a pathway for atp release . in fact , until recently search for the elusive pathways mediating non - lytic atp release had mainly concentrated on vesicle - mediated release or plasma membrane channels belonging to the connexin / pannexin family . comprising atp - generating systems ( p2x7 and other plasma membrane conduits ) , p2 receptors , ecto - atpases and p1 receptors . it is increasingly clear that the multiple facets of purinergic signalling and its profound pathophysiological implications can only be understood in the context of the atp signalosome . the first signal via toll - like receptors drives pro - il-1 and pro - il-18 expression and accumulation in the cytosol . oligomerisation of procaspase 1 leads to its proteolytic activation and the production of active caspase 1 which is involved in the proteolytic processing of pro - interleukins into their active form . p2x7 activation also triggers the proteolytic cleavage of plasma membrane proteins such as l - selectin , cd23 , tnf , cd27 , matrix metalloproteinase-9 and interleukin-6 receptor [ 5357 ] . have shown that p2x7 activation induces the proteolytic cleavage and shedding of the soluble fragment of the amyloid precursor protein ( sapp ) from neuroblastoma cells , in the absence of adam9 , 10 and 17 . the p2x7-dependent protease involved in the generation of sapp fragment was not identified but several pharmacological inhibitors suggested that a metalloprotease(s ) is involved in this processing pathway . therefore , the potential role of p2x7 in systemic or organ - specific auto - immune diseases has been tested in mice deficient for the p2x7 gene . in a monoclonal anti - collagen induced arthritis , less severe cartilage destructions and synovial inflammation as well as decrease in collagen cleavage products were found in p2x7-deficient mice suggesting that the lack of p2x7 leads to a decrease in pro - inflammatory cytokines such as il-1 known to be involved in arthritis severity . they did not find differences in the frequency of the genetic polymorphisms in patients and controls . found that the presence of rheumatoid factor and anti - mcv autoantibody was significantly associated with the 1513 a / c polymorphism , in ra patients . in contrast , atp - triggered monocytes from sle patients showed a significant decrease in il-1 production as compared to controls . thus , the observations made in ra patients are compatible with those made in the mouse model of arthritis even though the defects found in humans are moderate . interestingly , chen and brosnan found that p2x7 deficient animals develop more severe eae than wt animals . indeed , a significant increase in the number of lesions in the cns was found in p2x7 ko as compared to wt animals . overall , these data suggest that the decrease in apoptosis of lymphocytes in the cns plays a major role in the worsening of eae in p2x7 ko . the discrepancy between these studies might due to the usage of two different p2x7 ko lines : pfizer vs glaxosmithkline ( gsk ) p2x7 ko animals . have identified a p2x7 ( k ) isoform which is expressed in the gsk but not in the pfizer p2x7 ko . this p2x7 ( k ) variant was shown to have an 8-fold higher ligand sensitivity and to transduce signals more efficiently than the commonly expressed p2x7 ( a ) isoform . thus , these results showing that the gsk p2x7 ko mice might express a functional isoform of p2x7 in t lymphocytes suggest that in these ko animals the decrease in the eae incidence might be due to the presence of p2x7 ( k ) on t lymphocytes , which down modulates autoimmune responses . it is well established that cell populations different from lymphocytes express p2x7 in the cns . in addition , in animals undergoing eae , the production of endocannabinoid ( 2-ag ) was significantly decreased in the cns of p2x7 ko animals as compared to wt mice witting . thus , expression of p2x7 by microglial cells and astrocytes should protect from eae even though endocannabinoid production is partially inhibited by eae indepently of p2x7 . oligodendrocytes which are the target of autoimmune attacks in eae were shown to express functional p2x7 and triggering of this receptor lead to oligodendrocyte death in vitro and in vivo . interestingly , treatment of mice with pharmacological inhibitors of p2x7 inhibited chronic eae by decreasing demyelination . thus , p2x7 stimulation may increase tissue damage in the cns of ms patients as it does in mice suffering from eae . in four mouse models of inflammatory bowel disease gulbransen inhibition of p2x7 with its pharmacological inhibitor o - atp prevented myenteric neuronal death but did not block the pathological signs associated with colitis such as macroscopic damages and weight losses . adp - ribosylation of p2x7 induces its activation leading to ca influx , non selective pore formation and cell death . among liver mononuclear cells , nkt and t lymphocytes but not nk cells express adp - ribosyltranferase activity and are adp - ribosylated on p2x7 provided that nad is added . these experiments clearly show that p2x7 mediates opposite effects in cona - induced autoimmune hepatitis . recently , the role of p2x7 in the development of type 1 diabetes in non obese diabetic ( nod ) mice was evaluated by yi - guang chen et al . hence , the lack of cd38 leads to a decrease in p2x7 dependent nad - induced cell death . in nod.cd38 ko mice , the acceleration of type i diabetes was attributed to nad - induced cell death of cd invariant nkt cells and foxp3 regulatory t lymphocytes . cd38 ko mice , they found that the lack of p2x7 abolished accelerated development of diabetes . altogether , these results demonstrate that adp - ribosylation of p2x7 triggers nad - induced cell death of subpopulations of lymphocytes involved in the down modulation of type 1 diabetes in nod mice . the evaluation of p2x7 role in autoimmune diseases is complicated by its presence on various sub - populations of lymphocytes , on antigen presenting cells and macrophages and on cells which are the target of the autoimmune attack . in addition , the biological function of p2x7 on these cells varies , its stimulation can lead to cell death , cell proliferation , secretion of pro - inflammatory cytokines , production and release of neuroprotective endocannabinoids . thus , the production and use of p2x7 conditional ko animals is required to improve and clarify our knowledge on the role of p2x7 in autoimmune diseases . although major progress has been achieved over the past ten years in the field of purinergic signalling several exciting areas of research remain to be investigated . thus , the impact of this cassette on the p2x4 gene , closely linked to p2x7 , has not been evaluated . in addition , some of these drugs might be useful as therapeutic agents to block or stimulate p2x receptors in various pathophysiological conditions .

adolescent reproductive health ( arh ) outcomes in africa are the worst in the world.1 for this reason , the world health organization ( who ) has strongly promoted youth - friendly reproductive health services for over a decade ; however , targets for service access , uptake and funding remain unmet.2 3 infrastructural limitations including health system failures,4 5 extreme lack of human resources , irregular medicine supply,6 inaccessibility due to long distances,7 and inflexible work routines combine with poor attitudes of government health workers8 to limit the effectiveness of public sector arh service provision . against this background , close - to - community providers such as private drug vendors and traditional healers are often the first port of call for adolescents with reproductive health needs.9 such close - to - community providers are perceived to be more convenient than government service providers such as clinical officers based in dispensaries and health centres.10 11 for this reason , health planners have increasingly explored different strategies for community - based and lay reproductive and maternal health service provision.1215 however , it is unclear what capacity close - to - community agents have to provide effective reproductive health services for adolescents . we report the results of a study examining the experience , attitudes and capacity of a range of close - to - community providers of such services , as well as their readiness and ability to integrate with the mainstream health sector . for the purpose of this study , we define a close - to - community provider as any service provider at the community level offering one or more of a range of arh services including contraception , antenatal care , obstetric services , sexually transmitted infection ( sti ) and hiv services , counselling and support . we adopt the who definition of adolescence , namely anyone who is 1019 years of age.16 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . these providers were selected from communities that were stratified into rural , urban and high - risk clusters . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . fgds were facilitated by senior researchers from the tanzania national institute for medical research ( nimr ) mwanza . the fgds were conducted in february 2011 at central locations ( e.g. schools , health centres or village offices ) . village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled ethical approvals were obtained from the liverpool school of tropical medicine research ethics committee and the tanzania medical research coordinating committee . permission was obtained from administrative leaders at the regional , district and ward levels , and informed written consent was sought from all participants . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . discussion focused on skills , attitudes and practices in family planning and contraception , antenatal care and maternal delivery , hiv and sti prevention and treatment as well as post - abortion care . after each round of discussion the guide was further tuned to key themes based on reflection on the previous fgds . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . these providers were selected from communities that were stratified into rural , urban and high - risk clusters . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . fgds were facilitated by senior researchers from the tanzania national institute for medical research ( nimr ) mwanza . the fgds were conducted in february 2011 at central locations ( e.g. schools , health centres or village offices ) . village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled , additional participants could not be turned away . ethical approvals were obtained from the liverpool school of tropical medicine research ethics committee and the tanzania medical research coordinating committee . permission was obtained from administrative leaders at the regional , district and ward levels , and informed written consent was sought from all participants . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . discussion focused on skills , attitudes and practices in family planning and contraception , antenatal care and maternal delivery , hiv and sti prevention and treatment as well as post - abortion care . after each round of discussion the guide was further tuned to key themes based on reflection on the previous fgds . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we conducted 35 fgds with a total of 323 close - to - community providers distributed among nine cadres as shown in table 1 . six key themes were identified as follows : knowledge , skills and attitudes towards arharh services offered by close - to - community providersarh needs and close - to - community providers ' ability to provide themclose - to - community providers ' trainingcommunity referralpreferred referral service . knowledge , skills and attitudes towards arh arh services offered by close - to - community providers arh needs and close - to - community providers ' ability to provide them close - to - community providers ' training preferred referral service . summary of close - to - community providers views within the emerging themes arh , adolescent reproductive health ; art , antiretroviral therapy ; arv , antiretroviral ; fp , family planning ; phc , primary health care ; rh , reproductive health ; sti , sexually transmitted infection ; vct , voluntary counselling and testing . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . some maintained the belief that condoms were impregnated with hiv , while others believed that condoms could cause cancer.there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] drug shops mainly sell medicines . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . home - based care volunteers and social workers reported their main services to be palliative care , education and counselling . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the next most requested service was information on hiv prevention , pregnancy and family planning . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . youth - friendly service provision was also difficult for some due to lack of space.to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] some drug sellers complained of being consulted for sti treatment but unable to provide it , as they are not licensed to do so:we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. dispensaries and health centres ) . we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . referrals from drug shops to dispensaries were also reported:patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] participants discussed ideal community referral interventions to increase adolescents access to reproductive health services . they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] drug sellers also stated that linkages to dispensaries would be beneficial to them:we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . some maintained the belief that condoms were impregnated with hiv , while others believed that condoms could cause cancer.there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . so you can not tell someone who is 18 years old to use it , they ca n't accept because they want to have children . drug shops mainly sell medicines . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . home - based care volunteers and social workers reported their main services to be palliative care , education and counselling . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the next most requested service was information on hiv prevention , pregnancy and family planning . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . youth - friendly service provision was also difficult for some due to lack of space.to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] to be frank adolescents do not like our dispensary because of lack of space . there is no confidential room where i can take an adolescent and listen to her in private . adolescents are scared of coming because the moment they find someone they know , they tend to turn around and go back home . [ fgd # 33 , clinical officers ] some drug sellers complained of being consulted for sti treatment but unable to provide it , as they are not licensed to do so:we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] we ca n't treat stis . maybe the government should permit us to sell sti drugs so that when people do n't get them at the dispensary they can come and buy them from us . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. dispensaries and health centres ) . we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . referrals from drug shops to dispensaries were also reported:patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] patients come saying they have been sent by drug shops to get injections . [ fgd # 31 , clinical officers ] they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] drug sellers also stated that linkages to dispensaries would be beneficial to them:we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] we could refer any patient for prescription before we sell the medicines [ ... ] we need collaboration so that what we tell adolescents is accepted at the health centres when they go there . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . improved integration of close - to - community providers into formal health services has been identified as a strategy to strengthen health service provision.20 training and involvement of close - to - community provider cadres is included in the tanzania national strategic plan 20092015.21 however , our findings suggest that despite activities by the government of tanzania ministry of health and social welfare to engage this segment of the health sector22 the capacity of community - based providers to offer effective adolescent reproductive health services is poor . our study systematically explored views about arh across 10 cadres of close - to - community providers in nine communities in two districts in tanzania . the large number of fgds utilised in this study and the stratification of communities to include rural , urban and high - risk increases the representativeness of our findings , and makes this one of the largest studies of its kind.23 24 however , although large , representative and inclusive , this study is not without limitations . first , while we emphasised the need for confidentiality and set clear ground rules during each session , fgds are a communal activity and disclosure is dependent on the degree of trust generated within the discussion group . it is therefore possible that some close - to - community providers did not fully express their views out of embarrassment or fear of retribution . the use of the village executive officers as an entry point may also have led to unknown biases arising from local and interpersonal relationships ; however , we hope that the large number of participants and geographical breadth of the study has offset some of this bias . there was no gender or age separation within the cadre - specific groups as we believed service provision to be a topic that both male and female participants within a cadre could discuss openly ; however , the presence of older male participants could well have inhibited younger or female participants from speaking their minds extensively . whilst this did not appear to be the case from a review of field reports overall , our results suggest that levels of knowledge about arh among close - to - community providers remains poor and attitudes negative . misconceptions and mistrust are especially demonstrated through observed perceptions that hiv has been put into condoms to harm the public . these misconceptions date back to the very early days of the hiv epidemic and it is disappointing that they persist despite the substantial and varied community information campaigns that have been implemented over the past two decades.23 25 strongly held close - to - community provider views about adolescent sexual behaviour are based on cultural norms , which are difficult to change . there is a need to develop and model rights - based approaches to reduce judgemental views on adolescents sexuality . the perceived increase in demand for condoms among adolescents beyond the available supply could suggest that the number of adolescents seeking protection during sex is increasing . other hiv - prevention interventions have reported increases in this demand;26 27 however , such trends are difficult to interpret . whilst it may mean that decades of health promotion strategies are finally translating into increased condom use , it could also represent increasing numbers of sexual encounters among adolescents . however , our findings suggest that abortions are being sought by adolescents , potentially leading to increased numbers of unsafe abortions being carried out . whilst reported data of this kind are a good indicator of perceptions , they must be interpreted with caution in the absence of quantitative documentation of actual condoms dispensed or abortions conducted . we also found that close - to - community providers services are largely fragmented and under - resourced . the close - to - community providers are juggling hiv / sti , family planning and maternal health in large populations with very little capacity . they promote themselves as providers of these services , even though they are not in a position to offer them effectively . notwithstanding this , research shows that some close - to - community providers such as drug shops are more popular than mainstream health services.24 28 it means that close - to - community providers have the potential to powerfully influence health outcomes in the communities . a systematic review of evidence on health care workforce task - shifting demonstrated that issues identified in our study such as training and integration were key to improving health outcomes in communities where largely untrained and unsupported human resources are to be deployed.29 for example , dispensaries with staff who have diagnostic and prescribing skills often lack supplies of medicines,30 31 whereas drug shops where such skills are lacking may have stocks of these medicines . at the time of writing the ministry of health was upgrading drug stores into accredited drug distribution outlets ( addo ) with a licence to sell controlled medicines , and further development of the addo programme to include linkages with local dispensaries could significantly improve accessibility of skilled reproductive health services and effective treatment . our findings suggest that tbas may be one of the effective cadres in the community . in our fgds , tbas reported weekly participation in facility - based , in addition to house - to - house , visits . although evidence on population - level benefits of tba involvement in maternal health is equivocal,32 a recent study in southern tanzania reported that only 46.7% ( n=974 ) of women delivered in a health facility.33 we argue that the popularity of tbas at the community level can not be ignored . the regular use of their services , even at the institutional level as demonstrated in the present study , indicates a continued need for their role . improving the capability of tbas to provide arh services is an option worth considering in the context of tanzania . close - to - community providers potential to provide effective services is limited by their low capacity and skills , negative attitudes and lack of resources . other researchers have argued that training , integration and provision of resources to close - to - community providers is important if the health system achievements they contribute to are to be realised.20 our findings suggest that currently this training is lacking and that there have been few integration efforts to include the close - to - community providers in the health system or to address their lack of capacity and resources . close - to - community providers , known to be the first port of call for many adolescents seeking health care in tanzania , continue to have negative attitudes towards the provision of arh services , family planning and condom use . although they lack training , skills or resources to provide effective arh care , they are open to integration with the formal health sector . potentially therefore they could provide an entry point for expanded access to reproductive health services , but only with significant skills building and training to address negative attitudes .

objectivesyoung people in tanzania are known to access reproductive health services from a range of close - to - community providers outside formal health settings such as drug stores , village aids committees , traditional healers and traditional birth attendants ( tbas ) . however , questions remain about the quality of services such agents provide . this study investigated their capacity to provide adolescent reproductive health ( arh ) services and explored their readiness and ability to integrate with the mainstream health sector through community referral interventions.methodsthirty-five focus group discussions exploring close - to - community provider experiences and attitudes to arh service provision were carried out in two districts in northern tanzania . discussions were conducted in kiswahili , digitally recorded , verbatim - transcribed , translated and back - translated from swahili to english . a thematic analysis was conducted using nvivo 9.resultsthe major close - to - community cadres providing reproductive health services were drug stores , traditional healers , tbas and village health workers . they reported being the first port of call for adolescents seeking reproductive health services , but their knowledge of arh needs was poor . they had negative attitudes to , and lacked the necessary resources for , the provision of such services for adolescents . some were particularly unwilling to provide condom services and were prejudiced against adolescents using them . there was poor integration between the close - to - community providers and the formal health sector , further limiting their ability to provide adequate services.conclusionsalthough close - to - community providers are considered a key resource in the community , most have limited capacity to provide arh services . without capacity - building investments such as training and cooperation with the mainstream health sector , their contribution to positive reproductive health outcomes is limited , or could indeed lead to adverse outcomes .

Introduction Methods Study setting Study type and participant selection Ethical considerations Data collection and analysis Results Theme 1: Knowledge, skills and attitudes towards ARH Theme 2: ARH services offered by close-to-community providers Theme 3: ARH needs and close-to-community providers ability to meet them Theme 4: Close-to-community providers training Theme 5: Community referral Theme 6: Preferred referral service Discussion Conclusions

adolescent reproductive health ( arh ) outcomes in africa are the worst in the world.1 for this reason , the world health organization ( who ) has strongly promoted youth - friendly reproductive health services for over a decade ; however , targets for service access , uptake and funding remain unmet.2 3 infrastructural limitations including health system failures,4 5 extreme lack of human resources , irregular medicine supply,6 inaccessibility due to long distances,7 and inflexible work routines combine with poor attitudes of government health workers8 to limit the effectiveness of public sector arh service provision . against this background , close - to - community providers such as private drug vendors and traditional healers are often the first port of call for adolescents with reproductive health needs.9 such close - to - community providers are perceived to be more convenient than government service providers such as clinical officers based in dispensaries and health centres.10 11 for this reason , health planners have increasingly explored different strategies for community - based and lay reproductive and maternal health service provision.1215 however , it is unclear what capacity close - to - community agents have to provide effective reproductive health services for adolescents . we report the results of a study examining the experience , attitudes and capacity of a range of close - to - community providers of such services , as well as their readiness and ability to integrate with the mainstream health sector . for the purpose of this study , we define a close - to - community provider as any service provider at the community level offering one or more of a range of arh services including contraception , antenatal care , obstetric services , sexually transmitted infection ( sti ) and hiv services , counselling and support . we adopt the who definition of adolescence , namely anyone who is 1019 years of age.16 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . the fgds were conducted in february 2011 at central locations ( e.g. village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled ethical approvals were obtained from the liverpool school of tropical medicine research ethics committee and the tanzania medical research coordinating committee . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we aimed to determine which cadres of close - to - community providers were providing reproductive services to adolescents in nine communities in two districts ( magu and sengerema ) in mwanza region on the northwest shore of lake victoria ; what services they offered ; their skills and capacity to provide them ; their attitudes towards arh ; and their attitudes to cooperation with the mainstream health sector , especially referral of their clients to formal health facilities . we also included formal health service providers from government dispensaries and health centres to triangulate views on community referral and integration . cadre - specific focus group discussions ( fgds ) with 814 participants were conducted in order to capture the consensus view on the respective experiences and attitudes17 18 to arh . the fgds were conducted in february 2011 at central locations ( e.g. village executive officers invited eight people per cadre to participate in planned fgds ; however , word - of - mouth spread of information about the study led to greater numbers of participants than expected . in view of the distance they had travelled , additional participants could not be turned away . fgd guides were prepared , pretested in the field and revised to incorporate the views of pre - test participants . the discussions were digitally recorded , transcribed in kiswahili language and translated into english . using nvivo 9 software ( qsr international , doncaster , victoria , australia ) , the transcripts were analysed using a thematic framework based on nodes that were deductively drawn from predefined themes in the discussion guide.19 we conducted 35 fgds with a total of 323 close - to - community providers distributed among nine cadres as shown in table 1 . six key themes were identified as follows : knowledge , skills and attitudes towards arharh services offered by close - to - community providersarh needs and close - to - community providers ' ability to provide themclose - to - community providers ' trainingcommunity referralpreferred referral service . knowledge , skills and attitudes towards arh arh services offered by close - to - community providers arh needs and close - to - community providers ' ability to provide them close - to - community providers ' training preferred referral service . summary of close - to - community providers views within the emerging themes arh , adolescent reproductive health ; art , antiretroviral therapy ; arv , antiretroviral ; fp , family planning ; phc , primary health care ; rh , reproductive health ; sti , sexually transmitted infection ; vct , voluntary counselling and testing . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . [ fgd # 17 , traditional healers ] drug shops mainly sell medicines . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . [ fgd # 31 , clinical officers ] participants discussed ideal community referral interventions to increase adolescents access to reproductive health services . they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . most close - to - community providers were subject to a number of serious misconceptions and negative attitudes about arh . specifically , they had negative attitudes towards provision of reproductive health services to adolescents and expressed hostility to arh rights ; dismissing the right of adolescents to choose when and where to seek reproductive health services or when to use contraceptives as nonsense . [ fgd # 17 , traditional healers ] there are bugs in condoms they have been put in condoms to control our population through hiv infections . [ fgd # 17 , traditional healers ] such sentiments were also linked to doubts on the general purpose of family planning interventions : people in this village know that using family planning a person wo n't bear children ever again . [ fgd # 17 , traditional healers ] people in this village know that using family planning a person wo n't bear children ever again . however , whilst they are only licensed to sell non - prescription drugs and items such as analgesics and condoms , they reported treating stis and selling antibiotics . they felt that provision of guidance on hiv prevention including condom use , education and informal referral to health facilities in the community was their responsibility ; however , clinical officers from dispensaries did not corroborate that these referrals take place . in addition , village aids committee members stated that if consulted by adolescents on reproductive health issues they would disclose the discussion to parents . village health workers ( also known as community health workers ) reported providing counselling , distribution of condoms and referral to health facilities . traditional birth attendants conducted home births and provided advice to pregnant mothers . in one community , tbas worked 1 day per week at dispensaries helping with deliveries . traditional healers , the largest group of close - to - community providers , were the least likely to have had any form of training , despite the range of services that they claim to provide ; including infertility treatment , abortions , home births and treatment of stis using herbal remedies and charms . all close - to - community providers reported that the main reproductive health need of the adolescents was condoms . the close - to - community providers and dispensary clinicians also highlighted a number of specific reproductive health demands that they were not able to meet . for example , dispensaries could not offer complex family planning services such as sterilisation , and although they could not provide abortion due to its illegality in tanzania , its need amongst adolescents was reported to be on the rise . [ fgd # 17 , drugstore attendants ] close - to - community providers also felt that the demand for information and other reproductive health services could not be met as they lack the ability or resources . most close - to - community providers did not have any training except drugstore attendants and dispensary clinical officers who said they had certificates in drug dispensing and diplomas in clinical medicine , respectively . community health workers , home - based care volunteers and social workers reported receiving orientations from various civil society organisations promoting hiv prevention in the community , although the level and quality of such orientations to provide arh services was unclear . the other close - to - community providers had received no training or orientation , and all except dispensary staff felt that they had limited knowledge on arh and requested further training . unfortunately verification of the actual level of training attained by the close - to - community providers was beyond the scope of our study . our fgds explored the acceptability of establishing community referral mechanisms from close - to - community providers to formal health facilities ( i.e. we established that referral is done informally between close - to - community providers , for example , from village health workers to tbas . [ fgd # 31 , clinical officers ] they wanted linkages and connections with formal health facilities as well as recognition and integration into the health sector . dispensaries were also willing to work with drug shops saying:we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 8 , clinical officers ] we have skills but lack medicines at the dispensary , but drug shops have medicines they do not know how to prescribe , if we had a linkage , we could fill the gaps . [ fgd # 17 , drugstore attendants ] table 1 summarises the close - to - community providers views within the themes discussed above . improved integration of close - to - community providers into formal health services has been identified as a strategy to strengthen health service provision.20 training and involvement of close - to - community provider cadres is included in the tanzania national strategic plan 20092015.21 however , our findings suggest that despite activities by the government of tanzania ministry of health and social welfare to engage this segment of the health sector22 the capacity of community - based providers to offer effective adolescent reproductive health services is poor . our study systematically explored views about arh across 10 cadres of close - to - community providers in nine communities in two districts in tanzania . the large number of fgds utilised in this study and the stratification of communities to include rural , urban and high - risk increases the representativeness of our findings , and makes this one of the largest studies of its kind.23 24 however , although large , representative and inclusive , this study is not without limitations . it is therefore possible that some close - to - community providers did not fully express their views out of embarrassment or fear of retribution . the use of the village executive officers as an entry point may also have led to unknown biases arising from local and interpersonal relationships ; however , we hope that the large number of participants and geographical breadth of the study has offset some of this bias . there was no gender or age separation within the cadre - specific groups as we believed service provision to be a topic that both male and female participants within a cadre could discuss openly ; however , the presence of older male participants could well have inhibited younger or female participants from speaking their minds extensively . whilst this did not appear to be the case from a review of field reports overall , our results suggest that levels of knowledge about arh among close - to - community providers remains poor and attitudes negative . these misconceptions date back to the very early days of the hiv epidemic and it is disappointing that they persist despite the substantial and varied community information campaigns that have been implemented over the past two decades.23 25 strongly held close - to - community provider views about adolescent sexual behaviour are based on cultural norms , which are difficult to change . other hiv - prevention interventions have reported increases in this demand;26 27 however , such trends are difficult to interpret . however , our findings suggest that abortions are being sought by adolescents , potentially leading to increased numbers of unsafe abortions being carried out . whilst reported data of this kind are a good indicator of perceptions , they must be interpreted with caution in the absence of quantitative documentation of actual condoms dispensed or abortions conducted . we also found that close - to - community providers services are largely fragmented and under - resourced . the close - to - community providers are juggling hiv / sti , family planning and maternal health in large populations with very little capacity . they promote themselves as providers of these services , even though they are not in a position to offer them effectively . notwithstanding this , research shows that some close - to - community providers such as drug shops are more popular than mainstream health services.24 28 it means that close - to - community providers have the potential to powerfully influence health outcomes in the communities . a systematic review of evidence on health care workforce task - shifting demonstrated that issues identified in our study such as training and integration were key to improving health outcomes in communities where largely untrained and unsupported human resources are to be deployed.29 for example , dispensaries with staff who have diagnostic and prescribing skills often lack supplies of medicines,30 31 whereas drug shops where such skills are lacking may have stocks of these medicines . at the time of writing the ministry of health was upgrading drug stores into accredited drug distribution outlets ( addo ) with a licence to sell controlled medicines , and further development of the addo programme to include linkages with local dispensaries could significantly improve accessibility of skilled reproductive health services and effective treatment . our findings suggest that tbas may be one of the effective cadres in the community . in our fgds , tbas reported weekly participation in facility - based , in addition to house - to - house , visits . the regular use of their services , even at the institutional level as demonstrated in the present study , indicates a continued need for their role . improving the capability of tbas to provide arh services is an option worth considering in the context of tanzania . close - to - community providers potential to provide effective services is limited by their low capacity and skills , negative attitudes and lack of resources . other researchers have argued that training , integration and provision of resources to close - to - community providers is important if the health system achievements they contribute to are to be realised.20 our findings suggest that currently this training is lacking and that there have been few integration efforts to include the close - to - community providers in the health system or to address their lack of capacity and resources . close - to - community providers , known to be the first port of call for many adolescents seeking health care in tanzania , continue to have negative attitudes towards the provision of arh services , family planning and condom use . although they lack training , skills or resources to provide effective arh care , they are open to integration with the formal health sector . potentially therefore they could provide an entry point for expanded access to reproductive health services , but only with significant skills building and training to address negative attitudes .

misuse by health personnel of their privileged access to pharmaceuticals is a sensitive issue in the current context of scarce resources , promotion of generics , hiv epidemic and growing demand for health care . misappropriation is a widespread practice rarely explicitly acknowledged or documented , even in studies that have looked into the coping strategies of health professionals [ 1 - 4 ] . for example , in the average ugandan health facility drug leakage involving facility health workers as well as district health teams and health unit management committees is as high as 78% , with resale of drugs representing the greatest single source of income for health workers in most units . in many other developing countries all this represents a financial loss to the health care system , and affects health outcomes negatively . it also contributes to the growing sense of mistrust and disrespect for the health professions and their institutions . by its very nature misuse of access to pharmaceuticals an alternative is to ask health workers about what occurs in services without inquiring into their own behaviour . although basically a record of hearsay , such information can shed light on the type of misuse that occurs , and , more importantly , how this is perceived and valued by the personnel . it describes their perceptions of the importance , the nature and the justification of pilfering of drugs in these countries . the study used a mixed open - ended and closed questions self - administered questionnaire addressed to a convenience sample of individuals working at various levels in the health sector . together they cover a range of situations where health workers and patients interact or where decisions related to pharmaceutical policy are taken . in mozambique 27 individuals ( 11 doctors , three nurses , one pharmacist and 12 orderlies ) answered the questionnaire . there were 15 females and 12 males , ranging in age from 27 to 58 years ( median 38 years ) . all worked in the public sector : two in the ministry of health , two in the provincial health administration , one at district level , eight in hospitals and four in health centres . six doctors had university appointments . only 13 ( six doctors , three nurses and four orderlies ) said they also worked in the private for profit sector and one in the private not for profit sector . in cape verde 26 individuals ( four doctors , nine nurses , one pharmacist and 12 orderlies ) answered the questionnaire . there were 5 females and 21 males , ranging in age from 25 to 54 years ( median 35 years ) . all worked in the public sector : two at district level , 17 at hospital level , one at pharmacy level and two at health centre level . only six ( two doctors , nine nurses and nine orderlies ) reported working also in the private for profit sector and 1 doctor in the private not for profit sector . five ( two nurses and three orderlies ) worked in rural areas , the others in town . in mozambique 27 individuals ( 11 doctors , three nurses , one pharmacist and 12 orderlies ) answered the questionnaire . there were 15 females and 12 males , ranging in age from 27 to 58 years ( median 38 years ) . all worked in the public sector : two in the ministry of health , two in the provincial health administration , one at district level , eight in hospitals and four in health centres . six doctors had university appointments . only 13 ( six doctors , three nurses and four orderlies ) said they also worked in the private for profit sector and one in the private not for profit sector . in cape verde 26 individuals ( four doctors , nine nurses , one pharmacist and 12 orderlies ) answered the questionnaire . there were 5 females and 21 males , ranging in age from 25 to 54 years ( median 35 years ) . all worked in the public sector : two at district level , 17 at hospital level , one at pharmacy level and two at health centre level . only six ( two doctors , nine nurses and nine orderlies ) reported working also in the private for profit sector and 1 doctor in the private not for profit sector . five ( two nurses and three orderlies ) worked in rural areas , the others in town . all respondents in both countries claimed personal knowledge of instances of misappropriation of pharmaceuticals by health personnel for personal financial gain . this took various forms from outright stealing to requesting under - the - counter payments or overcharging . " the porter was searched by the security people at the gate , and they found several medicines in his handbag " , " in nyassa a health centre worker removed antibiotics from his workplace to send them across the border to malawi , in exchange for ... shoes , tv sets , video sets , hi - fi sets , etc . he opened his own shop in town on account of his dishonesty " , " the nurse replaced prescribed post - op pethidine with diclofenac or aspergic . he sold the pethidine to drug addicts afterwards " , " my mother went to a public hospital and the doctor prescribed injectables . at the hospital pharmacy , the pharmacy technician told us that the injections were out of stock and referred us to one nurse in one of the wards . we went there and the nurse sold us the injections for 300.000,00 meticais ( approximately us$ 20 ) " . at times respondents would report ' less objectionable ' practices : " the pharmacy technician overcharged the patients for drugs but kept only the difference for himself " . twenty - one respondents in mozambique and seven in cape verde included promotion of undesirable prescribing patterns by representatives of the pharmaceutical industry among the ways pharmaceuticals are used to generate personal income . according to the respondents , the range of unorthodox practices related to the handling of drugs in health facilities is widest among doctors and particularly those active in private practice ( table 1 ) . respondents mainly pointed at doctors and pharmacists , less at nurses except to say that nurses in private practice or orderlies in private pharmacies overprescribe . frequency with which each type of misuse was reported by 27 mozambican ( m ) and 26 cape verdean ( cv ) respondents . in both countries respondents said they had the impression that the phenomenon was generalised : " everyone knows that stealing of medicines and other medical supplies by health personnel is a common practice in our society " , " i think this is happening all the time in our country ... let me tell you , this type of things has been going on for a long time and it is not going to end all that easily " , " anyone going to the market or strolling down any street in town , can meet people selling medicines , usually not health workers , selling medicines . other stuff such as iud and other drugs find their way straight to private consulting rooms " . particularly in mozambique actual theft of drugs is said to be highly organised : " no one ever steals alone . this involves cooking the books to eliminate all evidence of stolen goods , bribing those in charge of supervision , reliable sales ' outlets ( at home , private pharmacies , in private clinics , in dumbanengs [ informal market outlets ] etc . ) . recently there is even talk of contracts between private clinics and public sector health workers to ensure the steady supply certain types of medicines . these circuits may even be under the control of people not associated with the health care sector , but rather with the import / export business " . nineteen of the mozambican respondents ( eight doctors , one nurse and 10 orderlies ) and thirteen in cape verde ( eight doctors , one nurse and 10 orderlies ) had the impression that such incidents were on the increase . most if not all the stories typically concerned public sector facilities , particularly hospitals : " ... these stories happen all the time in the hospitals , but no one talks about them because they are afraid . " a number of respondents were of the opinion that such practices , including theft , can at times be justified . the public sector does not function well and health professionals have to do whatever they can to help patients : " people who steal medicines do not do it to get rich , but rather as a favour " , " ... to help a sick neighbour " , " i can tell you what happened between an in - law of mine and a health auxiliary . my in - law went to a consultation , where he was prescribed brufen and amoxicillin . therefore this health auxiliary told him that he had a small supply at home and could sell him the necessary medicines " , " they do these things because there are patients that do not go to the hospital because of the long waiting times . they will rather go to these people to avoid wasting their time in the hospitals " . most respondents however , implicitly or explicitly condemned such practices , while still attempting to explain and/or justify them in various ways . an obvious explanation is that of " serious lack of motivation " and insufficient salaries : " economic reasons , and low salaries ... those are the reasons ... it is a means of surviving " ... " the true reasons for these practices are the low salaries paid by the government " . a considerable number of respondents interpreted these phenomena in moral terms , as a question of individual ethical characteristics : " i do think that it is mostly because of dishonesty and irresponsibility , " ... vanity , is often what drives people " , " i do think that it depends of the conscience of each one , even when the salary is very low , those who carry out their work honestly do not do these things " , " in my opinion it just reflects the bad attributes of people " , " lack of morals and of civic sense " . these are then related to a general breakdown of norms in society that is associated with liberalisation ( " the increase over the past five past years ... is due to the liberalisation of the market " , " nowadays medicine is seen as a commercial activity " ) where it is possible to exploit " the ignorance of our people " . most comments , however , did not focus on the behaviour of individuals as much as on service organisation and its management that provides the opportunity for misappropriation and misuse : " lack of effective control " , " low salary , lack of incentives , lack of control systems , lack of inspections , absence of systems to receive and keep the daily income resulting from drug sales " , " the lack of organisation of the pharmacies makes it easy to steal " , " defective organisation and lack of knowledge from the systems ' managers makes it easy for the prevaricators " . combined with " lack of penalties , disciplinary or legal " this results in " people doing whatever they want because they know they will not be punished " . all this takes place in an environment of laissez - faire : " everyone is trying to go on with their lives , therefore no one worries about disciplinary measures or about punishing the prevaricators " , with lack of career structure , workload and working conditions as attenuating circumstances . they are said to weaken the public sector health sector and damage people 's health : " the impact is always negative , not only for those who carry it out but also for the whole of society . this negative impact is visible in the weakening of the health structure , results in material damage and creates risks for the public 's health " . " the impact is negative , because many times we see a patient who tells us that he took some medicine bought at sucupira market . therefore , these practices create stock breaks and also they results in lack of response to medicines later prescribed by the doctor as a result of inappropriate doses of the antibiotics taken without a medical prescription " . respondents also saw consequences that went beyond the immediate health or financial implications , but touch on the symbolic and the political . " cape verde is a country where nobody pays for medicines because they have a social meaning . when these are stolen we are damaging the health units " . " ... systematic stealing of medicines creates a deficit in the public sector that results in serious damage to its already tarnished reputation " , and to the reputation of " the different professional groups : paramedics , nurses and porters ) . " patients may be believe there are thieves in the hospital " , and " the interaction patient - health personnel becomes just a commercial interaction " . besides overall legal measures -"first of all to define what is or it is not legal " , " to enforce the law in the health system of mozambique " , " to create conditions to make it possible to denounce these situations"- the solutions proposed fall into four categories . the first is to improve the management of the health units : " implement a rigorous system of control and management " , " train the managers " , " improve the management of the system " , " increase surveillance , supervision ... " , " improve management of the pharmaceutical sector , to improve its supervision in order to expedite the detection of problems " , " more controls not only in the public but also over the private sector ... " , " train people specifically on pharmaceutical management " , " i do think there should exist a stricter control of the sector right on from the central stores to the most peripheral sales outlets and hospital wards " , " in our hospital in praia we are computerising the pharmacy and standardising the package system ... " . the second group of measures proposed is to focus on the conditions of employment and working conditions of the human resources : " give appropriate incentives to the personnel ... and improve their working conditions " , " harmonise salaries with the cost of living " , " increase the number of trained pharmacists " , " study measures to reinvest the funds generated by the local pharmacy locally " . third , respondents feel one should inform the population " ... if people were better informed by the government , through the media , may be then people would become aware that these practices are damaging those carrying them out as well as innocent bystanders . " " inform the population not to use medicines unless prescribed by people with the competence to prescribe . " finally respondents feel one should appeal to personal and professional values : " meet with the staff to discuss strategies to reduce these practices " , " do it by giving the example of impeccable honesty " , " each person must take conscience of the wrongness in these practices " . basically the bulk of the claims are for better pay , more control and better management , but surprisingly almost nobody talks about " the application of sanctions , real sanctions " , or suggest alternative options , other than those currently in place , to better control prescribers . all respondents in both countries claimed personal knowledge of instances of misappropriation of pharmaceuticals by health personnel for personal financial gain . this took various forms from outright stealing to requesting under - the - counter payments or overcharging . " the porter was searched by the security people at the gate , and they found several medicines in his handbag " , " in nyassa a health centre worker removed antibiotics from his workplace to send them across the border to malawi , in exchange for ... shoes , tv sets , video sets , hi - fi sets , etc . he opened his own shop in town on account of his dishonesty " , " the nurse replaced prescribed post - op pethidine with diclofenac or aspergic . he sold the pethidine to drug addicts afterwards " , " my mother went to a public hospital and the doctor prescribed injectables . at the hospital pharmacy , the pharmacy technician told us that the injections were out of stock and referred us to one nurse in one of the wards . we went there and the nurse sold us the injections for 300.000,00 meticais ( approximately us$ 20 ) " . at times respondents would report ' less objectionable ' practices : " the pharmacy technician overcharged the patients for drugs but kept only the difference for himself " . twenty - one respondents in mozambique and seven in cape verde included promotion of undesirable prescribing patterns by representatives of the pharmaceutical industry among the ways pharmaceuticals are used to generate personal income . according to the respondents , the range of unorthodox practices related to the handling of drugs in health facilities is widest among doctors and particularly those active in private practice ( table 1 ) . respondents mainly pointed at doctors and pharmacists , less at nurses except to say that nurses in private practice or orderlies in private pharmacies overprescribe . frequency with which each type of misuse was reported by 27 mozambican ( m ) and 26 cape verdean ( cv ) respondents . in both countries respondents said they had the impression that the phenomenon was generalised : " everyone knows that stealing of medicines and other medical supplies by health personnel is a common practice in our society " , " i think this is happening all the time in our country ... let me tell you , this type of things has been going on for a long time and it is not going to end all that easily " , " anyone going to the market or strolling down any street in town , can meet people selling medicines , usually not health workers , selling medicines . other stuff such as iud and other drugs find their way straight to private consulting rooms " . particularly in mozambique actual theft of drugs is said to be highly organised : " no one ever steals alone . this involves cooking the books to eliminate all evidence of stolen goods , bribing those in charge of supervision , reliable sales ' outlets ( at home , private pharmacies , in private clinics , in dumbanengs [ informal market outlets ] etc . ) . recently there is even talk of contracts between private clinics and public sector health workers to ensure the steady supply certain types of medicines . these circuits may even be under the control of people not associated with the health care sector , but rather with the import / export business " . nineteen of the mozambican respondents ( eight doctors , one nurse and 10 orderlies ) and thirteen in cape verde ( eight doctors , one nurse and 10 orderlies ) had the impression that such incidents were on the increase . most if not all the stories typically concerned public sector facilities , particularly hospitals : " ... these stories happen all the time in the hospitals , but no one talks about them because they are afraid . " a number of respondents were of the opinion that such practices , including theft , can at times be justified . the public sector does not function well and health professionals have to do whatever they can to help patients : " people who steal medicines do not do it to get rich , but rather as a favour " , " ... to help a sick neighbour " , " i can tell you what happened between an in - law of mine and a health auxiliary . my in - law went to a consultation , where he was prescribed brufen and amoxicillin . therefore this health auxiliary told him that he had a small supply at home and could sell him the necessary medicines " , " they do these things because there are patients that do not go to the hospital because of the long waiting times . they will rather go to these people to avoid wasting their time in the hospitals " . most respondents however , implicitly or explicitly condemned such practices , while still attempting to explain and/or justify them in various ways . an obvious explanation is that of " serious lack of motivation " and insufficient salaries : " economic reasons , and low salaries ... those are the reasons ... it is a means of surviving " ... " the true reasons for these practices are the low salaries paid by the government " . a considerable number of respondents interpreted these phenomena in moral terms , as a question of individual ethical characteristics : " i do think that it is mostly because of dishonesty and irresponsibility , " ... vanity , is often what drives people " , " i do think that it depends of the conscience of each one , even when the salary is very low , those who carry out their work honestly do not do these things " , " in my opinion it just reflects the bad attributes of people " , " lack of morals and of civic sense " . these are then related to a general breakdown of norms in society that is associated with liberalisation ( " the increase over the past five past years ... is due to the liberalisation of the market " , " nowadays medicine is seen as a commercial activity " ) where it is possible to exploit " the ignorance of our people " . most comments , however , did not focus on the behaviour of individuals as much as on service organisation and its management that provides the opportunity for misappropriation and misuse : " lack of effective control " , " low salary , lack of incentives , lack of control systems , lack of inspections , absence of systems to receive and keep the daily income resulting from drug sales " , " the lack of organisation of the pharmacies makes it easy to steal " , " defective organisation and lack of knowledge from the systems ' managers makes it easy for the prevaricators " . combined with " lack of penalties , disciplinary or legal " this results in " people doing whatever they want because they know they will not be punished " . all this takes place in an environment of laissez - faire : " everyone is trying to go on with their lives , therefore no one worries about disciplinary measures or about punishing the prevaricators " , with lack of career structure , workload and working conditions as attenuating circumstances . they are said to weaken the public sector health sector and damage people 's health : " the impact is always negative , not only for those who carry it out but also for the whole of society . this negative impact is visible in the weakening of the health structure , results in material damage and creates risks for the public 's health " . " the impact is negative , because many times we see a patient who tells us that he took some medicine bought at sucupira market therefore , these practices create stock breaks and also they results in lack of response to medicines later prescribed by the doctor as a result of inappropriate doses of the antibiotics taken without a medical prescription " . respondents also saw consequences that went beyond the immediate health or financial implications , but touch on the symbolic and the political . " cape verde is a country where nobody pays for medicines because they have a social meaning . when these are stolen we are damaging the health units " . " ... systematic stealing of medicines creates a deficit in the public sector that results in serious damage to its already tarnished reputation " , and to the reputation of " the different professional groups : paramedics , nurses and porters ) . " patients may be believe there are thieves in the hospital " , and " the interaction patient - health personnel becomes just a commercial interaction " . besides overall legal measures -"first of all to define what is or it is not legal " , " to enforce the law in the health system of mozambique " , " to create conditions to make it possible to denounce these situations"- the solutions proposed fall into four categories . the first is to improve the management of the health units : " implement a rigorous system of control and management " , " train the managers " , " improve the management of the system " , " increase surveillance , supervision ... " , " improve management of the pharmaceutical sector , to improve its supervision in order to expedite the detection of problems " , " more controls not only in the public but also over the private sector ... " , " train people specifically on pharmaceutical management " , " i do think there should exist a stricter control of the sector right on from the central stores to the most peripheral sales outlets and hospital wards " , " in our hospital in praia we are computerising the pharmacy and standardising the package system ... " . the second group of measures proposed is to focus on the conditions of employment and working conditions of the human resources : " give appropriate incentives to the personnel ... and improve their working conditions " , " harmonise salaries with the cost of living " , " increase the number of trained pharmacists " , " study measures to reinvest the funds generated by the local pharmacy locally " . third , respondents feel one should inform the population " ... if people were better informed by the government , through the media , may be then people would become aware that these practices are damaging those carrying them out as well as innocent bystanders . " " inform the population not to use medicines unless prescribed by people with the competence to prescribe . " finally respondents feel one should appeal to personal and professional values : " meet with the staff to discuss strategies to reduce these practices " , " do it by giving the example of impeccable honesty " , " each person must take conscience of the wrongness in these practices " . basically the bulk of the claims are for better pay , more control and better management , but surprisingly almost nobody talks about " the application of sanctions , real sanctions " , or suggest alternative options , other than those currently in place , to better control prescribers . the social and professional culture within a profession has a major impact on its practice [ 10 - 12 ] . in the health field pressure from local practice styles is known to be particularly relevant in situations where professionals are uncertain about appropriate treatment norms it is likely that peer pressure works in the opposite direction as well , and the shifting of norms with regard to taking advantage of privileged access to pharmaceuticals illustrates this . individual coping strategies have , in some countries , become so prevalent that it is widely assumed that the very notion of civil services ethos has completely and possibly irreversibly disappeared . health workers apparently live a conflict between their self - image as honest civil servant wanting to do a decent job , and the brute facts of life that make them betray that aspiration . it suggests that , even in the difficult circumstances observed in many countries , behaviours that depart from traditional civil servant deontology have not been interiorised as a norm . first , it requires a clear separation of personal coping strategies from organised crime and institutional misconduct . second , it requires an understanding of the justification for and rationalisation of this type of behaviour , but seen in the light of the ambiguities revealed by the respondents to this survey . these ambiguities suggest that interventions to mitigate the erosion of proper conduct would be welcome . it is quite likely that a combination of dealing with the root causes and of a sequential combination of interventions ( regulatory enforcement , education and peer influence ) could be effective . the latter has been shown to work to control undesirable prescription behaviour by private pharmacies in hanoi , viet nam [ 16 - 19 ] . professional behaviour can be changed through " peer influence meetings " , particularly if the change is seen as building up public reputation and status . this points to the importance , in the absence of effective regulatory mechanisms , of the role of professional societies in ensuring peer - pressure mechanisms to reduce undesirable coping strategies , and illustrates that income topping up is not the principal or only driving force behind the coping strategies health professionals develop . in order to move forward , we need a better understanding of the mechanisms underlying these set of interventions , and the context in which they were or might be successful . none of them alone can be effective indeed and success stories of some categories of interventions relate probably more to the context in which they could emerge than merely their content . as pawson puts it : " what works for whom in what circumstances " is indeed a key question . a significant problem with individual coping strategies is the difficulty to assign individual responsibilities in situations where these are endemic . in these circumstances it might be relevant to introduce legislation that makes the head of an organization or department legally responsible for the actions of that body . this would be a way of increasing peer pressure and accountability and of signalling the boundaries of what is tolerable . this could then be combined with peer pressure groups to reduce undesirable coping strategies by supporting members to maintain their personal stance as well as by informing the public of their rights . making public the membership of such a group could be a way of identifying the non - members , an indirect way of increasing pressure . this study confirms that health workers in mozambique and cape verde do take advantage of their privileged access to pharmaceuticals . the respondents indicate that the phenomenon is widespread . that doctors are best placed to take advantage in a variety of ways is not really surprising . at a time of pressure for the public sector to create partnerships with the private sector , it is ironical to see to what extent " informal partnerships " already exist . apart from the economic loss to the state and to the patients , the long - term effects for staff morale and morality are far from negligible , as is , perhaps even more importantly , the effect on the public 's trust in their health system and its personnel . all the authors were involved in planning the research protocol , commenting on the results and reviewing the different drafts of the paper . wvland pfshared the responsibility for overall co - ordination of the work and writing the drafts that were circulated for comments . the work for this paper was supported by the inco - dev programme of the european commission ( contract number ic18ct980347 ) .

backgroundcoping strategies have , in some countries , become so prevalent that it has been widely assumed that the very notion of civil services ethos has completely and possibly irreversibly disappeared . this paper describes the importance and the nature of pilfering of drugs by health staff in mozambique and cape verde , as perceived by health professionals from these countries . their opinions provide pointers as to how to tackle these problems.methodsthis study is based on a self - administered questionnaire addressed to a convenience sample of health workers in mozambique and in cape verde.resultsthe study confirms that misuse of access to pharmaceuticals has become a key element in the coping strategies health personnel develop to deal with difficult living conditions . different professional groups ( mis)use their privileged access in different ways , but doctors diversify most . the study identifies the reasons given for misusing access to drugs , shows how the problem is perceived by the health workers , and discusses the implications for finding solutions to the problem.our findings reflect , from the health workers themselves , a conflict between their self image of what it means to be an honest civil servant who wants to do a decent job , and the brute facts of life that make them betray that image . the manifest unease that this provokes is an important observation as such.conclusionour findings suggest that , even in the difficult circumstances observed in many countries , behaviours that depart from traditional civil servant deontology have not been interiorised as a norm . this ambiguity indicates that interventions to mitigate the erosion of proper conduct would be welcome . the time to act is now , before small - scale individual coping grows into large - scale , well - organized crime .

Background Methods Population Results How access to pharmaceuticals boosts health workers' income Explaining and justifying The consequences What measures are necessary to minimise the negative impact of these practices? Discussion Conclusions Competing interests Authors' contributions Acknowledgements

misuse by health personnel of their privileged access to pharmaceuticals is a sensitive issue in the current context of scarce resources , promotion of generics , hiv epidemic and growing demand for health care . misappropriation is a widespread practice rarely explicitly acknowledged or documented , even in studies that have looked into the coping strategies of health professionals [ 1 - 4 ] . for example , in the average ugandan health facility drug leakage involving facility health workers as well as district health teams and health unit management committees is as high as 78% , with resale of drugs representing the greatest single source of income for health workers in most units . in many other developing countries all this represents a financial loss to the health care system , and affects health outcomes negatively . it also contributes to the growing sense of mistrust and disrespect for the health professions and their institutions . by its very nature misuse of access to pharmaceuticals an alternative is to ask health workers about what occurs in services without inquiring into their own behaviour . although basically a record of hearsay , such information can shed light on the type of misuse that occurs , and , more importantly , how this is perceived and valued by the personnel . it describes their perceptions of the importance , the nature and the justification of pilfering of drugs in these countries . the study used a mixed open - ended and closed questions self - administered questionnaire addressed to a convenience sample of individuals working at various levels in the health sector . all worked in the public sector : two in the ministry of health , two in the provincial health administration , one at district level , eight in hospitals and four in health centres . only 13 ( six doctors , three nurses and four orderlies ) said they also worked in the private for profit sector and one in the private not for profit sector . in cape verde 26 individuals ( four doctors , nine nurses , one pharmacist and 12 orderlies ) answered the questionnaire . all worked in the public sector : two at district level , 17 at hospital level , one at pharmacy level and two at health centre level . only six ( two doctors , nine nurses and nine orderlies ) reported working also in the private for profit sector and 1 doctor in the private not for profit sector . in mozambique 27 individuals ( 11 doctors , three nurses , one pharmacist and 12 orderlies ) answered the questionnaire . all worked in the public sector : two in the ministry of health , two in the provincial health administration , one at district level , eight in hospitals and four in health centres . only 13 ( six doctors , three nurses and four orderlies ) said they also worked in the private for profit sector and one in the private not for profit sector . in cape verde 26 individuals ( four doctors , nine nurses , one pharmacist and 12 orderlies ) answered the questionnaire . only six ( two doctors , nine nurses and nine orderlies ) reported working also in the private for profit sector and 1 doctor in the private not for profit sector . all respondents in both countries claimed personal knowledge of instances of misappropriation of pharmaceuticals by health personnel for personal financial gain . the porter was searched by the security people at the gate , and they found several medicines in his handbag " , " in nyassa a health centre worker removed antibiotics from his workplace to send them across the border to malawi , in exchange for ... shoes , tv sets , video sets , hi - fi sets , etc . he sold the pethidine to drug addicts afterwards " , " my mother went to a public hospital and the doctor prescribed injectables . we went there and the nurse sold us the injections for 300.000,00 meticais ( approximately us$ 20 ) " . twenty - one respondents in mozambique and seven in cape verde included promotion of undesirable prescribing patterns by representatives of the pharmaceutical industry among the ways pharmaceuticals are used to generate personal income . according to the respondents , the range of unorthodox practices related to the handling of drugs in health facilities is widest among doctors and particularly those active in private practice ( table 1 ) . in both countries respondents said they had the impression that the phenomenon was generalised : " everyone knows that stealing of medicines and other medical supplies by health personnel is a common practice in our society " , " i think this is happening all the time in our country ... let me tell you , this type of things has been going on for a long time and it is not going to end all that easily " , " anyone going to the market or strolling down any street in town , can meet people selling medicines , usually not health workers , selling medicines . particularly in mozambique actual theft of drugs is said to be highly organised : " no one ever steals alone . this involves cooking the books to eliminate all evidence of stolen goods , bribing those in charge of supervision , reliable sales ' outlets ( at home , private pharmacies , in private clinics , in dumbanengs [ informal market outlets ] etc . ) recently there is even talk of contracts between private clinics and public sector health workers to ensure the steady supply certain types of medicines . these circuits may even be under the control of people not associated with the health care sector , but rather with the import / export business " . nineteen of the mozambican respondents ( eight doctors , one nurse and 10 orderlies ) and thirteen in cape verde ( eight doctors , one nurse and 10 orderlies ) had the impression that such incidents were on the increase . most if not all the stories typically concerned public sector facilities , particularly hospitals : " ... these stories happen all the time in the hospitals , but no one talks about them because they are afraid . " the public sector does not function well and health professionals have to do whatever they can to help patients : " people who steal medicines do not do it to get rich , but rather as a favour " , " ... to help a sick neighbour " , " i can tell you what happened between an in - law of mine and a health auxiliary . they will rather go to these people to avoid wasting their time in the hospitals " . an obvious explanation is that of " serious lack of motivation " and insufficient salaries : " economic reasons , and low salaries ... those are the reasons ... it is a means of surviving " ... " the true reasons for these practices are the low salaries paid by the government " . a considerable number of respondents interpreted these phenomena in moral terms , as a question of individual ethical characteristics : " i do think that it is mostly because of dishonesty and irresponsibility , " ... vanity , is often what drives people " , " i do think that it depends of the conscience of each one , even when the salary is very low , those who carry out their work honestly do not do these things " , " in my opinion it just reflects the bad attributes of people " , " lack of morals and of civic sense " . these are then related to a general breakdown of norms in society that is associated with liberalisation ( " the increase over the past five past years ... is due to the liberalisation of the market " , " nowadays medicine is seen as a commercial activity " ) where it is possible to exploit " the ignorance of our people " . most comments , however , did not focus on the behaviour of individuals as much as on service organisation and its management that provides the opportunity for misappropriation and misuse : " lack of effective control " , " low salary , lack of incentives , lack of control systems , lack of inspections , absence of systems to receive and keep the daily income resulting from drug sales " , " the lack of organisation of the pharmacies makes it easy to steal " , " defective organisation and lack of knowledge from the systems ' managers makes it easy for the prevaricators " . this negative impact is visible in the weakening of the health structure , results in material damage and creates risks for the public 's health " . " therefore , these practices create stock breaks and also they results in lack of response to medicines later prescribed by the doctor as a result of inappropriate doses of the antibiotics taken without a medical prescription " . respondents also saw consequences that went beyond the immediate health or financial implications , but touch on the symbolic and the political . " cape verde is a country where nobody pays for medicines because they have a social meaning . when these are stolen we are damaging the health units " . " ... systematic stealing of medicines creates a deficit in the public sector that results in serious damage to its already tarnished reputation " , and to the reputation of " the different professional groups : paramedics , nurses and porters ) . " patients may be believe there are thieves in the hospital " , and " the interaction patient - health personnel becomes just a commercial interaction " . besides overall legal measures -"first of all to define what is or it is not legal " , " to enforce the law in the health system of mozambique " , " to create conditions to make it possible to denounce these situations"- the solutions proposed fall into four categories . the first is to improve the management of the health units : " implement a rigorous system of control and management " , " train the managers " , " improve the management of the system " , " increase surveillance , supervision ... " , " improve management of the pharmaceutical sector , to improve its supervision in order to expedite the detection of problems " , " more controls not only in the public but also over the private sector ... " , " train people specifically on pharmaceutical management " , " i do think there should exist a stricter control of the sector right on from the central stores to the most peripheral sales outlets and hospital wards " , " in our hospital in praia we are computerising the pharmacy and standardising the package system ... " . the second group of measures proposed is to focus on the conditions of employment and working conditions of the human resources : " give appropriate incentives to the personnel ... and improve their working conditions " , " harmonise salaries with the cost of living " , " increase the number of trained pharmacists " , " study measures to reinvest the funds generated by the local pharmacy locally " . basically the bulk of the claims are for better pay , more control and better management , but surprisingly almost nobody talks about " the application of sanctions , real sanctions " , or suggest alternative options , other than those currently in place , to better control prescribers . all respondents in both countries claimed personal knowledge of instances of misappropriation of pharmaceuticals by health personnel for personal financial gain . the porter was searched by the security people at the gate , and they found several medicines in his handbag " , " in nyassa a health centre worker removed antibiotics from his workplace to send them across the border to malawi , in exchange for ... shoes , tv sets , video sets , hi - fi sets , etc . he sold the pethidine to drug addicts afterwards " , " my mother went to a public hospital and the doctor prescribed injectables . at the hospital pharmacy , the pharmacy technician told us that the injections were out of stock and referred us to one nurse in one of the wards . twenty - one respondents in mozambique and seven in cape verde included promotion of undesirable prescribing patterns by representatives of the pharmaceutical industry among the ways pharmaceuticals are used to generate personal income . according to the respondents , the range of unorthodox practices related to the handling of drugs in health facilities is widest among doctors and particularly those active in private practice ( table 1 ) . in both countries respondents said they had the impression that the phenomenon was generalised : " everyone knows that stealing of medicines and other medical supplies by health personnel is a common practice in our society " , " i think this is happening all the time in our country ... let me tell you , this type of things has been going on for a long time and it is not going to end all that easily " , " anyone going to the market or strolling down any street in town , can meet people selling medicines , usually not health workers , selling medicines . particularly in mozambique actual theft of drugs is said to be highly organised : " no one ever steals alone . this involves cooking the books to eliminate all evidence of stolen goods , bribing those in charge of supervision , reliable sales ' outlets ( at home , private pharmacies , in private clinics , in dumbanengs [ informal market outlets ] etc . ) recently there is even talk of contracts between private clinics and public sector health workers to ensure the steady supply certain types of medicines . these circuits may even be under the control of people not associated with the health care sector , but rather with the import / export business " . nineteen of the mozambican respondents ( eight doctors , one nurse and 10 orderlies ) and thirteen in cape verde ( eight doctors , one nurse and 10 orderlies ) had the impression that such incidents were on the increase . most if not all the stories typically concerned public sector facilities , particularly hospitals : " ... these stories happen all the time in the hospitals , but no one talks about them because they are afraid . " the public sector does not function well and health professionals have to do whatever they can to help patients : " people who steal medicines do not do it to get rich , but rather as a favour " , " ... to help a sick neighbour " , " i can tell you what happened between an in - law of mine and a health auxiliary . my in - law went to a consultation , where he was prescribed brufen and amoxicillin . therefore this health auxiliary told him that he had a small supply at home and could sell him the necessary medicines " , " they do these things because there are patients that do not go to the hospital because of the long waiting times . they will rather go to these people to avoid wasting their time in the hospitals " . an obvious explanation is that of " serious lack of motivation " and insufficient salaries : " economic reasons , and low salaries ... those are the reasons ... it is a means of surviving " ... " the true reasons for these practices are the low salaries paid by the government " . a considerable number of respondents interpreted these phenomena in moral terms , as a question of individual ethical characteristics : " i do think that it is mostly because of dishonesty and irresponsibility , " ... vanity , is often what drives people " , " i do think that it depends of the conscience of each one , even when the salary is very low , those who carry out their work honestly do not do these things " , " in my opinion it just reflects the bad attributes of people " , " lack of morals and of civic sense " . these are then related to a general breakdown of norms in society that is associated with liberalisation ( " the increase over the past five past years ... is due to the liberalisation of the market " , " nowadays medicine is seen as a commercial activity " ) where it is possible to exploit " the ignorance of our people " . this negative impact is visible in the weakening of the health structure , results in material damage and creates risks for the public 's health " . " the impact is negative , because many times we see a patient who tells us that he took some medicine bought at sucupira market therefore , these practices create stock breaks and also they results in lack of response to medicines later prescribed by the doctor as a result of inappropriate doses of the antibiotics taken without a medical prescription " . respondents also saw consequences that went beyond the immediate health or financial implications , but touch on the symbolic and the political . " cape verde is a country where nobody pays for medicines because they have a social meaning . when these are stolen we are damaging the health units " . " ... systematic stealing of medicines creates a deficit in the public sector that results in serious damage to its already tarnished reputation " , and to the reputation of " the different professional groups : paramedics , nurses and porters ) . " patients may be believe there are thieves in the hospital " , and " the interaction patient - health personnel becomes just a commercial interaction " . besides overall legal measures -"first of all to define what is or it is not legal " , " to enforce the law in the health system of mozambique " , " to create conditions to make it possible to denounce these situations"- the solutions proposed fall into four categories . the first is to improve the management of the health units : " implement a rigorous system of control and management " , " train the managers " , " improve the management of the system " , " increase surveillance , supervision ... " , " improve management of the pharmaceutical sector , to improve its supervision in order to expedite the detection of problems " , " more controls not only in the public but also over the private sector ... " , " train people specifically on pharmaceutical management " , " i do think there should exist a stricter control of the sector right on from the central stores to the most peripheral sales outlets and hospital wards " , " in our hospital in praia we are computerising the pharmacy and standardising the package system ... " . the second group of measures proposed is to focus on the conditions of employment and working conditions of the human resources : " give appropriate incentives to the personnel ... and improve their working conditions " , " harmonise salaries with the cost of living " , " increase the number of trained pharmacists " , " study measures to reinvest the funds generated by the local pharmacy locally " . basically the bulk of the claims are for better pay , more control and better management , but surprisingly almost nobody talks about " the application of sanctions , real sanctions " , or suggest alternative options , other than those currently in place , to better control prescribers . in the health field pressure from local practice styles is known to be particularly relevant in situations where professionals are uncertain about appropriate treatment norms it is likely that peer pressure works in the opposite direction as well , and the shifting of norms with regard to taking advantage of privileged access to pharmaceuticals illustrates this . individual coping strategies have , in some countries , become so prevalent that it is widely assumed that the very notion of civil services ethos has completely and possibly irreversibly disappeared . health workers apparently live a conflict between their self - image as honest civil servant wanting to do a decent job , and the brute facts of life that make them betray that aspiration . it suggests that , even in the difficult circumstances observed in many countries , behaviours that depart from traditional civil servant deontology have not been interiorised as a norm . second , it requires an understanding of the justification for and rationalisation of this type of behaviour , but seen in the light of the ambiguities revealed by the respondents to this survey . these ambiguities suggest that interventions to mitigate the erosion of proper conduct would be welcome . the latter has been shown to work to control undesirable prescription behaviour by private pharmacies in hanoi , viet nam [ 16 - 19 ] . this points to the importance , in the absence of effective regulatory mechanisms , of the role of professional societies in ensuring peer - pressure mechanisms to reduce undesirable coping strategies , and illustrates that income topping up is not the principal or only driving force behind the coping strategies health professionals develop . in order to move forward , we need a better understanding of the mechanisms underlying these set of interventions , and the context in which they were or might be successful . a significant problem with individual coping strategies is the difficulty to assign individual responsibilities in situations where these are endemic . this would be a way of increasing peer pressure and accountability and of signalling the boundaries of what is tolerable . this could then be combined with peer pressure groups to reduce undesirable coping strategies by supporting members to maintain their personal stance as well as by informing the public of their rights . this study confirms that health workers in mozambique and cape verde do take advantage of their privileged access to pharmaceuticals . apart from the economic loss to the state and to the patients , the long - term effects for staff morale and morality are far from negligible , as is , perhaps even more importantly , the effect on the public 's trust in their health system and its personnel . the work for this paper was supported by the inco - dev programme of the european commission ( contract number ic18ct980347 ) .

the substitution of ambulatory care for inpatient hospital care has long been advocated as a safe and efficacious method of cost containment . procedures performed in ambulatory surgical units generally require fewer resources , in terms of professionals and facilities , than the same surgery performed on an inpatient basis . proponents of ambulatory surgery argue that quality is not compromised and patients are not exposed to the potential iatrogenic hazards of hospitalization . reimbursement and payment policies have had a considerable influence on the shift in surgery across alternative sites of care . private insurers who cover both inpatient and outpatient services can and do encourage the use of ambulatory surgery by offering special incentives to make it more attractive relative to inpatient surgery ( pauly and burns , 1984 ) . in addition , some companies have developed lists of operations that are reimbursed only on an outpatient basis unless the patient has complications ( nathanson , 1986 ) . under prospective payment systems based on diagnosis - related groups ( drg 's ) , hospitals are encouraged to operate more efficiently and are paid a fixed rate per case for inpatient services . hospitals may keep any excess of payments over costs , but must also absorb the loss if costs exceed payments . because outpatient services , including ambulatory surgery , are covered by a different method of reimbursement , financial considerations play an important role in the decision of where to treat patients . what is financially beneficial or disadvantageous to the hospital depends on the real resource cost of treating the patient in a given setting and the amount of the payment for services provided in that setting . in the medicare program , inpatient payment levels are reflected in the drg weight ; the higher the weight assigned a drg , the higher the payment for patients in that category . on the one hand , when the payment rate for a surgical drg is perceived to be too low to cover the costs of inpatient services , there is the incentive to do the procedure in an outpatient department . for example , under the medicare program , outpatient payments are still based on incurred cost ( carter and ginsberg , 1985 ) . in the case of lens extraction , the amount medicare has paid some hospitals for performing the surgery on an outpatient basis sometimes exceeds the inpatient drg rate . such procedures are prime candidates for outpatient shift under the prospective payment system ( pps ) . there is also the concern that patients who could be treated on an outpatient basis would be admitted as inpatients to generate short stays that might be more profitable under pps this incentive may be strong for surgical drg 's , in which procedures that can be performed in an ambulatory setting are often grouped with other procedures that require more followup care and are generally considered more appropriate for the inpatient setting . finally , choice of surgical setting , when based on financial incentives , rather than on patient morbidity considerations , might lead to adverse effects on quality of care ( hammons , brook , and newhouse , 1986 ) . this is a major concern of the prospective payment assessment commission ( propac ) , in light of substantial increases in outpatient utilization by medicare beneficiaries since the introduction of pps in 1983 ( prospective payment assessment commission , 1986 ) . the commission has recommended extending the responsibilities of the professional review organizations ( pro 's ) to include a review of outpatient surgery for selected procedures . rand researchers used internal documents from the health care financing administration ( hcfa ) and a physician consultant to prepare a list of drg 's that have a high potential for outpatient substitution ( carter and ginsberg , 1985 ) . no empirical data have previously been available for comparing this list with actual utilization by site of service . an empirical assessment is particularly difficult in the united states , because large health services data bases , covering residents of states or other well - defined populations ( such as medicare beneficiaries ) , do not combine inpatient and outpatient utilization data . information on individual persons must be merged across files that often have different formats and are maintained by different organizations . comparing utilization across settings for the medicare population may be particularly difficult in the future , because hcfa requires hospitals to use different coding systems to bill for inpatient ( international classification of diseases , 9th revision , clinical modification [ icd-9-cm ] ) and outpatient ( current procedural terminology [ cpt ] ) surgery ( shahoda , 1987 ) . in the universal health insurance system operating in manitoba , canada ( population 1 million ) , hospitals are funded on the basis of global budgets . hospital discharge abstracts are produced for all patients admitted to a hospital , whether for an inpatient stay or for an outpatient procedure . all diagnoses and procedures are coded using the icd-9-cm system . despite the differences in payment systems , there are many similarities between hospitalization patterns in manitoba and in the united states ( roos , 1984 ; roos and ramsey , 1987 ; roos and danzinger , 1986 ; roos and lyttle , 1985 ) . hospital discharge rates are very similar for individuals 65 years of age or over ( 398 discharges per 1,000 population in the united states and 403 per 1,000 in manitoba ) ( lohr , lohr , and brook , 1985 ) . manitoba discharge rates for the population overall are lower than those for the united states ( 141 per 1,000 versus 168 ) , although they are similar to discharge rates of hospitals in the western united states ( 144 per 1,000 ) ( lohr , lohr , and brook , 1985 ) . manitoba urban hospitals also represent reasonably progressive standards in terms of outpatient surgery . although comparable data for u.s . hospitals as a whole are lacking , manitoba hospitals can be compared with those of syracuse , new york , a city having aggressive standards for performing ambulatory surgery ( lagoe and milliren , 1986 ) . manitoba urban hospitals perform more procedures on an outpatient basis than a syracuse hospital with an on - site program , although somewhat fewer than a syracuse hospital with a freestanding ambulatory surgery unit ( roos , 1988 ) . the site of outpatient surgery does vary somewhat , however , across countries . with the exception of a few minor plastic surgical procedures , all outpatient surgery in manitoba there were 592 freestanding outpatient surgery centers open in 1986 , and they performed 14 percent of all outpatient procedures ( henderson , 1987 ) . from one perspective , manitoba is an ideal site for this research . because a drg - based payment system has never been used in manitoba , hospital admission policies and coding practices could not have been influenced by these payment concerns . in this article , we identify those drg categories that appear to have the most potential for inpatient - outpatient substitution . we then compare the manitoba results with the drg categories identified by researchers as having a potential for outpatient shift , using a nonempirical approach . finally , we focus on drg 's with a high potential for inpatient shift . of interest is whether the homogeneity of these groups could be improved with further subdivisions , using short - stay procedures . this latter analysis is meant to identify a new approach for refining the drg system , not to provide definitive guidelines . all 1982 - 84 acute inpatient and outpatient hospitalizations of manitobans 20 years of age or over were available to the project . ( medical and pediatric drg categories were excluded from the analysis , as were admissions classified under the miscellaneous drg category 470 . ) because the drg system in the united states has been applied largely to the medicare population , all analyses presented here focus on the manitoba population 65 years of age or over . discharges from manitoba 's 8 largest hospitals , each with 125 beds or more and occupancy rates of 70 percent or higher , were analyzed . all eight hospitals performed ambulatory surgery as part of a hospital - integrated program . after excluding hospitalizations resulting in deaths , there were 35,630 eligible discharges from these 8 hospitals during the 3-year period 1982 - 84 , with 18.0 percent of the patients undergoing treatment on an outpatient basis . patients treated in hospital walk - in clinics , the emergency room , or ambulatory care facilities were excluded , unless a previously scheduled procedure had taken place ( such as biopsy and laparoscopy ) . both inpatient and outpatient admissions were classified by icd-9-cm codes into surgical drg 's , using standard second - revision drg assignment software available from health systems international ( 1986 ) . these definitions are consistent with those reported in the september 3 , 1985 issue of the federal register . although the drg system requires a principal diagnosis ( the condition established after study to be chiefly responsible for occasioning admission of the patient to the hospital for care ) , the manitoba system recorded the primary diagnosis ( that diagnosis which describes the most significant condition for which the patient was hospitalized ) , as well as up to seven additional diagnoses . the extent to which any discrepancy between these two definitions affects the study 's generalizability to the united states is not known . however , internal studies at the u.s . veterans administration , cited by lloyd and rissing ( 1985 ) , noted only minute differences between primary and principal diagnoses . in a separate study of 1,100 medical records of australian inpatients , roberts , reid , and irwin ( 1985 ) found differences between the 2 diagnoses in 68 records ( 6.4 percent ) . forty - three of the differences in the 584 records with multiple diagnoses resulted in changes in drg assignment . first , hospital admissions are classified by drg and by length of stay ; that is , according to whether a patient was treated on an outpatient basis ( 0-day stays ) , was admitted for a 1 - 3-day stay , or was admitted for a 4-day stay or longer . patients discharged from the hospital after a 1 - 3-day stay were separately classified , because such patients might conceivably be treated on an outpatient basis . second , drg 's with a large proportion ( 35 percent or more ) of patients treated on an outpatient basis were identified as having a high potential for inpatient - outpatient substitution . these were divided into two groups : those with weights less than 1 and those with weights greater than or equal to 1 . drg 's in these two groups were considered candidates for outpatient and inpatient shift , respectively . a weight of 1 was chosen as a bound for reviewing groups for inpatient or outpatient shift , because drg 's with weights greater than 1 have mean costs per case ( and hence payment rates ) that are higher than the average across all drg 's for the medicare population . as one reviewer indicated , the point at which incentives exist to shift depends on an individual hospital 's costs for performing surgery in one site versus the other and the corresponding payment levels . third , patients in drg 's with a high potential for inpatient shift were classified by the procedure responsible for the patient 's drg assignment to determine if there are patterns that suggest methods of improving the system . if certain procedures in these high - weight drg 's are performed almost exclusively on an outpatient basis or during a 1 - 3-day stay , this would suggest that hospitals should be paid differently for such patients than for patients undergoing procedures almost always associated with a longer stay . finally , the results of our empirical assessment of the potential of individual drg 's for inpatient - outpatient substitution are compared with the results obtained by rand researchers carter and ginsberg ( 1985 ) . in table 1 , the proportion of cases in each major diagnostic category ( mdc ) are shown , according to the type of hospital admission ( outpatient , 1 - 3-day stay , or longer stay ) . the proportion of patients treated on an outpatient basis ranged from 66.1 percent to 0 ; patients with diseases and disorders of the skin , subcutaneous tissue , and breast were most likely to be treated on an outpatient basis and patients with burns or mental diseases least likely . in table 2 , the 35 surgical drg 's for which 35 percent or more of the cases in manitoba hospitals were treated on an outpatient basis in 1982 - 84 are identified . these procedures have been listed according to the percent treated on an outpatient basis and the associated drg weight ( the higher the weight , the more the hospital is paid for an inpatient case ) . in addition , procedures are flagged if the rand group identified this as a drg with potential for outpatient substitution . as expected , drg 's on this list , which included patients with complications or patients 70 years of age or over ( drg 's 269 , 259 , 154 , 152 , 157 , 401 ) have fewer patients admitted as outpatients than do the less - complex companion drg 's ( 270 , 260 , 155 , 153 , 158 , 402 ) . drg 's with a large proportion of outpatient admissions generally have low weights ( less than 1 ) . thus , 8 of the 11 drg categories with 60 percent or more cases treated on an outpatient basis ( the top 11 surgical drg 's listed in table 2 ) had weights of .762 or less ( including 5 with weights of .427 or less ) , reflecting relatively low payment rates under pps . hospitals might feel some pressure to treat cases in these drg categories in the outpatient setting ( thus causing outpatient shift ) , particularly if fewer resources are required to treat patients and if payment is on an incurred - cost basis . however , not all drg 's with a large proportion of cases treated on an outpatient basis have low weights and low payment rates . thus , drg 155 ( stomach , esophageal , and duodenal procedures ) has a weight of 1.791 , and drg 269 ( other skin , subcutaneous tissue , and breast operating room procedures ) has a weight of 1.133 , even though 74 percent and 91 percent , respectively , of these cases were treated on an outpatient basis in manitoba ( table 2 ) . overall , a wide variety of drg 's have a significant potential for inpatient - outpatient substitution ; a major incentive for inpatient shift or outpatient shift is the assigned weight . although not shown in table 2 , an additional 15 drg 's had 20 - 34 percent of their cases treated on an outpatient basis . in an attempt to identify cases with potential for inpatient shift , selected drg 's from table 2 these 9 drg 's had weights of 1.0 or greater and had at least 50 cases treated in manitoba urban hospitals in 1982 - 84 . the patients in each drg are classified according to whether they received treatment on an outpatient basis , during a 1 - 3-day stay , or during a longer inpatient admission . a large group of patients classified in each of the drg 's presented in table 3 was treated on an outpatient basis or during a short inpatient stay of 1 to 3 days . perhaps most impressively , fewer than two - thirds ( 53.8 percent ) of the patients classified in the very - high - weight , high - payment drg 154 were admitted for stays of 4 days or longer . this drg is for stomach , esophageal , and duodenal procedures for patient 70 years of age or over or patients with substantive comorbidities or complications . a large proportion of such patients had their procedures performed on an outpatient basis ( 45.3 percent ) . patients within each drg in table 3 were further subdivided , in an attempt to determine if particular procedures in each group lent themselves to treatment on an outpatient or short - stay basis . we also sought to determine if certain procedures were routinely associated with longer stays , potentially more appropriate to a high - weight drg . thus , in table 4 , patients in each of the nine high - weight drg groups are classified according to the specific procedure performed and type of stay . major surgical procedures are listed if 25 or more patients had this procedure and 30 percent or more of the patients were treated on an outpatient or short - stay basis . as can be seen in table 4 , a small group of primary procedures in several of these drg groups accounted for the large majority of outpatient and short - stay cases . depending on how the payment system is designed for some of these procedures , the code could represent either major or minor surgery . as an example , consider 45.15small bowel biopsy , not elsewhere classified . open biopsies belong in the surgical drg , and the closed biopsies belong in a nonsurgical or medical group . as a way of addressing this and other biopsy - related coding limitations , medicare requires that a specific non - operating - room code be substituted for an operating room code when the biopsy is closed this rule was not in effect in manitoba , so the small bowel biopsies performed there on an outpatient basis are most likely closed biopsies . the far - righthand column of table 2 flags those drg 's that the carter - ginsberg project labeled as drg 's with potential for outpatient substitution . of the 35 surgical drg 's for which manitoba hospitals treated 35 percent or more of their cases on an outpatient basis , only 11 were identified by the rand study . the other procedures identified in the rand study were procedures either not performed in manitoba on an outpatient basis ( as is true for prostatectomies ) or performed less frequently than the 35-percent criterion required for inclusion in table 2 . drg 's with a large proportion of outpatient admissions generally have low weights ( less than 1 ) . thus , 8 of the 11 drg categories with 60 percent or more cases treated on an outpatient basis ( the top 11 surgical drg 's listed in table 2 ) had weights of .762 or less ( including 5 with weights of .427 or less ) , reflecting relatively low payment rates under pps . hospitals might feel some pressure to treat cases in these drg categories in the outpatient setting ( thus causing outpatient shift ) , particularly if fewer resources are required to treat patients and if payment is on an incurred - cost basis . however , not all drg 's with a large proportion of cases treated on an outpatient basis have low weights and low payment rates . thus , drg 155 ( stomach , esophageal , and duodenal procedures ) has a weight of 1.791 , and drg 269 ( other skin , subcutaneous tissue , and breast operating room procedures ) has a weight of 1.133 , even though 74 percent and 91 percent , respectively , of these cases were treated on an outpatient basis in manitoba ( table 2 ) . overall , a wide variety of drg 's have a significant potential for inpatient - outpatient substitution ; a major incentive for inpatient shift or outpatient shift is the assigned weight . although not shown in table 2 , an additional 15 drg 's had 20 - 34 percent of their cases treated on an outpatient basis . in an attempt to identify cases with potential for inpatient shift , selected drg 's from table 2 are presented in table 3 . these 9 drg 's had weights of 1.0 or greater and had at least 50 cases treated in manitoba urban hospitals in 1982 - 84 . the patients in each drg are classified according to whether they received treatment on an outpatient basis , during a 1 - 3-day stay , or during a longer inpatient admission . a large group of patients classified in each of the drg 's presented in table 3 was treated on an outpatient basis or during a short inpatient stay of 1 to 3 days . perhaps most impressively , fewer than two - thirds ( 53.8 percent ) of the patients classified in the very - high - weight , high - payment drg 154 were admitted for stays of 4 days or longer . this drg is for stomach , esophageal , and duodenal procedures for patient 70 years of age or over or patients with substantive comorbidities or complications . a large proportion of such patients had their procedures performed on an outpatient basis ( 45.3 percent ) . patients within each drg in table 3 were further subdivided , in an attempt to determine if particular procedures in each group lent themselves to treatment on an outpatient or short - stay basis . we also sought to determine if certain procedures were routinely associated with longer stays , potentially more appropriate to a high - weight drg . thus , in table 4 , patients in each of the nine high - weight drg groups are classified according to the specific procedure performed and type of stay . major surgical procedures are listed if 25 or more patients had this procedure and 30 percent or more of the patients were treated on an outpatient or short - stay basis . as can be seen in table 4 , a small group of primary procedures in several of these drg groups accounted for the large majority of outpatient and short - stay cases . depending on how the payment system is designed for some of these procedures , the code could represent either major or minor surgery . as an example , consider 45.15small bowel biopsy , not elsewhere classified . open biopsies belong in the surgical drg , and the closed biopsies belong in a nonsurgical or medical group . as a way of addressing this and other biopsy - related coding limitations , medicare requires that a specific non - operating - room code be substituted for an operating room code when the biopsy is closed this rule was not in effect in manitoba , so the small bowel biopsies performed there on an outpatient basis are most likely closed biopsies . the far - righthand column of table 2 flags those drg 's that the carter - ginsberg project labeled as drg 's with potential for outpatient substitution . of the 35 surgical drg 's for which manitoba hospitals treated 35 percent or more of their cases on an outpatient basis , only 11 were identified by the rand study . the other procedures identified in the rand study were procedures either not performed in manitoba on an outpatient basis ( as is true for prostatectomies ) or performed less frequently than the 35-percent criterion required for inclusion in table 2 . in this article , we investigate the potential for inpatient and outpatient substitution by examining the proportion of cases in each drg treated on an outpatient or short - stay basis in eight manitoba hospitals . there was a lack of correspondence between the drg 's we identified as having substitution potential and the drg 's identified in the rand study ( carter and ginsberg , 1985 ) . some of the differences in the drg 's identified by the two approaches may be attributed to the methods used to evaluate substitution potential . the rand study appears to have selected those drg 's containing procedures known to be suitable for the ambulatory setting . for example , all of the procedures in drg 's 6 , 232 , 342 , 361 , and 362 are on blue cross and blue shield 's list of reimbursable outpatient surgery ( sieverts , 1984 ) . the drg 's identified in the rand study therefore tended to have short lengths of stay and relatively low weights . these drg 's are candidates for outpatient shift ; from a quality of care perspective , it is important to monitor these drg 's under pro outpatient review programs . however , lacking systematic data on outpatient surgery , the rand analysis also missed many low - weight drg categories in which a large proportion of cases are performed on an outpatient basis . some of these may have been ignored because they account for so few cases ( e.g. , drg 260subtotal mastectomy , and drg 313urethral procedures ) . by comparing the proportion of all hospital admissions with no overnight stay across drg 's , both the groups amenable to outpatient shift and those potentially susceptible to inpatient shift were identified . the data - based method used here flags those high - weight drg 's with patients treated on an ambulatory or short - stay basis as well as those treated during longer inpatient stays . such drg 's tend to have a heterogeneous mix of patients , and close examination of length of stay by primary procedure may help to sort out this heterogeneity . until these drg 's are so refined , hospitals can admit patients for selected procedures that could be performed on an ambulatory basis , thereby increasing their payments with few attendant expenses . if medicare 's prospective payment system is extended to hospital outpatient services , the incentives to shift treatment setting are likely to be radically changed . under the omnibus budget reconciliation act of 1986 ( public law 99 - 509 ) , the secretary of health and human services is required to develop and submit to congress a prospective payment system for ambulatory surgical procedures in hospitals on an outpatient basis by april 1 , 1989 , and a system of prospective payment for outpatient services other than ambulatory surgical procedures by january 1 , 1991 . as an interim method ( which began october 1 , 1987 ) , payments for outpatient hospital facility services associated with surgical procedures are the lesser of the amount the hospital would have received , based on reasonable costs , and a blend amount based on hospital cost ( 75 percent ) and the ambulatory surgical center ( asc ) rate ( 25 percent ) . in fiscal year 1989 , the blend will be based on 50 percent hospital cost and 50 percent asc rate . payments to asc 's are based on prospectively set rates for specific procedures described in the april 21 , 1987 federal register . the approach to refining drg 's proposed in this article complements the efforts of others to improve the drg classification system by developing measures of severity of illness . approaches emphasizing the disease process tend to be most useful in identifying the severely ill or most complex types of patients to treat . on the other hand , those at the other end of the spectrum short - stay , low - resource - use patients could be identified by examining the procedures chiefly responsible for the inpatient admission . we suggest that some drg 's have subgroups of patients ( short - stay outliers ) admitted principally for diagnostic purposes . for many of these patients , once the test is administered and the results evaluated , discharge occurs within 1 - 3 days . the identification and appropriate classification of such procedures would limit the amount of potential gain for the hospital through inpatient shift . this approach does not offer the extensive clinical refinement of systems such as patient management categories ( young , 1984 ) , disease staging ( gonella , hornbrook , and louis , 1984 ) , the medical illness severity grouping system ( brewster et al . , 1985 ) , acute physiology and chronic health evaluation ( wagner and draper , 1984 ) or the computerized severity of illness index ( horn and horn , 1986 ) . however , jencks and dobson 's ( 1987 ) recent review of these systems questions their ability to improve the basic drg system , particularly given the requirements of special data abstraction that three of these systems impose . the methodology proposed here can identify distinct and meaningful patient categories within the context of the drg system , without posing new coding requirements . understanding the potential role of inpatient - outpatient substitution in a drg - based payment system is critical . the implementation of the inpatient prospective payment system may be partly responsible for the increased shift in surgery to the outpatient setting . according to a recent american hospital association survey , the percent of all surgeries performed on an outpatient basis in community hospitals rose from approximately 24 percent in 1983 to more than 40 percent in 1986 ( american hospital association , 1987 ) . in this article , we demonstrate that large numbers of surgical drg 's lend themselves to inpatient - outpatient substitution . the potential impact of future alternative outpatient systems clearly needs to be assessed , using both inpatient and outpatient utilization data so that potential abuses can be detected and/or avoided . the following are the diagnosis - related group ( drg ) numbers and descriptions of the 23 procedures identified in the rand study but not included in table 2 : these drg 's represent medical rather than surgical drg 's and are therefore excluded from this analysis .

through analysis of data from the universal health insurance system in manitoba , canada , surgical diagnosis - related groups ( drg 's ) with the greatest potential for inpatient - outpatient substitution are identified . candidates for both inpatient shift and outpatient shift are discussed . it is also suggested that determination of the procedure chiefly responsible for hospital admission complements approaches to improve the drg classification system by measuring severity of illness . thus , health care planners ' efforts may be facilitated in establishing effective payment systems , though definitive guidelines are not provided .

Introduction Methods Results Outpatient shift Inpatient shift Comparison with RAND classifications Discussion Technical note

the substitution of ambulatory care for inpatient hospital care has long been advocated as a safe and efficacious method of cost containment . proponents of ambulatory surgery argue that quality is not compromised and patients are not exposed to the potential iatrogenic hazards of hospitalization . private insurers who cover both inpatient and outpatient services can and do encourage the use of ambulatory surgery by offering special incentives to make it more attractive relative to inpatient surgery ( pauly and burns , 1984 ) . under prospective payment systems based on diagnosis - related groups ( drg 's ) , hospitals are encouraged to operate more efficiently and are paid a fixed rate per case for inpatient services . what is financially beneficial or disadvantageous to the hospital depends on the real resource cost of treating the patient in a given setting and the amount of the payment for services provided in that setting . in the medicare program , inpatient payment levels are reflected in the drg weight ; the higher the weight assigned a drg , the higher the payment for patients in that category . on the one hand , when the payment rate for a surgical drg is perceived to be too low to cover the costs of inpatient services , there is the incentive to do the procedure in an outpatient department . in the case of lens extraction , the amount medicare has paid some hospitals for performing the surgery on an outpatient basis sometimes exceeds the inpatient drg rate . such procedures are prime candidates for outpatient shift under the prospective payment system ( pps ) . there is also the concern that patients who could be treated on an outpatient basis would be admitted as inpatients to generate short stays that might be more profitable under pps this incentive may be strong for surgical drg 's , in which procedures that can be performed in an ambulatory setting are often grouped with other procedures that require more followup care and are generally considered more appropriate for the inpatient setting . this is a major concern of the prospective payment assessment commission ( propac ) , in light of substantial increases in outpatient utilization by medicare beneficiaries since the introduction of pps in 1983 ( prospective payment assessment commission , 1986 ) . the commission has recommended extending the responsibilities of the professional review organizations ( pro 's ) to include a review of outpatient surgery for selected procedures . rand researchers used internal documents from the health care financing administration ( hcfa ) and a physician consultant to prepare a list of drg 's that have a high potential for outpatient substitution ( carter and ginsberg , 1985 ) . an empirical assessment is particularly difficult in the united states , because large health services data bases , covering residents of states or other well - defined populations ( such as medicare beneficiaries ) , do not combine inpatient and outpatient utilization data . comparing utilization across settings for the medicare population may be particularly difficult in the future , because hcfa requires hospitals to use different coding systems to bill for inpatient ( international classification of diseases , 9th revision , clinical modification [ icd-9-cm ] ) and outpatient ( current procedural terminology [ cpt ] ) surgery ( shahoda , 1987 ) . in the universal health insurance system operating in manitoba , canada ( population 1 million ) , hospitals are funded on the basis of global budgets . despite the differences in payment systems , there are many similarities between hospitalization patterns in manitoba and in the united states ( roos , 1984 ; roos and ramsey , 1987 ; roos and danzinger , 1986 ; roos and lyttle , 1985 ) . hospital discharge rates are very similar for individuals 65 years of age or over ( 398 discharges per 1,000 population in the united states and 403 per 1,000 in manitoba ) ( lohr , lohr , and brook , 1985 ) . with the exception of a few minor plastic surgical procedures , all outpatient surgery in manitoba there were 592 freestanding outpatient surgery centers open in 1986 , and they performed 14 percent of all outpatient procedures ( henderson , 1987 ) . because a drg - based payment system has never been used in manitoba , hospital admission policies and coding practices could not have been influenced by these payment concerns . in this article , we identify those drg categories that appear to have the most potential for inpatient - outpatient substitution . we then compare the manitoba results with the drg categories identified by researchers as having a potential for outpatient shift , using a nonempirical approach . finally , we focus on drg 's with a high potential for inpatient shift . this latter analysis is meant to identify a new approach for refining the drg system , not to provide definitive guidelines . all 1982 - 84 acute inpatient and outpatient hospitalizations of manitobans 20 years of age or over were available to the project . ( medical and pediatric drg categories were excluded from the analysis , as were admissions classified under the miscellaneous drg category 470 . ) because the drg system in the united states has been applied largely to the medicare population , all analyses presented here focus on the manitoba population 65 years of age or over . after excluding hospitalizations resulting in deaths , there were 35,630 eligible discharges from these 8 hospitals during the 3-year period 1982 - 84 , with 18.0 percent of the patients undergoing treatment on an outpatient basis . both inpatient and outpatient admissions were classified by icd-9-cm codes into surgical drg 's , using standard second - revision drg assignment software available from health systems international ( 1986 ) . these definitions are consistent with those reported in the september 3 , 1985 issue of the federal register . although the drg system requires a principal diagnosis ( the condition established after study to be chiefly responsible for occasioning admission of the patient to the hospital for care ) , the manitoba system recorded the primary diagnosis ( that diagnosis which describes the most significant condition for which the patient was hospitalized ) , as well as up to seven additional diagnoses . forty - three of the differences in the 584 records with multiple diagnoses resulted in changes in drg assignment . patients discharged from the hospital after a 1 - 3-day stay were separately classified , because such patients might conceivably be treated on an outpatient basis . second , drg 's with a large proportion ( 35 percent or more ) of patients treated on an outpatient basis were identified as having a high potential for inpatient - outpatient substitution . drg 's in these two groups were considered candidates for outpatient and inpatient shift , respectively . a weight of 1 was chosen as a bound for reviewing groups for inpatient or outpatient shift , because drg 's with weights greater than 1 have mean costs per case ( and hence payment rates ) that are higher than the average across all drg 's for the medicare population . as one reviewer indicated , the point at which incentives exist to shift depends on an individual hospital 's costs for performing surgery in one site versus the other and the corresponding payment levels . third , patients in drg 's with a high potential for inpatient shift were classified by the procedure responsible for the patient 's drg assignment to determine if there are patterns that suggest methods of improving the system . if certain procedures in these high - weight drg 's are performed almost exclusively on an outpatient basis or during a 1 - 3-day stay , this would suggest that hospitals should be paid differently for such patients than for patients undergoing procedures almost always associated with a longer stay . finally , the results of our empirical assessment of the potential of individual drg 's for inpatient - outpatient substitution are compared with the results obtained by rand researchers carter and ginsberg ( 1985 ) . in table 1 , the proportion of cases in each major diagnostic category ( mdc ) are shown , according to the type of hospital admission ( outpatient , 1 - 3-day stay , or longer stay ) . the proportion of patients treated on an outpatient basis ranged from 66.1 percent to 0 ; patients with diseases and disorders of the skin , subcutaneous tissue , and breast were most likely to be treated on an outpatient basis and patients with burns or mental diseases least likely . in table 2 , the 35 surgical drg 's for which 35 percent or more of the cases in manitoba hospitals were treated on an outpatient basis in 1982 - 84 are identified . in addition , procedures are flagged if the rand group identified this as a drg with potential for outpatient substitution . as expected , drg 's on this list , which included patients with complications or patients 70 years of age or over ( drg 's 269 , 259 , 154 , 152 , 157 , 401 ) have fewer patients admitted as outpatients than do the less - complex companion drg 's ( 270 , 260 , 155 , 153 , 158 , 402 ) . drg 's with a large proportion of outpatient admissions generally have low weights ( less than 1 ) . thus , 8 of the 11 drg categories with 60 percent or more cases treated on an outpatient basis ( the top 11 surgical drg 's listed in table 2 ) had weights of .762 or less ( including 5 with weights of .427 or less ) , reflecting relatively low payment rates under pps . hospitals might feel some pressure to treat cases in these drg categories in the outpatient setting ( thus causing outpatient shift ) , particularly if fewer resources are required to treat patients and if payment is on an incurred - cost basis . however , not all drg 's with a large proportion of cases treated on an outpatient basis have low weights and low payment rates . thus , drg 155 ( stomach , esophageal , and duodenal procedures ) has a weight of 1.791 , and drg 269 ( other skin , subcutaneous tissue , and breast operating room procedures ) has a weight of 1.133 , even though 74 percent and 91 percent , respectively , of these cases were treated on an outpatient basis in manitoba ( table 2 ) . overall , a wide variety of drg 's have a significant potential for inpatient - outpatient substitution ; a major incentive for inpatient shift or outpatient shift is the assigned weight . although not shown in table 2 , an additional 15 drg 's had 20 - 34 percent of their cases treated on an outpatient basis . in an attempt to identify cases with potential for inpatient shift , selected drg 's from table 2 these 9 drg 's had weights of 1.0 or greater and had at least 50 cases treated in manitoba urban hospitals in 1982 - 84 . a large group of patients classified in each of the drg 's presented in table 3 was treated on an outpatient basis or during a short inpatient stay of 1 to 3 days . perhaps most impressively , fewer than two - thirds ( 53.8 percent ) of the patients classified in the very - high - weight , high - payment drg 154 were admitted for stays of 4 days or longer . a large proportion of such patients had their procedures performed on an outpatient basis ( 45.3 percent ) . thus , in table 4 , patients in each of the nine high - weight drg groups are classified according to the specific procedure performed and type of stay . major surgical procedures are listed if 25 or more patients had this procedure and 30 percent or more of the patients were treated on an outpatient or short - stay basis . as a way of addressing this and other biopsy - related coding limitations , medicare requires that a specific non - operating - room code be substituted for an operating room code when the biopsy is closed this rule was not in effect in manitoba , so the small bowel biopsies performed there on an outpatient basis are most likely closed biopsies . the far - righthand column of table 2 flags those drg 's that the carter - ginsberg project labeled as drg 's with potential for outpatient substitution . of the 35 surgical drg 's for which manitoba hospitals treated 35 percent or more of their cases on an outpatient basis , only 11 were identified by the rand study . the other procedures identified in the rand study were procedures either not performed in manitoba on an outpatient basis ( as is true for prostatectomies ) or performed less frequently than the 35-percent criterion required for inclusion in table 2 . drg 's with a large proportion of outpatient admissions generally have low weights ( less than 1 ) . thus , 8 of the 11 drg categories with 60 percent or more cases treated on an outpatient basis ( the top 11 surgical drg 's listed in table 2 ) had weights of .762 or less ( including 5 with weights of .427 or less ) , reflecting relatively low payment rates under pps . hospitals might feel some pressure to treat cases in these drg categories in the outpatient setting ( thus causing outpatient shift ) , particularly if fewer resources are required to treat patients and if payment is on an incurred - cost basis . however , not all drg 's with a large proportion of cases treated on an outpatient basis have low weights and low payment rates . thus , drg 155 ( stomach , esophageal , and duodenal procedures ) has a weight of 1.791 , and drg 269 ( other skin , subcutaneous tissue , and breast operating room procedures ) has a weight of 1.133 , even though 74 percent and 91 percent , respectively , of these cases were treated on an outpatient basis in manitoba ( table 2 ) . overall , a wide variety of drg 's have a significant potential for inpatient - outpatient substitution ; a major incentive for inpatient shift or outpatient shift is the assigned weight . although not shown in table 2 , an additional 15 drg 's had 20 - 34 percent of their cases treated on an outpatient basis . in an attempt to identify cases with potential for inpatient shift , selected drg 's from table 2 are presented in table 3 . these 9 drg 's had weights of 1.0 or greater and had at least 50 cases treated in manitoba urban hospitals in 1982 - 84 . a large group of patients classified in each of the drg 's presented in table 3 was treated on an outpatient basis or during a short inpatient stay of 1 to 3 days . perhaps most impressively , fewer than two - thirds ( 53.8 percent ) of the patients classified in the very - high - weight , high - payment drg 154 were admitted for stays of 4 days or longer . thus , in table 4 , patients in each of the nine high - weight drg groups are classified according to the specific procedure performed and type of stay . major surgical procedures are listed if 25 or more patients had this procedure and 30 percent or more of the patients were treated on an outpatient or short - stay basis . as a way of addressing this and other biopsy - related coding limitations , medicare requires that a specific non - operating - room code be substituted for an operating room code when the biopsy is closed this rule was not in effect in manitoba , so the small bowel biopsies performed there on an outpatient basis are most likely closed biopsies . the far - righthand column of table 2 flags those drg 's that the carter - ginsberg project labeled as drg 's with potential for outpatient substitution . of the 35 surgical drg 's for which manitoba hospitals treated 35 percent or more of their cases on an outpatient basis , only 11 were identified by the rand study . the other procedures identified in the rand study were procedures either not performed in manitoba on an outpatient basis ( as is true for prostatectomies ) or performed less frequently than the 35-percent criterion required for inclusion in table 2 . in this article , we investigate the potential for inpatient and outpatient substitution by examining the proportion of cases in each drg treated on an outpatient or short - stay basis in eight manitoba hospitals . there was a lack of correspondence between the drg 's we identified as having substitution potential and the drg 's identified in the rand study ( carter and ginsberg , 1985 ) . some of the differences in the drg 's identified by the two approaches may be attributed to the methods used to evaluate substitution potential . the rand study appears to have selected those drg 's containing procedures known to be suitable for the ambulatory setting . for example , all of the procedures in drg 's 6 , 232 , 342 , 361 , and 362 are on blue cross and blue shield 's list of reimbursable outpatient surgery ( sieverts , 1984 ) . the drg 's identified in the rand study therefore tended to have short lengths of stay and relatively low weights . these drg 's are candidates for outpatient shift ; from a quality of care perspective , it is important to monitor these drg 's under pro outpatient review programs . by comparing the proportion of all hospital admissions with no overnight stay across drg 's , both the groups amenable to outpatient shift and those potentially susceptible to inpatient shift were identified . the data - based method used here flags those high - weight drg 's with patients treated on an ambulatory or short - stay basis as well as those treated during longer inpatient stays . such drg 's tend to have a heterogeneous mix of patients , and close examination of length of stay by primary procedure may help to sort out this heterogeneity . until these drg 's are so refined , hospitals can admit patients for selected procedures that could be performed on an ambulatory basis , thereby increasing their payments with few attendant expenses . as an interim method ( which began october 1 , 1987 ) , payments for outpatient hospital facility services associated with surgical procedures are the lesser of the amount the hospital would have received , based on reasonable costs , and a blend amount based on hospital cost ( 75 percent ) and the ambulatory surgical center ( asc ) rate ( 25 percent ) . payments to asc 's are based on prospectively set rates for specific procedures described in the april 21 , 1987 federal register . the approach to refining drg 's proposed in this article complements the efforts of others to improve the drg classification system by developing measures of severity of illness . on the other hand , those at the other end of the spectrum short - stay , low - resource - use patients could be identified by examining the procedures chiefly responsible for the inpatient admission . we suggest that some drg 's have subgroups of patients ( short - stay outliers ) admitted principally for diagnostic purposes . the identification and appropriate classification of such procedures would limit the amount of potential gain for the hospital through inpatient shift . , 1985 ) , acute physiology and chronic health evaluation ( wagner and draper , 1984 ) or the computerized severity of illness index ( horn and horn , 1986 ) . however , jencks and dobson 's ( 1987 ) recent review of these systems questions their ability to improve the basic drg system , particularly given the requirements of special data abstraction that three of these systems impose . the methodology proposed here can identify distinct and meaningful patient categories within the context of the drg system , without posing new coding requirements . understanding the potential role of inpatient - outpatient substitution in a drg - based payment system is critical . the implementation of the inpatient prospective payment system may be partly responsible for the increased shift in surgery to the outpatient setting . in this article , we demonstrate that large numbers of surgical drg 's lend themselves to inpatient - outpatient substitution . the potential impact of future alternative outpatient systems clearly needs to be assessed , using both inpatient and outpatient utilization data so that potential abuses can be detected and/or avoided . the following are the diagnosis - related group ( drg ) numbers and descriptions of the 23 procedures identified in the rand study but not included in table 2 : these drg 's represent medical rather than surgical drg 's and are therefore excluded from this analysis .

the research was performed as part of an ongoing clinical trial ( nct01461213 ) approved by the national ethics committee and adhered to the declaration of helsinki ( 2013 ) . high - resolution spectral - domain oct was obtained using the spectralis hra+oct system ( heidelberg engineering gmbh , heidelberg , germany ) with 37 horizontal volume scans covering 30 15 centered over the foveola . enhanced - depth imaging mode was activated during oct capture and a simultaneous 30 30 infrared confocal slo image was captured automatically . in the same sitting , a 30 30 bluepeak laser fundus af image was captured using the same optics according to the manufacturer 's standard operating procedure , including focusing in the red - free reflectance mode and automated real - time ( art ' ) alignment of at least eight single images to create a mean image . precise point - to - point alignment of corresponding locations between oct and af images was performed in the heidelberg eye explorer software ( heyex ; heidelberg engineering gmbh ) in two ways using the slo image as an intermediary . heyex has a sophisticated in - built image recognition and alignment capability , which allows tracking of eye movements during image acquisition . this function is also available during image analysis when a marker annotating the 30 30 slo image attached to the oct may be copied and pasted onto the corresponding 30 30 af image using common landmarks as reference points in heyex . we confirmed the precision of this method of image alignment using an alternative manual technique . this consisted of choosing two fixed reference points ( e.g. , small vessel branch points ) common to both the slo and af images . the distance between the two reference points can be measured precisely on each image using the heyex marker tool . a point of interest on an oct image is automatically displayed on the linked slo image as a crosshair in heyex . a pair of circles centered at the two reference points could then be drawn on the slo image under high magnification such that they intersect exactly at the crosshair . the diameters of the circles were automatically displayed by heyex and used to calculate the predicted diameters of corresponding circles on the af image by multiplying through the conversion factor . the intersection of the two circles of predicted diameters drawn on the af image would then correspond exactly with the point of interest on the oct . six choroideremia patients ( aged 3565 years ) underwent gene therapy : 0.6 to 1.0 10 genome particles of a recombinant adeno - associated viral vector encoding rep1 ( aav2-rep1 ) was delivered by pars plana vitrectomy and subretinal injection , as reported previously . serial images pre- , 1 month post- , and 6 months postgene therapy were obtained at the same locations using the autorescan function of the spectralis by selecting the baseline image as the reference scan . all outer retinal tubulations ( cystic structures ) on each b - scan of the oct were identified and mapped to corresponding positions on the af image using the point - to - point image alignment technique described above . subfoveal choroidal thickness ( sfct ) was defined as the distance from the outer border of the rpe to the inner scleral border directly below the foveal dip on edi - oct . subfoveal choroidal thickness was measured in 23 choroideremia patients and 20 aged - matched male patients without significant retinal or choroidal pathology . all subjects had refractions between 5.5 and + 5.0 diopters ( d ) . for data analysis , choroideremia patients were further divided into three groups based on foveal outer retinal integrity being preserved , disrupted , or lost on oct . a kruskal - wallis test was performed to assess the significance of any differences between the groups . manual and automated point - to - point alignment between outer retinal morphology on the oct and the edges of af islands showed consistent correspondence between the edge of af and the beginning of an acute decline in outer nuclear layer ( onl ) thickness on the oct , which diminished to zero in a triangular slope over approximately 200 m ( fig . , image alignment analysis was performed using oct and af images from 78 eyes of 39 patients with choroideremia . of these , the images of 49 eyes were suitable for alignment . twenty - nine eyes were excluded from the analysis for the following reasons : ( 1 ) no outer retina remaining on oct ( due to end stage degeneration , n = 15 ) , ( 2 ) poor image quality ( due to poor fixation , n = 8) , and ( 3 ) intact outer retina extending beyond the edge of captured image ( usually in early disease , n = 6 ) ( table ) . when the transfoveal af island widths ( y ) measured on af images were compared with those predicted ( y ' ) based on the width of intact onl on oct ( x ) in all 49 eyes ( see schematic illustration in fig . 1d ) , the median difference was zero ( range , 65 to + 62 m ) . in 29 ( 59% ) eyes , an exact correspondence was found between the predicted and measured transfoveal af island width . the overall spearman rank correlation coefficient ( rho ) between measured and predicted transfoveal af width was 0.9992 . the external limiting membrane ( elm ) and the ellipsoid zone ( ez ) generally terminated at the same location as where the onl thickness began to show a sharp decline . the ez and elm were often ill defined at the edge of degeneration due to a disrupted appearance on the oct , and were less reliable predictors of the location of the af border . panels ( a c ) show representative images from three choroideremia patients . by plotting pairs of intersecting circles of proportional diameters from fixed reference points ( e.g. , vessel branch points ) on the af and slo images , precisely corresponding point locations could be identified between the af and oct b - scans , and vice versa . the intersection between two red circles on the af image marking the left - hand border of rpe degeneration ( ai ) , with high magnification view shown ( aii ) , corresponds to the position on the oct indicated by a vertical red line ( aiv ) , while the intersection between green circles marking the right - hand border of rpe degeneration ( ai , aiii ) corresponds to a second position on the oct indicated by a vertical green line ( av ) . the edges of surviving rpe on the oct coincide with the start of an acute decline in onl height , which diminishes to zero in a triangular transition zone , denoted by horizontal yellow bars . immediately beyond the loss of onl , outer retinal tubulations scale bars : 200 m . a summary of the characteristic oct to af image alignment features is provided in ( d ) . correlation between the transfoveal island width ( y ) measured on af images and those predicted based on oct outer retinal width ( x ) was performed in 49 choroideremia eyes ( data summarized in the table ) . choroideremia patient data in six eyes of three patients aged 16 , 22 , and 23 years , the residual af islands consisted of an ovoid central zone of relatively preserved af and a peripheral zone of mottled af ( fig . 2 ) . when these af images were aligned to their respective octs , the regions with preserved af were found to correspond to areas of the retina with preserved ez integrity , while the surrounding region of mottled af corresponded to areas with disrupted ez . the horizontal diameters of the central ovoid zones with preserved af were individual - specific but non age - dependent : 3050 m od/3180 m os ( age 16 ) , 3250 m od/3050 m os ( age 22 ) , and 2400 m od/2400 m os ( age 23 ) . the mean eccentricities of the zones of preserved af in relation to the anatomic foveal dip were 1533 m ( sem = 179 ) nasally , 1339 ( sem = 157 ) temporally , and 1436 ( sem = 70 ) overall , which are consistent with these zones representing areas of relatively high cone density . while such af zonal transition was seen in three of the youngest choroideremia patients in the cohort ( presumably a phenotype associated with an early stage of disease ) , the rest of the eyes presented with more homogeneous mottled af over the entire island , associated with generalized ez disruption . correlation between the pattern of af and integrity of the ez . in three young choroideremia patients aged 16 ( a ) , 22 ( b ) , and 23 years ( c ) who have large residual islands remaining on af imaging , central ovoid zones with relatively preserved af were observed , which aligned with regions of preserved ez integrity on the oct ( green arrow bars ) . the size and eccentricities of these ovoid zones in relation to the anatomical foveal dip ( white arrows ) the rest of the islands showed mottled af , which corresponded to disrupted ez ( yellow arrow bars ) . note that in ( a ) ( od & os ) and ( c ) ( os ) , at least one edge of the af island fell outside the oct image boundaries , therefore the foveola has been used as one of the reference points during image alignment . outer retinal tubulations ( ort ) have previously been observed in advanced amd and other degenerative retinal conditions . the mechanism of tubule formation is unclear , although it has been speculated to represent an arrangement of degenerating photoreceptors with potential contribution from adjacent rpe or glial elements . in our cohort of choroideremia patients , tubulations were found to be present to a variable extent on the oct of the majority of eyes across the age range and disease severity ( fig . 3 ) : 71 of 78 ( 91% ) eyes examined demonstrated tubulations ; one eye was not assessable due to poor image quality ( table ) . the six eyes that did not show any tubulations were all at end - stage degeneration with no outer retinal structure remaining . when mapped onto af images , the tubulations seen on oct b - scans were universally located beyond the edges of the af islands ( therefore , overlying bare bruch 's membrane ) , with a mean distance of approximately 400 to 500 m from the nearest island edge , although some were significantly farther away ( fig . 3d ) . even though tubulations appear to be isolated and cystic in appearance on oct b - scans , two - dimensional analysis of their locations showed that many were in fact contiguous with the main island of surviving outer retina via pseudodendritic ' tubular extensions ( fig . 4 ) . these thin pseudodendrites appear to lie over areas devoid of rpe ( by the absence of af ) but are detectable by their retinal pigmentation on high magnification infrared slo images . distribution of outer retinal cysts in six patients with choroideremia aged 16 to 71 years ( [ a f ] in ascending age order ) . the distribution of tubulations could be inferred from the positions of all outer retinal cysts ( orcs ) , which represent cross - sections of tubulations , seen on all b - scans of the oct . for each eye , all orcs were mapped to corresponding locations on the af image ( shown as white dots ) . the table displays the mean distance of orcs from the nearest edge of af island ( sd ) and genetic mutations in the chm gene for each patient . outer retinal cysts on oct represent cross - sections through outer retinal tubulations following rpe loss at the edges of degeneration . plotting the locations of orcs ( white dots ) seen on every b - scan of the oct ( horizontal green lines ) over the corresponding high magnification infrared slo image revealed that the orcs lie over thin pseudodendrites ' containing retinal pigment along the edges of the surviving retina ( outlined in dotted blue ) . these pseudodendrites do not match the background autofluorescence of the choroidal vasculature ( top panel ) but represent distinct tubular outer retinal structures . because tubulations likely represent rearrangement of surviving photoreceptors , we asked whether their distribution would be affected by subretinal injections in gene therapy , because this would provide us with indirect evidence as to whether or not they might have retained synapses to the overlying retina . when the locations of tubulations were compared between baseline , 1 month , and 6 months post aav.rep1 gene therapy , it could be seen that the general distributions of tubulations were not significantly affected by surgically induced retinal detachments ( fig . moreover , while a slight dip in the number of outer retinal cysts seen in some eyes at 1 month could be attributed to mechanical disturbance from the retinal detachment , there was no statistically significant fluctuation over the 6-month period ( linear regression : f(5,6 ) = 1.29 , p = 0.38 ; supplementary fig . s1 ) . these would be in keeping with tubulations maintaining some physical attachment to the inner retina , thereby potentially retaining some visual function over these anatomical anomalies . gene therapy using recombinant aav2-rep1 vector for choroideremia were performed on six eyes ( a f ) in which the residual af islands were surgically detached by subretinal injections . the locations of outer retinal cysts ( white dots ) seen on all b - scans of the octs , which represent cross - sections of tubulations , have been mapped onto the corresponding af images at baseline ( left column ) , 1 month post ( center column ) and 6 months post ( right column ) gene therapy . although the chm gene defect is known to cause degeneration , most patients start life with good vision . we were therefore interested to know if there might be any developmental anomalies in choroideremia patients and/or if the subfoveal choroid thinning begins before significant foveal retinal degeneration is evident . subfoveal choroidal thickness was measured in 42 eyes of 23 choroideremia patients ( mean age 37.5 years ; range , 1263 ) and 40 eyes of 20 age - matched healthy male controls ( mean age 37.6 years ; range , 1362 ; supplementary table s1 ) . the mean refraction of chm eyes was 0.92 d ( sem 0.37 ) and control eyes 0.28 d ( sem 0.34 ) . unpaired 2-tailed t - test showed no statistically significant difference between the refractions of the two groups ( p = 0.21 ) . the mean sfct in healthy controls ( 302.0 m 4.8 sem ) was found to be significantly greater than that in chm eyes with preserved ez ( 179.7 m 17.2 sem ; p also , the sfct of chm eyes with disrupted ez ( 191.9 m 10.8 sem ) was significantly greater than those with lost ez ( 43.1 m 6.5 sem ; p < 0.0001 ) . however , no significant difference in sfct was found between chm eyes associated with preserved and disrupted ez ( p = 0.8251 ) . scatter plot of sfct as measured on enhanced - depth oct in choroideremia patients with preserved , disrupted or lost ellipsoid zone ( as illustrated by representative oct images above ) versus age and refractive error - matched healthy controls . the mean of each group ( in black ) is shown with 1 sd on either side ( in gray ) . manual and automated point - to - point alignment between outer retinal morphology on the oct and the edges of af islands showed consistent correspondence between the edge of af and the beginning of an acute decline in outer nuclear layer ( onl ) thickness on the oct , which diminished to zero in a triangular slope over approximately 200 m ( fig . , image alignment analysis was performed using oct and af images from 78 eyes of 39 patients with choroideremia . of these , the images of 49 eyes were suitable for alignment . twenty - nine eyes were excluded from the analysis for the following reasons : ( 1 ) no outer retina remaining on oct ( due to end stage degeneration , n = 15 ) , ( 2 ) poor image quality ( due to poor fixation , n = 8) , and ( 3 ) intact outer retina extending beyond the edge of captured image ( usually in early disease , n = 6 ) ( table ) . when the transfoveal af island widths ( y ) measured on af images were compared with those predicted ( y ' ) based on the width of intact onl on oct ( x ) in all 49 eyes ( see schematic illustration in fig . 1d ) , the median difference was zero ( range , 65 to + 62 m ) . in 29 ( 59% ) eyes , an exact correspondence was found between the predicted and measured transfoveal af island width . the overall spearman rank correlation coefficient ( rho ) between measured and predicted transfoveal af width was 0.9992 . the external limiting membrane ( elm ) and the ellipsoid zone ( ez ) generally terminated at the same location as where the onl thickness began to show a sharp decline . the ez and elm were often ill defined at the edge of degeneration due to a disrupted appearance on the oct , and were less reliable predictors of the location of the af border . panels ( a c ) show representative images from three choroideremia patients . by plotting pairs of intersecting circles of proportional diameters from fixed reference points ( e.g. , vessel branch points ) on the af and slo images , precisely corresponding point locations could be identified between the af and oct b - scans , and vice versa . the intersection between two red circles on the af image marking the left - hand border of rpe degeneration ( ai ) , with high magnification view shown ( aii ) , corresponds to the position on the oct indicated by a vertical red line ( aiv ) , while the intersection between green circles marking the right - hand border of rpe degeneration ( ai , aiii ) corresponds to a second position on the oct indicated by a vertical green line ( av ) . the edges of surviving rpe on the oct coincide with the start of an acute decline in onl height , which diminishes to zero in a triangular transition zone , denoted by horizontal yellow bars . immediately beyond the loss of onl , outer retinal tubulations scale bars : 200 m . a summary of the characteristic oct to af image alignment features is provided in ( d ) . correlation between the transfoveal island width ( y ) measured on af images and those predicted based on oct outer retinal width ( x ) was performed in 49 choroideremia eyes ( data summarized in the table ) . choroideremia patient data in six eyes of three patients aged 16 , 22 , and 23 years , the residual af islands consisted of an ovoid central zone of relatively preserved af and a peripheral zone of mottled af ( fig . 2 ) . when these af images were aligned to their respective octs , the regions with preserved af were found to correspond to areas of the retina with preserved ez integrity , while the surrounding region of mottled af corresponded to areas with disrupted ez . the horizontal diameters of the central ovoid zones with preserved af were individual - specific but non age - dependent : 3050 m od/3180 m os ( age 16 ) , 3250 m od/3050 m os ( age 22 ) , and 2400 m od/2400 m os ( age 23 ) . the mean eccentricities of the zones of preserved af in relation to the anatomic foveal dip were 1533 m ( sem = 179 ) nasally , 1339 ( sem = 157 ) temporally , and 1436 ( sem = 70 ) overall , which are consistent with these zones representing areas of relatively high cone density . while such af zonal transition was seen in three of the youngest choroideremia patients in the cohort ( presumably a phenotype associated with an early stage of disease ) , the rest of the eyes presented with more homogeneous mottled af over the entire island , associated with generalized ez disruption . correlation between the pattern of af and integrity of the ez . in three young choroideremia patients aged 16 ( a ) , 22 ( b ) , and 23 years ( c ) who have large residual islands remaining on af imaging , central ovoid zones with relatively preserved af were observed , which aligned with regions of preserved ez integrity on the oct ( green arrow bars ) . the size and eccentricities of these ovoid zones in relation to the anatomical foveal dip ( white arrows ) the rest of the islands showed mottled af , which corresponded to disrupted ez ( yellow arrow bars ) . note that in ( a ) ( od & os ) and ( c ) ( os ) , at least one edge of the af island fell outside the oct image boundaries , therefore the foveola has been used as one of the reference points during image alignment . outer retinal tubulations ( ort ) have previously been observed in advanced amd and other degenerative retinal conditions . the mechanism of tubule formation is unclear , although it has been speculated to represent an arrangement of degenerating photoreceptors with potential contribution from adjacent rpe or glial elements . in our cohort of choroideremia patients , tubulations were found to be present to a variable extent on the oct of the majority of eyes across the age range and disease severity ( fig . 3 ) : 71 of 78 ( 91% ) eyes examined demonstrated tubulations ; one eye was not assessable due to poor image quality ( table ) . the six eyes that did not show any tubulations were all at end - stage degeneration with no outer retinal structure remaining . when mapped onto af images , the tubulations seen on oct b - scans were universally located beyond the edges of the af islands ( therefore , overlying bare bruch 's membrane ) , with a mean distance of approximately 400 to 500 m from the nearest island edge , although some were significantly farther away ( fig . 3d ) . even though tubulations appear to be isolated and cystic in appearance on oct b - scans , two - dimensional analysis of their locations showed that many were in fact contiguous with the main island of surviving outer retina via pseudodendritic ' tubular extensions ( fig . 4 ) . these thin pseudodendrites appear to lie over areas devoid of rpe ( by the absence of af ) but are detectable by their retinal pigmentation on high magnification infrared slo images . distribution of outer retinal cysts in six patients with choroideremia aged 16 to 71 years ( [ a f ] in ascending age order ) . the distribution of tubulations could be inferred from the positions of all outer retinal cysts ( orcs ) , which represent cross - sections of tubulations , seen on all b - scans of the oct . for each eye , all orcs were mapped to corresponding locations on the af image ( shown as white dots ) . the table displays the mean distance of orcs from the nearest edge of af island ( sd ) and genetic mutations in the chm gene for each patient . outer retinal cysts on oct represent cross - sections through outer retinal tubulations following rpe loss at the edges of degeneration . plotting the locations of orcs ( white dots ) seen on every b - scan of the oct ( horizontal green lines ) over the corresponding high magnification infrared slo image revealed that the orcs lie over thin pseudodendrites ' containing retinal pigment along the edges of the surviving retina ( outlined in dotted blue ) . these pseudodendrites do not match the background autofluorescence of the choroidal vasculature ( top panel ) but represent distinct tubular outer retinal structures . because tubulations likely represent rearrangement of surviving photoreceptors , we asked whether their distribution would be affected by subretinal injections in gene therapy , because this would provide us with indirect evidence as to whether or not they might have retained synapses to the overlying retina . when the locations of tubulations were compared between baseline , 1 month , and 6 months post aav.rep1 gene therapy , it could be seen that the general distributions of tubulations were not significantly affected by surgically induced retinal detachments ( fig . , while a slight dip in the number of outer retinal cysts seen in some eyes at 1 month could be attributed to mechanical disturbance from the retinal detachment , there was no statistically significant fluctuation over the 6-month period ( linear regression : f(5,6 ) = 1.29 , p = 0.38 ; supplementary fig . s1 ) . these would be in keeping with tubulations maintaining some physical attachment to the inner retina , thereby potentially retaining some visual function over these anatomical anomalies . gene therapy using recombinant aav2-rep1 vector for choroideremia were performed on six eyes ( a f ) in which the residual af islands were surgically detached by subretinal injections . the locations of outer retinal cysts ( white dots ) seen on all b - scans of the octs , which represent cross - sections of tubulations , have been mapped onto the corresponding af images at baseline ( left column ) , 1 month post ( center column ) and 6 months post ( right column ) gene therapy . although the chm gene defect is known to cause degeneration , most patients start life with good vision . we were therefore interested to know if there might be any developmental anomalies in choroideremia patients and/or if the subfoveal choroid thinning begins before significant foveal retinal degeneration is evident . subfoveal choroidal thickness was measured in 42 eyes of 23 choroideremia patients ( mean age 37.5 years ; range , 1263 ) and 40 eyes of 20 age - matched healthy male controls ( mean age 37.6 years ; range , 1362 ; supplementary table s1 ) . the mean refraction of chm eyes was 0.92 d ( sem 0.37 ) and control eyes 0.28 d ( sem 0.34 ) . unpaired 2-tailed t - test showed no statistically significant difference between the refractions of the two groups ( p = 0.21 ) . the mean sfct in healthy controls ( 302.0 m 4.8 sem ) was found to be significantly greater than that in chm eyes with preserved ez ( 179.7 m 17.2 sem ; p also , the sfct of chm eyes with disrupted ez ( 191.9 m 10.8 sem ) was significantly greater than those with lost ez ( 43.1 m 6.5 sem ; p < 0.0001 ) . however , no significant difference in sfct was found between chm eyes associated with preserved and disrupted ez ( p = 0.8251 ) . scatter plot of sfct as measured on enhanced - depth oct in choroideremia patients with preserved , disrupted or lost ellipsoid zone ( as illustrated by representative oct images above ) versus age and refractive error - matched healthy controls . the mean of each group ( in black ) is shown with 1 sd on either side ( in gray ) . the chm gene product , rep1 ( rab escort protein 1 ) , regulates the prenylation of rab gtpases , which play important roles in intracellular vesicular trafficking . deficiency of rep1 is thought to impair the transport of proteins from the golgi apparatus to the outer segments in photoreceptors , as well as the phagocytosis and degradation of shed outer segments by rpe cells . cell type - specific knockouts of chm in mice have shown the potential for independent degeneration of photoreceptor and rpe layers , but the rate of degeneration was accelerated when both layers were defective simultaneously , presumably due to interdependent cell survival . our data revealed a precise anatomical correspondence between rpe and photoreceptor degeneration : the edge of rpe loss colocalized with a sharp decline in onl thickness in the majority of eyes with chm . small sporadic discrepancies were occasionally observed , which were equivalent to the size of fewer than four rpe cells each with a diameter of approximately 15 m . these could be attributed , in part , to temperature and calibration - dependent alignment errors between simultaneous infrared and tomographic scans from the heidelberg spectralis system itself . in the narrow transition zone between intact and degenerate outer retina , the onl was consistently seen to taper in a triangular fashion , a feature that has previously been termed an we hypothesize that this transition zone represents degenerating photoreceptors that reduce in height as they lose outer segments following the death of the interdigitating rpe cells , eventually leading to cell death . this would be in keeping with histopathologic observations from postmortem female carriers of choroideremia . the correlation between rpe and photoreceptor integrity was not only observed at the advancing border of degeneration , but also within the islands of surviving outer retina / rpe . ovoid zones of parafoveal preservation of af were seen in some of the youngest choroideremia patients in the cohort , which correlated with transitions in the integrity of the ez . moreover , the size and eccentricities of the ovoid zones were consistent with representing areas of relatively high cone density previously observed using histology and adaptive optics . taken together , the data provides indirect evidence for relative sparing of cones ( and their underlying rpe ) in the early stages of choroideremia . the early cone sparing may be explained by the low rep1 expression in cones versus high expression in rods . as the disease progresses , the central preservation is lost , suggesting that cone degeneration in choroideremia may be secondary to rod or rpe degeneration . it would also explain the clinical finding of early - onset nyctalopia , but preservation of visual acuity and color perception until late stage degeneration in choroideremia patients . we found no significant difference between the subfoveal choroidal thickness underlying retina with preserved or disrupted ez . this suggests that choroidal degeneration may be a secondary phenomenon , which lags significantly behind photoreceptor ( and rpe ) degeneration . this would be in keeping with observations from postmortem female carriers in which choroidal thinning was only seen in areas of severe retinal degeneration . we noted , however , that sfct was reduced in early choroideremia eyes compared with controls , at a time when the ez remained relatively intact . although choroidal thickness is affected by age , sex , and refractive error , these factors were all similar between the chm and control eyes in the study . a possible explanation would be that the impairment of rpe due to rep1 deficiency also impairs the ability of the rpe to influence development of the choroidal vasculature long before the onset of degeneration . alternatively , rep1 deficiency may be associated with a primary choroidal abnormality , although this is unlikely based on two observations : ( 1 ) chm carrier females with patchy retinal degeneration due to x - inactivation do not show corresponding patchy choroidal abnormality , and ( 2 ) chm patients occasionally present with secondary choroidal neovascularization , which suggests relatively intact choroidal angiogenesis . nevertheless the reduced choroidal thickness at baseline did not appear to have any obvious effect on visual acuity in the early stages of the disease . choroidal thinning has also been described as a secondary phenomenon in dry amd , presumably as a result of degeneration of the rpe . hence , choroidal thinning in chm appears to occur through a dual mechanism of reduced baseline thickness , which is probably not clinically significant , but which then progresses once there is loss of the overlying rpe as part of the late disease process . not all photoreceptors are lost beyond the edge of rpe degeneration , as some appear to survive by forming outer retinal tubulations , which could extend a considerable distance from the main surviving outer retinal island . we found tubulations in the majority of choroideremia eyes , except those with near total outer retinal loss . while they appear cystic on oct b - scans , mapping their locations to high magnification slo images showed that they most likely represent cross - sections through long tubular structures jutting out from the edges of outer retinal degeneration . tubulations have been described in a range of degenerative retinal disorders , including amd , stargardt disease , central serous retinopathy , serpiginous choroiditis , maternally inherited diabetes and deafness , gyrate atrophy , and choroideremia . they likely represent rearrangement of photoreceptors that have lost their outer segments following the degeneration of the underlying rpe cells , but are still close enough to the residual choroid to derive oxygenation and other diffusible substrates required for metabolic survival . this would be consistent with rpe degeneration being a key feature of all the conditions above . the fact that tubulations represent residual photoreceptors is in keeping with histologic analysis of tubulations in advanced amd , which showed cones lacking outer segments . apart from a slight dip in the total number of outer retinal cysts at 1 month post gene therapy ( likely due to physical disturbance from the surgical retinal detachment ) , the overall numbers and distributions of tubulations were stable over a 6-month period . while this does not completely exclude the possibility of some tubulations disappearing and forming at similar locations , the data provide indirect evidence that tubulations represent stable outer retinal structures , which retain some physical attachment to the retina , rather than being inflammatory in origin . the preservation of tubulations following gene therapy could also be an indication that the surgery did not cause significant disruption of outer retinal integrity , a concern due to possible stretching of the retina during subretinal injection . because tubulations lie over bare bruch 's membrane and are often contiguous with the main retinal island , one might expect retinal detachment to be induced with greater ease at these locations compared with more adherent degenerative retina elsewhere . what remains to be seen , however , is whether or not visual function can be restored in these tubulations following gene therapy and more importantly , whether or not this represents a degree of degeneration in choroideremia that may be arrested .

purposeto evaluate the relationships between rpe , photoreceptor , and choroidal degeneration in choroideremia.methodsenhanced-depth imaging optical coherence tomography ( edi - oct ) , scanning laser ophthalmoscopy ( slo ) , and autofluorescence ( af ) were performed on 39 patients ( 78 eyes ) with choroideremia . the edges of surviving outer retina on oct and residual af were aligned . the distribution of outer retinal tubulations was mapped over a range of ages ( 1671 years ) , and comparison made between pre- and postsubretinal gene therapy . subfoveal choroidal thickness ( sfct ) was compared between 23 choroideremia patients ( 42 eyes ) and 20 age- and refraction - matched male controls ( 40 eyes).resultsthe edges of rpe af aligned with a reduction in outer nuclear layer thickness ( spearman 's rho = 0.9992 ) . correlation was also found between the quality of af and integrity of ellipsoid zone within islands of surviving retina . tubulations existed in 71 of 78 ( 91% ) eyes with choroideremia and remained stable following gene therapy . subfoveal choroidal thickness was reduced at baseline in choroideremia ( 179.7 17.2 m ) compared with controls ( 302.0 4.8 m ; p < 0.0001 ) , but did not undergo significant thinning until end - stage retinal degeneration ( 43.1 6.5 m).conclusionsthe data suggest that rpe loss is the primary cause of photoreceptor degeneration in choroideremia . the choroid is thinner than controls from early stages , in keeping with a mild developmental defect . photoreceptors appear to lose outer segments following loss of underlying rpe and form tubulations at the edges of degeneration . the preservation of tubulations over time and after subretinal injection would be consistent with these structures maintaining attachment to the inner retina and hence being potentially light responsive ( clinicaltrials.gov , nct01461213 ) .

Methods Results Correlation Between AF and OCT Morphology Tubulations and the Effects of Gene Therapy Correlation Between Choroidal Thickness and Ellipsoid Zone Discussion Supplementary Material

six choroideremia patients ( aged 3565 years ) underwent gene therapy : 0.6 to 1.0 10 genome particles of a recombinant adeno - associated viral vector encoding rep1 ( aav2-rep1 ) was delivered by pars plana vitrectomy and subretinal injection , as reported previously . subfoveal choroidal thickness ( sfct ) was defined as the distance from the outer border of the rpe to the inner scleral border directly below the foveal dip on edi - oct . subfoveal choroidal thickness was measured in 23 choroideremia patients and 20 aged - matched male patients without significant retinal or choroidal pathology . manual and automated point - to - point alignment between outer retinal morphology on the oct and the edges of af islands showed consistent correspondence between the edge of af and the beginning of an acute decline in outer nuclear layer ( onl ) thickness on the oct , which diminished to zero in a triangular slope over approximately 200 m ( fig . , image alignment analysis was performed using oct and af images from 78 eyes of 39 patients with choroideremia . twenty - nine eyes were excluded from the analysis for the following reasons : ( 1 ) no outer retina remaining on oct ( due to end stage degeneration , n = 15 ) , ( 2 ) poor image quality ( due to poor fixation , n = 8) , and ( 3 ) intact outer retina extending beyond the edge of captured image ( usually in early disease , n = 6 ) ( table ) . in 29 ( 59% ) eyes , an exact correspondence was found between the predicted and measured transfoveal af island width . the ez and elm were often ill defined at the edge of degeneration due to a disrupted appearance on the oct , and were less reliable predictors of the location of the af border . the intersection between two red circles on the af image marking the left - hand border of rpe degeneration ( ai ) , with high magnification view shown ( aii ) , corresponds to the position on the oct indicated by a vertical red line ( aiv ) , while the intersection between green circles marking the right - hand border of rpe degeneration ( ai , aiii ) corresponds to a second position on the oct indicated by a vertical green line ( av ) . the edges of surviving rpe on the oct coincide with the start of an acute decline in onl height , which diminishes to zero in a triangular transition zone , denoted by horizontal yellow bars . immediately beyond the loss of onl , outer retinal tubulations scale bars : 200 m . correlation between the transfoveal island width ( y ) measured on af images and those predicted based on oct outer retinal width ( x ) was performed in 49 choroideremia eyes ( data summarized in the table ) . choroideremia patient data in six eyes of three patients aged 16 , 22 , and 23 years , the residual af islands consisted of an ovoid central zone of relatively preserved af and a peripheral zone of mottled af ( fig . the mean eccentricities of the zones of preserved af in relation to the anatomic foveal dip were 1533 m ( sem = 179 ) nasally , 1339 ( sem = 157 ) temporally , and 1436 ( sem = 70 ) overall , which are consistent with these zones representing areas of relatively high cone density . correlation between the pattern of af and integrity of the ez . in three young choroideremia patients aged 16 ( a ) , 22 ( b ) , and 23 years ( c ) who have large residual islands remaining on af imaging , central ovoid zones with relatively preserved af were observed , which aligned with regions of preserved ez integrity on the oct ( green arrow bars ) . 3 ) : 71 of 78 ( 91% ) eyes examined demonstrated tubulations ; one eye was not assessable due to poor image quality ( table ) . the six eyes that did not show any tubulations were all at end - stage degeneration with no outer retinal structure remaining . when mapped onto af images , the tubulations seen on oct b - scans were universally located beyond the edges of the af islands ( therefore , overlying bare bruch 's membrane ) , with a mean distance of approximately 400 to 500 m from the nearest island edge , although some were significantly farther away ( fig . even though tubulations appear to be isolated and cystic in appearance on oct b - scans , two - dimensional analysis of their locations showed that many were in fact contiguous with the main island of surviving outer retina via pseudodendritic ' tubular extensions ( fig . these thin pseudodendrites appear to lie over areas devoid of rpe ( by the absence of af ) but are detectable by their retinal pigmentation on high magnification infrared slo images . distribution of outer retinal cysts in six patients with choroideremia aged 16 to 71 years ( [ a f ] in ascending age order ) . the distribution of tubulations could be inferred from the positions of all outer retinal cysts ( orcs ) , which represent cross - sections of tubulations , seen on all b - scans of the oct . outer retinal cysts on oct represent cross - sections through outer retinal tubulations following rpe loss at the edges of degeneration . because tubulations likely represent rearrangement of surviving photoreceptors , we asked whether their distribution would be affected by subretinal injections in gene therapy , because this would provide us with indirect evidence as to whether or not they might have retained synapses to the overlying retina . when the locations of tubulations were compared between baseline , 1 month , and 6 months post aav.rep1 gene therapy , it could be seen that the general distributions of tubulations were not significantly affected by surgically induced retinal detachments ( fig . moreover , while a slight dip in the number of outer retinal cysts seen in some eyes at 1 month could be attributed to mechanical disturbance from the retinal detachment , there was no statistically significant fluctuation over the 6-month period ( linear regression : f(5,6 ) = 1.29 , p = 0.38 ; supplementary fig . these would be in keeping with tubulations maintaining some physical attachment to the inner retina , thereby potentially retaining some visual function over these anatomical anomalies . gene therapy using recombinant aav2-rep1 vector for choroideremia were performed on six eyes ( a f ) in which the residual af islands were surgically detached by subretinal injections . the locations of outer retinal cysts ( white dots ) seen on all b - scans of the octs , which represent cross - sections of tubulations , have been mapped onto the corresponding af images at baseline ( left column ) , 1 month post ( center column ) and 6 months post ( right column ) gene therapy . we were therefore interested to know if there might be any developmental anomalies in choroideremia patients and/or if the subfoveal choroid thinning begins before significant foveal retinal degeneration is evident . subfoveal choroidal thickness was measured in 42 eyes of 23 choroideremia patients ( mean age 37.5 years ; range , 1263 ) and 40 eyes of 20 age - matched healthy male controls ( mean age 37.6 years ; range , 1362 ; supplementary table s1 ) . the mean sfct in healthy controls ( 302.0 m 4.8 sem ) was found to be significantly greater than that in chm eyes with preserved ez ( 179.7 m 17.2 sem ; p also , the sfct of chm eyes with disrupted ez ( 191.9 m 10.8 sem ) was significantly greater than those with lost ez ( 43.1 m 6.5 sem ; p < 0.0001 ) . scatter plot of sfct as measured on enhanced - depth oct in choroideremia patients with preserved , disrupted or lost ellipsoid zone ( as illustrated by representative oct images above ) versus age and refractive error - matched healthy controls . manual and automated point - to - point alignment between outer retinal morphology on the oct and the edges of af islands showed consistent correspondence between the edge of af and the beginning of an acute decline in outer nuclear layer ( onl ) thickness on the oct , which diminished to zero in a triangular slope over approximately 200 m ( fig . , image alignment analysis was performed using oct and af images from 78 eyes of 39 patients with choroideremia . twenty - nine eyes were excluded from the analysis for the following reasons : ( 1 ) no outer retina remaining on oct ( due to end stage degeneration , n = 15 ) , ( 2 ) poor image quality ( due to poor fixation , n = 8) , and ( 3 ) intact outer retina extending beyond the edge of captured image ( usually in early disease , n = 6 ) ( table ) . in 29 ( 59% ) eyes , an exact correspondence was found between the predicted and measured transfoveal af island width . the ez and elm were often ill defined at the edge of degeneration due to a disrupted appearance on the oct , and were less reliable predictors of the location of the af border . the intersection between two red circles on the af image marking the left - hand border of rpe degeneration ( ai ) , with high magnification view shown ( aii ) , corresponds to the position on the oct indicated by a vertical red line ( aiv ) , while the intersection between green circles marking the right - hand border of rpe degeneration ( ai , aiii ) corresponds to a second position on the oct indicated by a vertical green line ( av ) . the edges of surviving rpe on the oct coincide with the start of an acute decline in onl height , which diminishes to zero in a triangular transition zone , denoted by horizontal yellow bars . immediately beyond the loss of onl , outer retinal tubulations scale bars : 200 m . correlation between the transfoveal island width ( y ) measured on af images and those predicted based on oct outer retinal width ( x ) was performed in 49 choroideremia eyes ( data summarized in the table ) . choroideremia patient data in six eyes of three patients aged 16 , 22 , and 23 years , the residual af islands consisted of an ovoid central zone of relatively preserved af and a peripheral zone of mottled af ( fig . the horizontal diameters of the central ovoid zones with preserved af were individual - specific but non age - dependent : 3050 m od/3180 m os ( age 16 ) , 3250 m od/3050 m os ( age 22 ) , and 2400 m od/2400 m os ( age 23 ) . the mean eccentricities of the zones of preserved af in relation to the anatomic foveal dip were 1533 m ( sem = 179 ) nasally , 1339 ( sem = 157 ) temporally , and 1436 ( sem = 70 ) overall , which are consistent with these zones representing areas of relatively high cone density . correlation between the pattern of af and integrity of the ez . in three young choroideremia patients aged 16 ( a ) , 22 ( b ) , and 23 years ( c ) who have large residual islands remaining on af imaging , central ovoid zones with relatively preserved af were observed , which aligned with regions of preserved ez integrity on the oct ( green arrow bars ) . 3 ) : 71 of 78 ( 91% ) eyes examined demonstrated tubulations ; one eye was not assessable due to poor image quality ( table ) . the six eyes that did not show any tubulations were all at end - stage degeneration with no outer retinal structure remaining . when mapped onto af images , the tubulations seen on oct b - scans were universally located beyond the edges of the af islands ( therefore , overlying bare bruch 's membrane ) , with a mean distance of approximately 400 to 500 m from the nearest island edge , although some were significantly farther away ( fig . even though tubulations appear to be isolated and cystic in appearance on oct b - scans , two - dimensional analysis of their locations showed that many were in fact contiguous with the main island of surviving outer retina via pseudodendritic ' tubular extensions ( fig . these thin pseudodendrites appear to lie over areas devoid of rpe ( by the absence of af ) but are detectable by their retinal pigmentation on high magnification infrared slo images . distribution of outer retinal cysts in six patients with choroideremia aged 16 to 71 years ( [ a f ] in ascending age order ) . the distribution of tubulations could be inferred from the positions of all outer retinal cysts ( orcs ) , which represent cross - sections of tubulations , seen on all b - scans of the oct . outer retinal cysts on oct represent cross - sections through outer retinal tubulations following rpe loss at the edges of degeneration . plotting the locations of orcs ( white dots ) seen on every b - scan of the oct ( horizontal green lines ) over the corresponding high magnification infrared slo image revealed that the orcs lie over thin pseudodendrites ' containing retinal pigment along the edges of the surviving retina ( outlined in dotted blue ) . because tubulations likely represent rearrangement of surviving photoreceptors , we asked whether their distribution would be affected by subretinal injections in gene therapy , because this would provide us with indirect evidence as to whether or not they might have retained synapses to the overlying retina . when the locations of tubulations were compared between baseline , 1 month , and 6 months post aav.rep1 gene therapy , it could be seen that the general distributions of tubulations were not significantly affected by surgically induced retinal detachments ( fig . , while a slight dip in the number of outer retinal cysts seen in some eyes at 1 month could be attributed to mechanical disturbance from the retinal detachment , there was no statistically significant fluctuation over the 6-month period ( linear regression : f(5,6 ) = 1.29 , p = 0.38 ; supplementary fig . these would be in keeping with tubulations maintaining some physical attachment to the inner retina , thereby potentially retaining some visual function over these anatomical anomalies . gene therapy using recombinant aav2-rep1 vector for choroideremia were performed on six eyes ( a f ) in which the residual af islands were surgically detached by subretinal injections . the locations of outer retinal cysts ( white dots ) seen on all b - scans of the octs , which represent cross - sections of tubulations , have been mapped onto the corresponding af images at baseline ( left column ) , 1 month post ( center column ) and 6 months post ( right column ) gene therapy . we were therefore interested to know if there might be any developmental anomalies in choroideremia patients and/or if the subfoveal choroid thinning begins before significant foveal retinal degeneration is evident . subfoveal choroidal thickness was measured in 42 eyes of 23 choroideremia patients ( mean age 37.5 years ; range , 1263 ) and 40 eyes of 20 age - matched healthy male controls ( mean age 37.6 years ; range , 1362 ; supplementary table s1 ) . the mean sfct in healthy controls ( 302.0 m 4.8 sem ) was found to be significantly greater than that in chm eyes with preserved ez ( 179.7 m 17.2 sem ; p also , the sfct of chm eyes with disrupted ez ( 191.9 m 10.8 sem ) was significantly greater than those with lost ez ( 43.1 m 6.5 sem ; p < 0.0001 ) . scatter plot of sfct as measured on enhanced - depth oct in choroideremia patients with preserved , disrupted or lost ellipsoid zone ( as illustrated by representative oct images above ) versus age and refractive error - matched healthy controls . our data revealed a precise anatomical correspondence between rpe and photoreceptor degeneration : the edge of rpe loss colocalized with a sharp decline in onl thickness in the majority of eyes with chm . in the narrow transition zone between intact and degenerate outer retina , the onl was consistently seen to taper in a triangular fashion , a feature that has previously been termed an we hypothesize that this transition zone represents degenerating photoreceptors that reduce in height as they lose outer segments following the death of the interdigitating rpe cells , eventually leading to cell death . the correlation between rpe and photoreceptor integrity was not only observed at the advancing border of degeneration , but also within the islands of surviving outer retina / rpe . ovoid zones of parafoveal preservation of af were seen in some of the youngest choroideremia patients in the cohort , which correlated with transitions in the integrity of the ez . it would also explain the clinical finding of early - onset nyctalopia , but preservation of visual acuity and color perception until late stage degeneration in choroideremia patients . this would be in keeping with observations from postmortem female carriers in which choroidal thinning was only seen in areas of severe retinal degeneration . although choroidal thickness is affected by age , sex , and refractive error , these factors were all similar between the chm and control eyes in the study . nevertheless the reduced choroidal thickness at baseline did not appear to have any obvious effect on visual acuity in the early stages of the disease . not all photoreceptors are lost beyond the edge of rpe degeneration , as some appear to survive by forming outer retinal tubulations , which could extend a considerable distance from the main surviving outer retinal island . while they appear cystic on oct b - scans , mapping their locations to high magnification slo images showed that they most likely represent cross - sections through long tubular structures jutting out from the edges of outer retinal degeneration . tubulations have been described in a range of degenerative retinal disorders , including amd , stargardt disease , central serous retinopathy , serpiginous choroiditis , maternally inherited diabetes and deafness , gyrate atrophy , and choroideremia . they likely represent rearrangement of photoreceptors that have lost their outer segments following the degeneration of the underlying rpe cells , but are still close enough to the residual choroid to derive oxygenation and other diffusible substrates required for metabolic survival . the fact that tubulations represent residual photoreceptors is in keeping with histologic analysis of tubulations in advanced amd , which showed cones lacking outer segments . apart from a slight dip in the total number of outer retinal cysts at 1 month post gene therapy ( likely due to physical disturbance from the surgical retinal detachment ) , the overall numbers and distributions of tubulations were stable over a 6-month period . while this does not completely exclude the possibility of some tubulations disappearing and forming at similar locations , the data provide indirect evidence that tubulations represent stable outer retinal structures , which retain some physical attachment to the retina , rather than being inflammatory in origin . the preservation of tubulations following gene therapy could also be an indication that the surgery did not cause significant disruption of outer retinal integrity , a concern due to possible stretching of the retina during subretinal injection . what remains to be seen , however , is whether or not visual function can be restored in these tubulations following gene therapy and more importantly , whether or not this represents a degree of degeneration in choroideremia that may be arrested .

prodiginines , such as prodigiosin ( 1 ) , undecylprodiginine ( 2 ) , streptorubin b ( 3 ) , and metacycloprodiginine ( 4 ) ( figure 1 ) , are a family of linear and cyclic oligopyrrole red - pigmented compounds with anticancer , antimalarial , and immunosuppressant activities . the biosynthesis of members of this class of natural products has been extensively studied . the biosynthesis of 2 and 3 in streptomyces coelicolor a(3)2 is directed by the red gene cluster and proceeds via a bifurcated pathway that culminates with the formation of two subunits , 2-undecylpyrrole ( 2-up ) and 4-methoxy-[2,2-bipyrrole]-5-carboxaldehyde ( mbc ) ( scheme 1 ) . redh then catalyzes the condensation of these two subunits to generate 2 , and in the final step of the pathway a non - heme iron rieske - oxygense , redg , catalyzes the oxidative cyclization of the alkyl chain of 2 to generate the prodiginine 3 ( scheme 1 ) . a redg homologue in s. longispororuber , named mcpg , catalyzes the cyclization of 2 to 4 ( figure 1 ) . structures of naturally occurring prodiginines ( 14 ) and marineosins ( 5 and 6 ) . red color is for parts of the structure derived from acetate ; green , from glycine and l - serine ; blue , from l - proline . a = adenylation domain , ks = ketide synthase , at = acyltransferase , gt = glycine transferase , st = serine transferase , hbp = 4-hydroxy-5-hydroxymethyl-2,2-bipyrrole , 2-up = 2-undecylpyrrole subunit , mbc = 4-methoxy-[2,2-bipyrrole]-5-carboxaldehyde subunit . marineosins a ( 5 ) and b ( 6 ) ( figure 1 ) are two prodiginine analogues isolated from the marine streptomyces sp . cnq-617 and were shown to have strong and selective anticancer activity . marineosins are novel prodiginines in that they have a different regioselectivity for the alkyl chain cyclization , a partially reduced tripyrrole nucleus , and an oxygen . spiroaminal moieties are present in a number of marine - derived natural products such as crambescidin 800 and crambescidin 816 ( compounds with strong antiviral and antimalarial activities ) but are generally rare in natural products . the intriguing structure and biological activity of marineosins has spurred efforts toward total synthesis attempts . although synthesis of spiroaminal containing compounds has been reported by several research groups , the total synthesis of marineosins is still incomplete . the ability to access marineosin analogues and evaluate their biological activity is thus currently elusive . the unique structure of marineosins can not be directly explained on the basis of the current knowledge of prodiginines biosynthesis and has spurred two disparate biosynthetic hypotheses . the first hypothesis was formation of an enone - undecylprodiginine intermediate that undergoes a hetero diels alder cyclization to yield marineosins a ( 5 ) and b ( 6 ) ( supplementary figure s1 ) . the proposed hetero - diels alder cyclization was subsequently found to be energetically unfavorable . a second hypothesis proposed that a homologue of the non - heme iron rieske oxygenase , redg must be encoded by the marineosin gene cluster ( supplementary figure s2 ) . this redg - homologue consecutively catalyzes the oxidative cyclization of the alkyl chain of 2 to form the macrocyclic ring and hydroxylation of the alkyl chain to initiate the formation of the spiroaminal ring of marineosin . finally , the spiroaminal ring formation is induced by a series of transformations , such as a 1,5-sigmatropic hydride shift , cyclization , and tautomerization ( supplementary figure s2 ) . to date , no experimentally supported biosynthetic pathway for the marineosins has been reported . in this study we sought to identify the marineosin ( mar ) gene cluster , in order to both provide the tools to study the biosynthetic process and access novel marineosin analogues for biological testing . we report cloning , sequencing , and analysis of this cluster and successful production of marineosin by expression of this cluster in a heterologous host . through construction of gene mutants , the final steps in the marineosin biosynthetic process in addition , a number of new marineosin intermediates and shunt metabolites have been purified and characterized , and their antimalarial activity was determined . nmr spectra were measured in acetone - d6 using a bruker amx-600 mhz spectrometer calibrated using residual acetone as an internal standard . high resolution mass spectra analyses and tandem ms analyses were performed using thermoelectron ltq - orbitrap high resolution mass spectrometer . lc ms / uv analyses were performed using a microtof - q mass spectrometer equipped with an agilent 1100 series hplc with mwd detector . hplc purification of marineosin and analogues was performed using either a waters hplc equipped with a dwd detector or an agilent hplc equipped with a mwd detector . topo - pcr8 vector and chemically competent oneshot top10 e. coli were purchased from life technologies and were used in routine cloning and subcloning of plasmid and cosmid dna . e. coli strains used in red / et - mediated recombination were provided by the john innes institute . a list of major strains and cosmids generated in this study can be found in supplementary tables s1 and s2 . all antibiotics , solvents , and hplc grade solvents were purchased from either sigma - aldrich or fisher - scientific . pcr amplifications were carried out using either platinum high - fidelity ( life technologies ) or phusion high - fidelity polymerase ( new england biolabs ) . genomic and plasmid dna purification , restriction analysis , transformation , and conjugation of e. coli and s. venezuelae strains were performed as described . a list of primers used throughout this study can be found in supplementary table s3 . cnq-617 led to the identification of 8a7 cosmid carrying the entire mar gene cluster . the mar cluster was sequenced and annotated and the complete sequence was deposited into genbank ( accession kf711829 ) . the ampicillin - resistance gene bla of 8a7 cosmid was replaced with orit , c31 int , and aac(3)iv to allow for site - specific integration of 8a7 cosmid into the attb site of s. venezuelae genome using red / et - mediated recombination ( supplementary figure s3 ) . gene replacement was confirmed via pcr , and the engineered cosmid was named pmar . the donor e. coli strain harboring the pmar cosmid e. coli et12537/puz8002/pmar was conjugated with wild type s. venezuelae to generate s. venezuelae jnd2 expressing the marineosin gene cluster . for more information about construction of the genomic library seed cultures were used to inoculate 1-l fermentations ( in 4-l baffled flasks ) of s. venezuelae jnd2 at 1% ratio . cultures were then incubated at 30 c and 220 rpm for 7 days , and cells were harvested by centrifugation at 7000 rpm for 10 min . marineosin a ( 5 ) was purified using hplc as specified in supplementary table s4 and s5 . the h nmr , ms , tandem - ms , and lc ms analyses of 5 were compared to that of a standard marineosin sample . the mara and marg genes in the pmar cosmid were replaced with the spectinomycin resistance marker aada gene using red / et mediated recombination to generate pmaraand pmarg cosmids , respectively . pij788 plasmid was used as a template for the amplification of the aada gene using recombination primers specified in supplementary table s3 . gene replacements were confirmed via pcr amplification using outer primers ( supplementary table s3 ) . this was subsequently conjugated with wild type s. venezuelae to generate s. venezuelae jnd2a strain . fresh protoplasts of wild type s. venezuelae were transformed with pmarg cosmid to generate s. venezuelae jnd2g strain . seed cultures of s. venezuelae jnd2a were used to inoculate 60 and 100 ml of scm media in 300- and 500-ml baffled flasks , respectively , supplied with the appropriate antibiotics . seed cultures were used at 1% ratio . it should be noted that no production was observed when larger fermentations were used . the cells were harvested as before , and the cells were extracted with acetone followed by sonication . the filtered , dried crude acetone extract was dissolved in methanol and was partially purified by hplc using a gradient specified in supplementary table s6 . peaks with retention times of 15 and 21 min , corresponding to 16-ketopremarineosin a ( 10 ) and premarineosin a ( 9 ) , respectively , were collected and evaporated under vacuum , and residual water was eliminated via lyophilization . the semipure compounds were subjected to another round of hplc purification using the same specified conditions to yield 1.0 and 0.5 mg of 10 and 9 , respectively . seed cultures were used to inoculate 1-l fermentations ( in 4-l baffled flasks ) of s. venezuelae jnd2g at 1% ratio , and then the fermentations were incubated at 30 c , 220 rpm for 7 days . the acetone extract was dried under vacuum , and the residual water was extracted with ethyl acetate 3 times . the dried extract was dissolved in methanol and purified according to the gradient specified in supplementary table s7a . the peak with retention time of 25 min was collected and evaporated under vacuum , and the residual water was removed by lyophilization . the partially purified sample was dissolved in chloroform and loaded onto 20 cm 20 cm preparative silica gel 60a tlc plates ( whatman ) that were developed using 50% ethyl acetate / hexane 3 times . the top and bottom layers were excised from the tlc plate , extracted with acetone , and then dried under vacuum to yield semipure 23-ketoundecylprodiginine ( 8) and 23-hydroxyundecylprodiginine ( 7 ) , respectively . the individual compounds 7 and 8 were obtained in pure form after final hplc purification using the gradient specified in supplementary table s7b . spores of s. venezuelae jnd2 were propagated onto spa agar containing 50 g / ml apramycin , and spores of s. venezuelae jnd2g and jnd2a were propagated onto spa agar plates containing 50 g / ml apramycin and spectinomycin antibiotics . fresh spores were used to inoculate seed cultures of 10 ml of scm media supplied with the appropriate antibiotics , and the cultures were incubated at 30 c and 220 rpm for 72 h. the cells were harvested by centrifugation at 4000 rpm for 5 min and then extracted with acetone and sonication . the acetone extract was dried under vacuum , and then the crude extract was dissolved in methanol ( 0.5 mg / ml ) to be used in ms and lc ms / uv analysis according to protocols in supplementary tables s8s10 . analyses were done in comparison to extracts of wild type s. venezuelae prepared under the same conditions . we hypothesized that the streptorubin b biosynthetic pathway ( scheme 1 ) and marineosin pathway would have strong similarities and that the differences between the two structures would likely be in the formation of the 2-up subunit ( scheme 1 ) and in the latter stages of the biosynthetic process . the similarities in the structures suggested they likely follow an identical route to the mbc subunit . the biosynthesis of the mbc subunit of undecylprodiginine ( 2 ) in s. coelicolor a(3)2 starts with the oxidation of the amino acid l - proline into pyrroline in a step catalyzed by the acyl - coa dehydrogenase redw ( scheme 1 ) . thus a p - labeled redw probe was used to screen a supercos1 genomic library of streptomyces sp . cnq-617 ( as described in the experimental section ) . a secondary screen to determine if these cosmids contained other red homologues was then carried out . degenerate pcr primers for redg , which encodes a non - heme iron rieske oxygenase ( redg ) that catalyzes oxidative cyclization of undecylprodiginine ( 2 ) into streptorubin b ( 3 ) , were used on the basis that there must be a redg homologue in the marineosin pathway . the 15 positive cosmids from redw screening of the genomic cosmid library of streptomyces cnq-617 were thus screened using marg_fwd and marg_rev primers ( supplementary table s3 ) , leading to the identification of the 8a7 cosmid . shotgun sequencing of the 8a7 insert revealed 21 genes with strong homology to the red biosynthetic genes . not only was every gene involved in the streptorubin biosynthetic process present in the putative mar gene cluster , but the gene organization was identical ( figure 2 ) . the homology between the red and mar gene products varied between 53 and 79% ( table 1 ) . the lowest homology ( less than 60% ) was noticed among regulatory and hypothetical proteins such as redd redy , redz , redv , and redu and their respective mar homologues . the highest homology was noticed between redr and redp and their respective mar homologues , marr and marp ( 79% and 75% , respectively ) . it should be noted that redp and redr initiate the biosynthesis of the 2-up subunit of 2 ( scheme 1 ) , suggesting that the mar and red pathways are probably identical at these steps . other proteins involved in the biosynthesis of the 2-up subunit such as redq , redj , and redl share a homology of 6169% relative to their mar counterparts ( table 1 ) . most of the genes involved in the biosynthesis of the mbc subunit of 2 ( scheme 1 ) such as redw , redo , redn , redm , and redi share a homology of 6979% with the mar proteins ( table 1 ) . the pep - utilizing redh protein ( responsible for the condensation of the 2-up and mbc subunits ) has a homology of 70% to marh , while the rieske oxygenase redg ( responsible for the oxidative cyclization of 2 to 3 ) has a homology of 64% to the marg homologue ( table 1 ) . sequencing of the 8a7 insert also revealed the presence of 6 kbp of primary metabolic genes at the 3 end of the mar gene cluster . at the far end of the cluster and before these primary metabolic genes was a gene that we designated mara . a conserved domain analysis of the primary amino acid sequence of mara using conserved domain database ( cdd ) suggests that mara belongs to the hot - dog superfamily of proteins that is functionally divided mainly into dehydratases / hydratases and thioesterases . no other apparent homologies to known proteins or conserved domains were noticed from the primary amino acid sequence of mara ( supplementary figure s4 ) . comparison between the mar gene cluster ( top panel ) and the red cluster ( bottom ) . red color is for regulatory proteins ; green , involved in the biosynthesis of the mbc subunit ; dark blue , involved in the biosynthesis of the 2-up subunit ; light blue , condensation of the mbc and 2-up subunits ; orange , oxidative cyclization ; yellow , pyrrole reduction ; gray , membrane protein ; black , hypothetical proteins . spa agar plates showing change in phenotype of wild type s. venezuelae ( left ) from gray to red color as a result of introduction of the mar gene cluster , yielding the jnd2 strain ( right ) . the striking similarity between the putative mar cluster and the red cluster and in particular the presence of homologues of every biosynthetic enzyme raised the question of whether it encoded marineosin biosynthesis ( rather than undecylprodiginine biosynthesis ) , and if so , whether all of the necessary genes were present . to investigate this , we sought to move the entire mar gene cluster into a heterologous host to determine the products encoded by the cluster . to facilitate this transfer , the bla gene of 8a7 cosmid was replaced with attp attachment site , integrase gene of phage c31 , and aac(3)iv gene ( conferring resistance to apramycin ) . the resulting pmar cosmid was capable of site - specific integration into another streptomycete . the pmar cosmid was transformed into e. coli et12537 harboring puz8002 plasmid to be used in conjugation with wild type s. venezuelae , to ultimately provide the apramycin resistant strain s. venezuelae jnd2 ( supplementary figure s3 ) . the engineered jnd2 strain grew and sporulated in a manner similar to the wild type s. venezuelae but was characterized by a distinctive red color ( figure 3 ) . the marineosins , by virtue of the partially reduced middle pyrrole , do not have a red color . the jnd2 strain was grown under standard shake flask conditions for production of the natural products pikromycin and methymycin . an ms profile of the acetone extract of jnd2 strain showed accumulation of not only these natural products but also a compound not present in wild type s. venezuelae with [ m + h ] m / z 410.28 , identical to that reported for marineosin ( supplementary figure s5 ) . analysis by hplc revealed that the compound has the same retention time ( supplementary figure s6 ) and uv profiles ( max 320 nm ) as a marineosin standard . the compound was further purified , and its identity was confirmed as marineosin a ( 5 ) by spectroscopic analysis including ms / ms and h nmr ( supplementary figures s7s9 ) . the marineosin a ( 5 ) yields from the jnd2 strain were approximately 5 mg / l . yields of marineosins from streptomyces cnq-617 in contrast were reported as 0.5 mg / l . a compound with the same mass [ m + h ] m / z 410.3 was also observed in a very minor quantity after the peak of marineosin a ( 5 ) ( figure 4 ) and was not purified and characterized but is likely marineosin b ( 6 ) . the production of marineosin a ( 5 ) and b ( 6 ) in the jnd2 strain provides unequivocal evidence that the mar cluster encodes marineosin biosynthesis and that this pathway proceeds in a manner analogous at many steps to that of other prodiginines . in addition to marineosins , the lc ms / uv profile of the jnd2 strain ( figure 4 ) indicates the accumulation of a myriad of pathway intermediates and , possibly , shunt - metabolites . there were no reports of additional compounds being observed in the streptomyces cnq-617 host , and the presence of these may be a result of either higher production yields or other factors associated with expression in s. venenzuelae . two groups of compounds are accumulated due to expression of the marineosin gene cluster . the first group consists of red - colored compounds ( 7 , 8 , and mixture of other compounds ) with max of 530 nm ( blue chromatogram ) , while the second group consists of marineosin a ( 5 ) , b ( 6 ) , premarineosin a and b ( 9 and 11 , respectively ) , and shunt metabolites ( 10 and 12 ) with max of 320 nm ( orange chromatogram ) . the manner in which key structural differences of marineosins arise was not clear from an examination of the mar gene cluster alone . to answer these questions , we looked at the additional compounds generated by the jnd2 strain and by derivatives of the jnd2 strain in which key mar genes had been replaced . two groups of compounds were identified in the jnd2 strain , a group of conjugated red - colored compounds with max of 530 nm characteristic to prodiginines and a group of nonconjugated compounds with max of 320 nm similar to that of marineosin ( figure 4 ) . we posited that if the red - colored compounds in the jnd2 strain are pathway intermediates ( rather than shunt metabolites ) , the reduction of middle pyrrole ( ring b ) and hence loss of aromatic ring conjugation of marineosin would be one of the final steps in the biosynthetic process , and thus the biosynthesis of marineosin would proceed via an intermediate analogue of 2 . the main compounds within this group were 7 and 8 ( figure 5 ) with [ m + h ] m / z 410.28 , and m / z 408.26 , respectively . compounds with the same mass and retention time were subsequently purified from a jnd2g mutant strain ( see below ) and shown to be 23-hydroxyundecylprodiginine ( 7 ) and 23-ketoundecylprodiginine ( 8) , respectively . no detectable levels of production of undecylprodiginine ( 2 ) were observed in extracts of jnd2 fermentations . the group of compounds characterized with max of 320 nm comprises marineosin a ( 5 ) and b ( 6 ) and the four other analogues 9 , 10 , 11 , and 12 ( figure 5 ) , with [ m + h ] m / z 408.26 , 422.24 , 408.26 , and 424.26 with corresponding molecular formulas of c25h34o2n3 , c25h32o3n3 , c25h34o2n3 , and c25h34o3n3 . compounds with the same mass and retention time as 9 and 10 were subsequently isolated from a jnd2a mutant and were shown to be premarineosin a ( 9 ) and a 16-ketopremarineosin a ( 10 ) , respectively . compounds 11 and 12 were not purified and characterized but are likely premarineosin b and 16-hydroxypremarineosin a , respectively ( figure 5 ) . structures of isolated compounds 710 and expected compounds 11 and 12 from s. venezuelae jnd2g and jnd2a . the most notable difference between the mar and red gene clusters is the presence of mara . we posited that this was required for marineosin biosynthesis , and that mara might catalyze formation of the spiroaminal ring via an intramolecular hydroalkoxylation reaction . to test this hypothesis we replaced mara in pmar cosmid with the spectinomycin resistance aada gene . the resulting pmara was transformed into e. coli et12537/puz8002 and then used in conjugation with wild type s. venezuelae to provide s. venezuelae jnd2a . this strain is both apramycin- and spectinomycin - resistant and has a fainter red color when compared to either the jnd2 or jnd2g strains ( see below ) . marineosin production was not observed in this mutant demonstrating that mara is essential for biosynthesis of marineosin ( supplementary figure s10a ) . however , lc ms analysis of the acetone extract of jnd2a strain ( supplementary figure s10b ) indicates the accumulation of two major and one minor compounds with max 320 nm , premarineosin a ( 9 ) , 16-ketopremarineosin a ( 10 ) , and premarineosin b ( 11 ) with [ m + h ] m / z 408.2660 , m / z 422.2450 , and m / z 408.26 and corresponding molecular formulas c25h34o2n3 , c25h32o3n3 and c25h34o2n3 respectively ( supplementary figure s1114 ) . these three compounds were also observed in the acetone extract of jnd2 strain ( figure 4 ) . larger scale fermentations of s. venezuelae jnd2a permitted both 9 and 10 to be purified and characterized by ir , ms , and extensive 2d nmr experiments . the mass spectral analysis of 9 indicated two fewer hydrogens than the marineosins ( 5 and 6 ) , while 10 has four fewer hydrogens and additional oxygen . the ir spectra of 9 and 10 are almost identical except for the presence of a peak at 1705 cm in the ir spectrum of 10 indicating the presence of a carbonyl ( c = o ) group ( supplementary figure s15 ) . one of the significant differences in the h nmr spectra of 9 , 10 , and marineosin is the appearance of six aromatic protons in both 9 and 10 instead of the five protons in marineosin . cosy and tocsy correlations among aromatic protons at c-1 , c-2 , and c-3 as well as those at c-11 and c-12 confirm that the six protons form the tripyrrole nucleus ( figure 5 ) . the cosy correlations between protons at c-9 and c-21 that are extended to c-20 and c-22 in the tocsy of both 9 and 10 confirm the presence of a macrocyclic ring between c-9 and c-21 ( figure 5 ) . the same macrocyclic ring in 10 was confirmed by the hmbc correlations between the proton at c-9 and carbons c-7 , c-8 , c-10 , c-11 , c-20 , c-21 , and c-22 ( figure 6 ) . the hsqc data ( supplementary figure s19 ) suggest that compounds 9 and 10 have eight and seven methylenes in macrocyclic and spiroaminal rings , respectively , which suggests the keto group of 10 has replaced one of the methylene carbons of 9 . in compound 10 , hmbc correlations between a carbonyl carbon at c 211.3 ppm and protons at c-17 and c-14 suggested that the carbonyl group observed in the ir spectrum of 10 exists in the vicinity of c-17 and c-14 ( figure 6 ) . the tocsy spectrum of 10 shows strong correlations between protons at c-14 and c-15 , but these correlations are not extended to any other proton in the macrocyclic skeleton of 10 , suggesting that c-15 is next to either a heteroatom or a quaternary carbon . also , protons at c-15 and c-17 in 10 are slightly shifted downfield relative to their counterparts in 9 ( supplementary table s14 ) . therefore , the keto group of 10 was assigned at c-16 ( figure 6 ) . the methoxy group of 9 and 10 is connected to c-7 on the basis of hmbc correlations from och3 - 25 to c-7 and h-9 to c-7 . the c-7 quaternary carbon appears in the most downfield region , which suggests the double bond exists between c-6 and c-7 ( ring b is not reduced ) ; this is the only significant difference between marineosins ( 5 and 6 ) and premarineosin a ( 9 ) . the proton at c-9 demonstrated hmbc correlations to a quaternary carbon at c-8 , suggesting the presence of a spiro carbon , and the oxygen exists between c-8 and c-23 in both 9 and 10 . full spectral data and correlations of 9 and 10 are presented in detail in supporting information ( figures s1124 and tables s1114 ) . collectively this work is consistent with a structure for 9 identical to that of marineosin a ( 5 ) , but with a double bond present between c-6 and c-7 . a compound ( 11 ) with the same mass [ m + h ] m / z 408.26 ( figure 5 ) was observed in a very minor quantity after the peak of 9 in the lc ms / uv of both jnd2a ( supplementary figure s10b ) and jnd2 strain ( figure 4 ) . although compound 11 was not purified due to low quantity , it is likely to be a diasteriomer of 9 . the production of 9 clearly demonstrates that mara is not required for formation of the spiroaminal ring . rather , mara is likely required for reduction of the c6c7 olefin ( although a clear nadh / nadph binding site could not be identified from the predicted primary mara sequence ) as the last step in the marineosin biosynthetic pathway and led us to name these compounds premarineosin a ( 9 ) and premarineosin b ( 11 ) ( scheme 2 ) . the presence of diasteromeric premarineosins indicate that the alternative stereochemical configuration at the spiroaminal carbon ( c8 ) is established before the final biosynthetic step . efforts thus far to obtain a soluble recombinant mara to verify this activity in vitro have been unsuccessful . the analyses also show that compound 10 is a 16-ketopremarineosin a , which probably arises from c16-hydroxylation and oxidation of premarineosin a. compound 12 ( figures 4 and 5 ) was also not purified due to low quantity but is likely the 16-hydroxypremarineosin a. both of these compounds are likely shunt metabolites of the main marineosin pathway and may arise either from the action of mar proteins or enzymes present in the s. venezuelae host . selected hc hmbc correlations for 8 and 10 . the formation of premarineosins ( 9 and 11 ) with loss of mara demonstrated that another gene product must be responsible for formation of the spiroaminal ring . furthermore , this step , formation of the c8o c23 bond , and introduction of the hydroxyl group must occur at an earlier step in the pathway . we hypothesized that marg would likely be responsible for one if not several of these steps . this hypothesis was based on the observation that marg has homology ( 64% ) to redg , which catalyzes the oxidative cyclization in the streptorubin b pathway ( in this case a cyclization between c-11 and c-20 of 2 ) , and was consistent with an earlier hypothesis that a redg homologue introduces the hydroxyl group in the marineosin structure . thus we sought the replacement of marg gene in the pmar cosmid with aada gene , which confers resistance to spectinomycin via red / et - mediated recombination , to generate the pmarg cosmid . the pmarg cosmid was subsequently transformed into protoplasts of s. venezuelae to generate the jnd2g strain . the new strain is both apramycin- and spectinomycin - resistant and is characterized by a dark red color when grown on both solid and liquid media . ms analysis of the acetone extract of jnd2g strain indicates the accumulation of two major red - colored compounds , 23-hydroxyundecylprodiginine ( 7 ) and 23-ketoundecylprodiginine ( 8) ( figure 5 ) , with [ m + h ] m / z 410.2807 and 408.2622 , which correspond to the molecular formulae c25h36o2n3 and c25h34o2n3 respectively . compounds 7 and 8 were also observed in the acetone extract of jnd2 strain ( figure 4 and supplementary figure s25 ) . marineosin production in this mutant was not observed , which demonstrates that marg , like mara , is essential for marineosin biosynthesis . premarineosins ( 9 and 11 ) and 16-ketopremarineosin a ( 10 ) were also not observed in the jnd2g strain . indeed , lc ms / uv analysis of jnd2g strain demonstrates the accumulation of only red - colored metabolites ( supplementary figure s26 ) . these observations provide clear evidence that the biosynthesis of marineosin proceeds as a conjugated tripyrrole intermediate that is reduced in a step subsequent to marg - catalyzed cyclization . the pure compounds 7 and 8 were obtained by consecutive chromatography on preparative hplc and preparative tlc . extensive 1d , 2d nmr studies and side by side comparison with 2 were carried out to elucidate the structures of 7 and 8 ( figure 5 ) . the h nmr spectra of 7 and 8 show seven protons at the aromatic region and a singlet at h 3.91 ppm ( 3h ) and h 4.12 ppm ( 3h ) , respectively . this is similar to h nmr data of 2 ( supplementary table s15 ) , where these proton signals are components of three pyrrole rings with ome on the middle pyrrole . one of the significant differences in the h nmr spectra of 7 and 2 is the appearance of an extra multiplet at h 3.67 ppm ( 1h ) and one doublet at h 1.09 ppm ( 3h ) in 7 , instead of the triplet of terminal methyl in 2 ( supplementary figure s31 and table s15 ) . the multiplet at h 3.67 ppm ( c-23 ) has correlations with h 1.09 ppm ( c-24 ) and h 1.34 ppm ( c-22 ) . the h 1.09 ppm ( c-24 ) doublet has only one correlation with 3.67 ppm ( c-23 ) in the cosy spectrum of 7 ( supplementary figure s32 ) , supporting the position of the -oh group at c-23 . tocsy correlations among protons at c-21 , c-22 , c-23 , and c-24 also supported the position of the -oh group at c-23 ( supplementary figure s33 ) . the significant difference in the h nmr spectra of 8 and 2 is the appearance of one new triplet at h 2.46 ppm ( 2h ) , and one singlet at h 2.04 ppm ( 3h ) in 8 , instead of the triplet of terminal methyl in 2 ( supplementary tables s15 and s16 ) . the hsqc spectra confirmed the new methylene at 42.0 ppm and terminal methyl at 26.9 ppm . in the hmbc spectrum , h 2.46 ppm ( 2h ) protons show correlations with c-24 ( h 2.04 , c 26.9 ppm ) , c-23 ( carbonyl carbon , c 207.1 ppm ) , and c-21 ( h 1.30 , c 23.6 ppm ) ; conversely , h 2.04 ppm ( 3h ) protons show a strong correlation with carbonyl carbon c 207.1 ( c-23 ) ( figure 6 ) ( supplementary table s16 ) . in addition , the h h correlations between protons of c-22 and c-24 in cosy and tocsy are missing , which strongly supports the carbonyl functional group position at c-23 ( supplementary figures s36 and s37 ) . together these data support the structure assignment of 23-hydroxyundecylprodiginine ( 7 ) and 23-ketoundecylprodiginine ( 8) . in addition , total syntheses of 7 and 8 were also undertaken and these compounds were shown to be spectroscopically and chromatographically identical ( to be reported elsewhere ) . the accumulation of 23-hydroxy- ( 7 ) , and 23-keto - undecylprodiginines ( 8) ( figure 5 ) as a result of marg gene deletion indicates that marg is essential for the formation of macrocyclic and spiro - tetrahydropyran - aminal rings but is not responsible for the introduction of the oxygen of the spiro - tetrahydropyran ring as postulated by snider and co - workers . sequence alignments of marg with other c c bond - forming non - heme iron rieske oxygenases such as redg , mcpg , and rphg involved in the biosynthesis of natural products 3 , 4 , and roseophilin , respectively , together with hydroxylating non - heme iron rieske oxygneases show that all have conserved n - terminal cxh and cxxh motifs . however , marg and its homologues have an exhx4h motif at their c - terminal end rather than the dxhx4h motif characteristic to rieske hydroxylases ( supplementary figure s40 ) . an aspartate to glutamate mutation of the dxhx4h motif of naphthalene dioxygenase yields an inactive enzyme . it might be that the exhx4h motif in marg and its homologues is essential to avoid side hydroxylation reactions and achieve selective c h bond activation and specific c c bond formation . the lack of spiroaminal ring - containing intermediates in the jnd2g strain demonstrates that the spiroaminal ring formation occurs in the last steps of biosynthesis and must either be part of a reaction catalyzed by marg or requires the marg catalyzed formation of c9c21 macrocycle prior to spiroaminal ring closure ( scheme 2 ) . preliminary experiments feeding 7 to a s. venezuelae host with a marg expression plasmid have demonstrated production of 9 and 10 . this data supports the role of marg in catalyzing formation of premarineosins directly from 7 and indicates 8 is a shunt metabolite ( the deuterium label would be lost in a pathway that proceeded through 8) ( supplementary figure s41 ) . determination of the major compounds that accumulate in s. venezuelae jnd2 and the mix of these that are found in the jnd2g and jnd2a strains provide the first experimental evidence to delineate key steps in the marineosin biosynthetic pathway . the data do not support either previous hypotheses , in which the pathway either passes through an enone analogue of undecylprodiginine or involves a hydroxylation of undecylprodiginine by a redg homologue ( marg ) . rather the pathway involves formation of 23-hydroxyundecylprodiginine ( 7 ) , in a step that precedes the reactions catalyzed by marg . the hydroxyl ( -oh ) group of 7 can be either introduced early in the pathway due to the hydroxylation of the 2-up subunit by an unidentified oxidase ( route 1 , scheme 2 ) , via recruitment of 2-hydroxybutyric acid starter unit by marp ( route 2 , scheme 2 ) or introduced later via hydroxylation of 2 ( route 3 , scheme 2 ) . compound 2 was never detected in any of the strains generated in this study . this observation does not prove that incorporation of the hydroxyl group of 7 occurs early in the pathway as it may be that hydroxylation of 2 occurs faster than its formation ( so the intermediate can not be detected ) . in any case , the gene product(s ) required for introduction of the hydroxyl group remain unknown . the 23-ketoundecylprodiginine ( 8) is an oxidative product of 7 and is a shunt metabolite . the marineosin pathway proceeds from 7 to premarineosins ( 9 and 11 ) in a step that requires marg ( scheme 2 ) . the pathway most likely proceeds from 7 , after an oxidative c9c21 cyclization ( analogous to the oxidative cyclization reaction catalyzed by redg ) . in this case there would be a subsequent intramolecular hydroalkoxylation at c-8 ( which may be spontaneous or catalyzed by marg ) . the 16-keto analogue , 10 , of 9 is most likely a shunt metabolite that comes from hydroxylation and oxidation of 9 . a 16-keto analogue of either 5 or 7 was not observed in any of the strains . in the proposed pathway the stereochemistry at c-8 is established during the conversion of 7 to 9 and 11 . if the proposed pathway ( intramolecular hydroalkoxylation at c-8 ) proceeds enzymatically ( catalyzed by marg ) , then the premarineosin a ( 9 ) might be the intermediate of marineosins a and b. it is noteworthy that the premarineosin b ( 11 ) most likely arises from an inversion of the aminal nitrogen of premarineosin a ( 9 ) under pathway conditions where the two isomers differ only in stereochemistry at the spiro aminal carbon ( c-8 ) . the premarineosin a ( 9 ) exists in higher ratio owing to an anomeric effect as demonstrated for marineosin a ( 5 ) , and this trend follows in the higher production of marineosin a ( 5 ) in the jnd2 strain . the final step of the pathway is a mara catalyzed reduction of premarineosins ( 9 and 11 ) ( scheme 2 ) . the generation of marineosin pathway intermediates and shunt metabolites provided new compounds to test for biological activity . additional compounds may be generated via established methodologies such as mutasynthesis and creation of hybrid pathways . our interest has been the antimalarial activity of prodiginine - type compounds . in a continuation of this work we determined the biological activity of 9 , 10 , and the compounds were evaluated for antimalarial activity against the chloroquine - sensitive ( cq ) d6 and the chloroquine - resistant ( cq ) dd2 and 7g8 strains of p. falciparum with chloroquine ( cq ) as a reference drug . premarineosin a ( 9 ) showed potent antimalarial activity against d6 ( ic50 = 2.3 nm ) , dd2 ( ic50 = 12 nm ) , and 7g8 ( ic50 = 1.5 nm ) strains and was more effective than chloroquine ( d6 ( ic50 = 14.3 nm ) , dd2 ( ic50 = 110 nm ) , and 7g8 ( ic50 = 131 nm ) ) or other tested compounds . the activity of 9 compares favorably with the most potent synthetic and naturally occurring prodiginines . the activity of marineosin a ( 5 ) against d6 ( ic50 = 138 nm ) , dd2 ( ic50 = 127 nm ) , and 7g8 ( ic50 = 199 nm ) was poorer than that of 9 , demonstrating that the oxidized middle pyrrole ring is required for maximal activity . the presence of a keto group in the macrocyclic ring of 10 also decreases the antimalarial activity ( d6 ( ic50 = 138 nm ) , dd2 ( ic50 = 127 nm ) , and 7g8 ( ic50 = 199 nm ) ) by 100 times when compared to 9 . the ability of 9 to inhibit the hepatocellular hepg2 cancer cell line was used to assess its cytotoxicity and hence selectivity . compound 9 has an ic50 = 4169 nm against hepg2 cells , giving it a large therapeutic index ranging from 347 ( for the dd2 strain ) to 2779 ( for the 7g8 strain ) , indicating very selective antimalarial activity . the final steps of the marineosin biosynthetic pathway have now been experimentally established . the genes and tools for an in - depth investigation of a new class of reduced prodiginines as well as spiroaminal natural products have been discovered and developed . notable in this regard is the class of c c bond - forming non - heme rieske iron monoxygenases such as marg , which could be involved in the biosynthesis of other spiroaminal natural products . new marineosin and undecylprodiginine analogues have been identified , characterized , and purified , and routes to obtain additional compounds are possible . the generation of marineosin pathway absent the final mara catalyzed step has led to the isolation and identification of a novel spiroaminal prodiginine with potent antimalarial activities .

the marine streptomyces sp . cnq-617 produces two diastereomers , marineosins a and b. these are structurally related to alkyl prodiginines , but with a more complex cyclization and an unusual spiroaminal skeleton . we report the identification of the mar biosynthetic gene cluster and demonstrate production of marineosins through heterologous expression in a s. venezuelae host named jnd2 . the mar cluster shares the same gene organization and has high homology to the genes of the red cluster ( which directs the biosynthesis of undecylprodiginine ) but contains an additional gene , named mara . replacement of mara in the jnd2 strain leads to the accumulation of premarineosin , which is identical to marineosin with the exception that the middle pyrrole ( ring b ) has not been reduced . the final step of the marineosin pathway is thus a mara catalyzed reduction of this ring . replacement of marg ( a homologue of redg that directs undecylprodiginine cyclization to give streptorubin b ) in the jnd2 strain leads to the loss of all spiroaminal products and the accumulation of 23-hydroxyundecylprodiginine and a shunt product , 23-ketoundecylprodiginine . marg thus catalyzes the penultimate step of the marineosin pathway catalyzing conversion of 23-hydroxyundecylprodiginine to premarineosin . the preceding steps of the biosynthetic marineosin pathway likely mirror that in the red - directed biosynthetic process , with the exception of the introduction of the hydroxyl functionality required for spiroaminal formation . this work presents the first experimentally supported scheme for biosynthesis of marineosin and provides a new biologically active molecule , premarineosin .

Introduction Experimental Section Results and Discussion Conclusions

the biosynthesis of 2 and 3 in streptomyces coelicolor a(3)2 is directed by the red gene cluster and proceeds via a bifurcated pathway that culminates with the formation of two subunits , 2-undecylpyrrole ( 2-up ) and 4-methoxy-[2,2-bipyrrole]-5-carboxaldehyde ( mbc ) ( scheme 1 ) . redh then catalyzes the condensation of these two subunits to generate 2 , and in the final step of the pathway a non - heme iron rieske - oxygense , redg , catalyzes the oxidative cyclization of the alkyl chain of 2 to generate the prodiginine 3 ( scheme 1 ) . marineosins a ( 5 ) and b ( 6 ) ( figure 1 ) are two prodiginine analogues isolated from the marine streptomyces sp . the first hypothesis was formation of an enone - undecylprodiginine intermediate that undergoes a hetero diels alder cyclization to yield marineosins a ( 5 ) and b ( 6 ) ( supplementary figure s1 ) . a second hypothesis proposed that a homologue of the non - heme iron rieske oxygenase , redg must be encoded by the marineosin gene cluster ( supplementary figure s2 ) . in this study we sought to identify the marineosin ( mar ) gene cluster , in order to both provide the tools to study the biosynthetic process and access novel marineosin analogues for biological testing . we report cloning , sequencing , and analysis of this cluster and successful production of marineosin by expression of this cluster in a heterologous host . through construction of gene mutants , the final steps in the marineosin biosynthetic process in addition , a number of new marineosin intermediates and shunt metabolites have been purified and characterized , and their antimalarial activity was determined . cnq-617 led to the identification of 8a7 cosmid carrying the entire mar gene cluster . we hypothesized that the streptorubin b biosynthetic pathway ( scheme 1 ) and marineosin pathway would have strong similarities and that the differences between the two structures would likely be in the formation of the 2-up subunit ( scheme 1 ) and in the latter stages of the biosynthetic process . the biosynthesis of the mbc subunit of undecylprodiginine ( 2 ) in s. coelicolor a(3)2 starts with the oxidation of the amino acid l - proline into pyrroline in a step catalyzed by the acyl - coa dehydrogenase redw ( scheme 1 ) . degenerate pcr primers for redg , which encodes a non - heme iron rieske oxygenase ( redg ) that catalyzes oxidative cyclization of undecylprodiginine ( 2 ) into streptorubin b ( 3 ) , were used on the basis that there must be a redg homologue in the marineosin pathway . the 15 positive cosmids from redw screening of the genomic cosmid library of streptomyces cnq-617 were thus screened using marg_fwd and marg_rev primers ( supplementary table s3 ) , leading to the identification of the 8a7 cosmid . shotgun sequencing of the 8a7 insert revealed 21 genes with strong homology to the red biosynthetic genes . not only was every gene involved in the streptorubin biosynthetic process present in the putative mar gene cluster , but the gene organization was identical ( figure 2 ) . it should be noted that redp and redr initiate the biosynthesis of the 2-up subunit of 2 ( scheme 1 ) , suggesting that the mar and red pathways are probably identical at these steps . other proteins involved in the biosynthesis of the 2-up subunit such as redq , redj , and redl share a homology of 6169% relative to their mar counterparts ( table 1 ) . most of the genes involved in the biosynthesis of the mbc subunit of 2 ( scheme 1 ) such as redw , redo , redn , redm , and redi share a homology of 6979% with the mar proteins ( table 1 ) . a conserved domain analysis of the primary amino acid sequence of mara using conserved domain database ( cdd ) suggests that mara belongs to the hot - dog superfamily of proteins that is functionally divided mainly into dehydratases / hydratases and thioesterases . comparison between the mar gene cluster ( top panel ) and the red cluster ( bottom ) . red color is for regulatory proteins ; green , involved in the biosynthesis of the mbc subunit ; dark blue , involved in the biosynthesis of the 2-up subunit ; light blue , condensation of the mbc and 2-up subunits ; orange , oxidative cyclization ; yellow , pyrrole reduction ; gray , membrane protein ; black , hypothetical proteins . spa agar plates showing change in phenotype of wild type s. venezuelae ( left ) from gray to red color as a result of introduction of the mar gene cluster , yielding the jnd2 strain ( right ) . the striking similarity between the putative mar cluster and the red cluster and in particular the presence of homologues of every biosynthetic enzyme raised the question of whether it encoded marineosin biosynthesis ( rather than undecylprodiginine biosynthesis ) , and if so , whether all of the necessary genes were present . the engineered jnd2 strain grew and sporulated in a manner similar to the wild type s. venezuelae but was characterized by a distinctive red color ( figure 3 ) . the jnd2 strain was grown under standard shake flask conditions for production of the natural products pikromycin and methymycin . an ms profile of the acetone extract of jnd2 strain showed accumulation of not only these natural products but also a compound not present in wild type s. venezuelae with [ m + h ] m / z 410.28 , identical to that reported for marineosin ( supplementary figure s5 ) . a compound with the same mass [ m + h ] m / z 410.3 was also observed in a very minor quantity after the peak of marineosin a ( 5 ) ( figure 4 ) and was not purified and characterized but is likely marineosin b ( 6 ) . the production of marineosin a ( 5 ) and b ( 6 ) in the jnd2 strain provides unequivocal evidence that the mar cluster encodes marineosin biosynthesis and that this pathway proceeds in a manner analogous at many steps to that of other prodiginines . in addition to marineosins , the lc ms / uv profile of the jnd2 strain ( figure 4 ) indicates the accumulation of a myriad of pathway intermediates and , possibly , shunt - metabolites . there were no reports of additional compounds being observed in the streptomyces cnq-617 host , and the presence of these may be a result of either higher production yields or other factors associated with expression in s. venenzuelae . the first group consists of red - colored compounds ( 7 , 8 , and mixture of other compounds ) with max of 530 nm ( blue chromatogram ) , while the second group consists of marineosin a ( 5 ) , b ( 6 ) , premarineosin a and b ( 9 and 11 , respectively ) , and shunt metabolites ( 10 and 12 ) with max of 320 nm ( orange chromatogram ) . the manner in which key structural differences of marineosins arise was not clear from an examination of the mar gene cluster alone . two groups of compounds were identified in the jnd2 strain , a group of conjugated red - colored compounds with max of 530 nm characteristic to prodiginines and a group of nonconjugated compounds with max of 320 nm similar to that of marineosin ( figure 4 ) . we posited that if the red - colored compounds in the jnd2 strain are pathway intermediates ( rather than shunt metabolites ) , the reduction of middle pyrrole ( ring b ) and hence loss of aromatic ring conjugation of marineosin would be one of the final steps in the biosynthetic process , and thus the biosynthesis of marineosin would proceed via an intermediate analogue of 2 . the group of compounds characterized with max of 320 nm comprises marineosin a ( 5 ) and b ( 6 ) and the four other analogues 9 , 10 , 11 , and 12 ( figure 5 ) , with [ m + h ] m / z 408.26 , 422.24 , 408.26 , and 424.26 with corresponding molecular formulas of c25h34o2n3 , c25h32o3n3 , c25h34o2n3 , and c25h34o3n3 . compounds with the same mass and retention time as 9 and 10 were subsequently isolated from a jnd2a mutant and were shown to be premarineosin a ( 9 ) and a 16-ketopremarineosin a ( 10 ) , respectively . marineosin production was not observed in this mutant demonstrating that mara is essential for biosynthesis of marineosin ( supplementary figure s10a ) . however , lc ms analysis of the acetone extract of jnd2a strain ( supplementary figure s10b ) indicates the accumulation of two major and one minor compounds with max 320 nm , premarineosin a ( 9 ) , 16-ketopremarineosin a ( 10 ) , and premarineosin b ( 11 ) with [ m + h ] m / z 408.2660 , m / z 422.2450 , and m / z 408.26 and corresponding molecular formulas c25h34o2n3 , c25h32o3n3 and c25h34o2n3 respectively ( supplementary figure s1114 ) . the c-7 quaternary carbon appears in the most downfield region , which suggests the double bond exists between c-6 and c-7 ( ring b is not reduced ) ; this is the only significant difference between marineosins ( 5 and 6 ) and premarineosin a ( 9 ) . collectively this work is consistent with a structure for 9 identical to that of marineosin a ( 5 ) , but with a double bond present between c-6 and c-7 . a compound ( 11 ) with the same mass [ m + h ] m / z 408.26 ( figure 5 ) was observed in a very minor quantity after the peak of 9 in the lc ms / uv of both jnd2a ( supplementary figure s10b ) and jnd2 strain ( figure 4 ) . rather , mara is likely required for reduction of the c6c7 olefin ( although a clear nadh / nadph binding site could not be identified from the predicted primary mara sequence ) as the last step in the marineosin biosynthetic pathway and led us to name these compounds premarineosin a ( 9 ) and premarineosin b ( 11 ) ( scheme 2 ) . the analyses also show that compound 10 is a 16-ketopremarineosin a , which probably arises from c16-hydroxylation and oxidation of premarineosin a. compound 12 ( figures 4 and 5 ) was also not purified due to low quantity but is likely the 16-hydroxypremarineosin a. both of these compounds are likely shunt metabolites of the main marineosin pathway and may arise either from the action of mar proteins or enzymes present in the s. venezuelae host . furthermore , this step , formation of the c8o c23 bond , and introduction of the hydroxyl group must occur at an earlier step in the pathway . this hypothesis was based on the observation that marg has homology ( 64% ) to redg , which catalyzes the oxidative cyclization in the streptorubin b pathway ( in this case a cyclization between c-11 and c-20 of 2 ) , and was consistent with an earlier hypothesis that a redg homologue introduces the hydroxyl group in the marineosin structure . thus we sought the replacement of marg gene in the pmar cosmid with aada gene , which confers resistance to spectinomycin via red / et - mediated recombination , to generate the pmarg cosmid . ms analysis of the acetone extract of jnd2g strain indicates the accumulation of two major red - colored compounds , 23-hydroxyundecylprodiginine ( 7 ) and 23-ketoundecylprodiginine ( 8) ( figure 5 ) , with [ m + h ] m / z 410.2807 and 408.2622 , which correspond to the molecular formulae c25h36o2n3 and c25h34o2n3 respectively . these observations provide clear evidence that the biosynthesis of marineosin proceeds as a conjugated tripyrrole intermediate that is reduced in a step subsequent to marg - catalyzed cyclization . the h 1.09 ppm ( c-24 ) doublet has only one correlation with 3.67 ppm ( c-23 ) in the cosy spectrum of 7 ( supplementary figure s32 ) , supporting the position of the -oh group at c-23 . the accumulation of 23-hydroxy- ( 7 ) , and 23-keto - undecylprodiginines ( 8) ( figure 5 ) as a result of marg gene deletion indicates that marg is essential for the formation of macrocyclic and spiro - tetrahydropyran - aminal rings but is not responsible for the introduction of the oxygen of the spiro - tetrahydropyran ring as postulated by snider and co - workers . sequence alignments of marg with other c c bond - forming non - heme iron rieske oxygenases such as redg , mcpg , and rphg involved in the biosynthesis of natural products 3 , 4 , and roseophilin , respectively , together with hydroxylating non - heme iron rieske oxygneases show that all have conserved n - terminal cxh and cxxh motifs . preliminary experiments feeding 7 to a s. venezuelae host with a marg expression plasmid have demonstrated production of 9 and 10 . determination of the major compounds that accumulate in s. venezuelae jnd2 and the mix of these that are found in the jnd2g and jnd2a strains provide the first experimental evidence to delineate key steps in the marineosin biosynthetic pathway . the hydroxyl ( -oh ) group of 7 can be either introduced early in the pathway due to the hydroxylation of the 2-up subunit by an unidentified oxidase ( route 1 , scheme 2 ) , via recruitment of 2-hydroxybutyric acid starter unit by marp ( route 2 , scheme 2 ) or introduced later via hydroxylation of 2 ( route 3 , scheme 2 ) . this observation does not prove that incorporation of the hydroxyl group of 7 occurs early in the pathway as it may be that hydroxylation of 2 occurs faster than its formation ( so the intermediate can not be detected ) . in any case , the gene product(s ) required for introduction of the hydroxyl group remain unknown . the marineosin pathway proceeds from 7 to premarineosins ( 9 and 11 ) in a step that requires marg ( scheme 2 ) . if the proposed pathway ( intramolecular hydroalkoxylation at c-8 ) proceeds enzymatically ( catalyzed by marg ) , then the premarineosin a ( 9 ) might be the intermediate of marineosins a and b. it is noteworthy that the premarineosin b ( 11 ) most likely arises from an inversion of the aminal nitrogen of premarineosin a ( 9 ) under pathway conditions where the two isomers differ only in stereochemistry at the spiro aminal carbon ( c-8 ) . the premarineosin a ( 9 ) exists in higher ratio owing to an anomeric effect as demonstrated for marineosin a ( 5 ) , and this trend follows in the higher production of marineosin a ( 5 ) in the jnd2 strain . the final step of the pathway is a mara catalyzed reduction of premarineosins ( 9 and 11 ) ( scheme 2 ) . in a continuation of this work we determined the biological activity of 9 , 10 , and the compounds were evaluated for antimalarial activity against the chloroquine - sensitive ( cq ) d6 and the chloroquine - resistant ( cq ) dd2 and 7g8 strains of p. falciparum with chloroquine ( cq ) as a reference drug . the activity of marineosin a ( 5 ) against d6 ( ic50 = 138 nm ) , dd2 ( ic50 = 127 nm ) , and 7g8 ( ic50 = 199 nm ) was poorer than that of 9 , demonstrating that the oxidized middle pyrrole ring is required for maximal activity . the final steps of the marineosin biosynthetic pathway have now been experimentally established . notable in this regard is the class of c c bond - forming non - heme rieske iron monoxygenases such as marg , which could be involved in the biosynthesis of other spiroaminal natural products . the generation of marineosin pathway absent the final mara catalyzed step has led to the isolation and identification of a novel spiroaminal prodiginine with potent antimalarial activities .

asthma and copd are chronic obstructive lung diseases that affect millions of patients and add a huge burden to health care systems worldwide.13 based on current treatment guidelines , both diseases are treated with maintenance medications delivered daily by inhaler devices ( usually a combination of bronchodilators and inhaled corticosteroids).4,5 however , it is common for patients under prolonged treatment to have poor adherence to medication.6,7 patients adherence to treatment can be influenced by their overall state of being , as well as by the frequency of dosing , their expectations regarding treatment of their disease and its outcomes , and to a great extent by the quality and ease of use of the device . a wide choice of inhaler devices with different drug combinations exist nowadays that offer excellent therapeutic results , when used efficiently.8 however , there is still space for improvement regarding device - related attributes that are considered important to the patients , such as ease of use , size , and portability . it has been suggested that allowing the patient to choose the inhalation device might help to increase acceptance of therapy and adherence.912 the idea is that involving the patient in the decision - making process will result in enhancing patient satisfaction with the inhaler device , which in turn will lead to increased adherence , better clinical outcomes , and reduced health care expenditures.10 recent evidence - based guidelines for device selection and outcomes of aerosol therapy have pointed out the importance of tailoring the device to the patient.8 the global initiative for asthma ( gina ) guidelines recognize that individual patient preference , convenience , and ease of use may influence not only the efficiency of drug delivery but also patients adherence to treatment and long - term control . moreover , the gina guidelines suggest that patients should see several inhaler devices and participate in the decision over which is best for them.4 patient satisfaction is by no means a new concept and its clinical significance became evident a long time ago.13 in this context , patient satisfaction can be defined as the extent to which the inhaler fulfills the patient requirements , in terms of his / her expectations of the device features.11 there is evidence supporting the association between inhaler satisfaction and patient adherence , as well as improved clinical outcome . patients who use their preferred inhaler device may obtain a greater degree of satisfaction with therapy , while lack of satisfaction with treatment is associated with a worst disease course and poorer disease control in patients with asthma.14,15 moreover , both asthma and copd patients are more likely to be compliant and experience better outcomes when they are satisfied with their inhalers.10,11 this study was an open , noninterventional , multicenter , parallel , 4-week clinical trial designed to compare patient satisfaction with three different commercially available dry powder inhalers ( dpis ) delivering inhaled corticosteroid / long - acting 2-agonist fixed combinations : seretide diskus ( dk ) , a multidose blister dpi ( glaxosmithkline plc , london , uk ) ; rolenium elpenhaler ( eh ) , a single - dose blister dpi ( elpen pharmaceuticals co. inc . , pikermi , greece ) ; and symbicort turbuhaler ( th ) , a multidose reservoir dpi ( astrazeneca plc , london , uk ) . this study was conducted in a real - world clinical setting in a large pool of patients with both asthma and copd already treated with any of the three studied dpis for a rather long period of time , using a recently validated greek version of the fsi-10 questionnaire.16 this study was a prospective , observational , 4-week , open , noninterventional , multicenter , parallel clinical study performed in 79 private practice and hospital centers across greece between january and september 2011 . the primary outcome of the study was patient satisfaction with the inhaled device used and the secondary outcomes were treatment efficacy ( according to lung function measurements ) and safety . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were recruited in the study . demographic characteristics and distribution of patients with asthma and copd stratified by disease severity and inhaler device are presented in table 1 . male or female patients of > 18 years of age with a history of either copd or asthma for at least 6 months and under fixed combination therapy with inhaled budesonide / formoterol or fluticasone / salmeterol were included in the study . the study s primary outcome was to assess patients satisfaction with the dpi devices they were already familiarized within a real - world clinical setting . therefore , patients had to have achieved mastery in any of the three studied dpis for at least 2 months before enrollment , in order to be enrolled in the study . disease severity for asthma and copd was defined according to current gina and global initiative for chronic obstructive lung disease guidelines , respectively.4,5 all patients were informed regarding the study procedures and objectives and signed an informed consent form before study enrollment . the study was approved by the athens chest hospital ethics committee as well as the corresponding institutional ethics committees and was registered with clinicaltrials.gov ( nct01475045 ) . during the first visit ( day 0 ) , patients were informed about the study and a written consent was obtained . demographics and medical history were obtained and patients were categorized in one of the three available groups , depending on the inhaler device they were using . the attending physician assessed directly the patients inhalation technique , and in cases of critical errors , instructions for proper use were given . during the second visit ( week 4 ) , patients were interviewed regarding possible exacerbations about the use of rescue medication or the addition of oral corticosteroids , as well as the occurrence of any adverse events . patients were instructed on how to answer the questions of the fsi-10 questionnaire and asked that they do so honestly on their own . patient satisfaction with their corresponding dpi was evaluated by means of fsi-10 questionnaire , which was adapted in linguistic and cultural terms for greek patients.16 the fsi-10 is a self - completed questionnaire designed to assess patients opinion regarding the satisfaction and usability of the inhalers , irrespective of the medication or formulation used.17 it consists of ten questions , each with five possible responses on a five - point likert scale ( very , fairly , somewhat , not very , and hardly at all ) scored from 5 to 1 , respectively ( supplementary material ) . the total score can range between 10 and 50 , with higher scores corresponding to higher level of feeling of patient satisfaction from the inhaler . normality was assessed using dagostino pearson omnibus normality tests . to establish the homogeneity index of the fsi-10 questionnaire , the pearson s correlation coefficient regarding the scores on each question and the total score was calculated to assess the relationship between questions . reliability was assessed in terms of internal consistency according to the cronbach s , considering that values > 0.8 reflected good internal consistency . one - way analysis of variance followed by bonferroni post hoc correction or the pearson chi - square test with appropriate post hoc analysis were used to compare fsi-10 total scores and individual question frequencies between the tested inhaler devices . two - way analysis of variance was used for subgroup analysis with respect to age and disease severity . statistical software packages were used for data analysis and graph preparation ( spss version 20 ; spss inc . , chicago , il , usa , and prism version 5 ; graphpad software , inc . , la jolla , ca , usa , respectively ) . this study was a prospective , observational , 4-week , open , noninterventional , multicenter , parallel clinical study performed in 79 private practice and hospital centers across greece between january and september 2011 . the primary outcome of the study was patient satisfaction with the inhaled device used and the secondary outcomes were treatment efficacy ( according to lung function measurements ) and safety . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were recruited in the study . demographic characteristics and distribution of patients with asthma and copd stratified by disease severity and inhaler device are presented in table 1 . male or female patients of > 18 years of age with a history of either copd or asthma for at least 6 months and under fixed combination therapy with inhaled budesonide / formoterol or fluticasone / salmeterol were included in the study . the study s primary outcome was to assess patients satisfaction with the dpi devices they were already familiarized within a real - world clinical setting . therefore , patients had to have achieved mastery in any of the three studied dpis for at least 2 months before enrollment , in order to be enrolled in the study . disease severity for asthma and copd was defined according to current gina and global initiative for chronic obstructive lung disease guidelines , respectively.4,5 all patients were informed regarding the study procedures and objectives and signed an informed consent form before study enrollment . the study was approved by the athens chest hospital ethics committee as well as the corresponding institutional ethics committees and was registered with clinicaltrials.gov ( nct01475045 ) . during the first visit ( day 0 ) , patients were informed about the study and a written consent was obtained . demographics and medical history were obtained and patients were categorized in one of the three available groups , depending on the inhaler device they were using . the attending physician assessed directly the patients inhalation technique , and in cases of critical errors , instructions for proper use were given . during the second visit ( week 4 ) , patients were interviewed regarding possible exacerbations about the use of rescue medication or the addition of oral corticosteroids , as well as the occurrence of any adverse events . patients were instructed on how to answer the questions of the fsi-10 questionnaire and asked that they do so honestly on their own . patient satisfaction with their corresponding dpi was evaluated by means of fsi-10 questionnaire , which was adapted in linguistic and cultural terms for greek patients.16 the fsi-10 is a self - completed questionnaire designed to assess patients opinion regarding the satisfaction and usability of the inhalers , irrespective of the medication or formulation used.17 it consists of ten questions , each with five possible responses on a five - point likert scale ( very , fairly , somewhat , not very , and hardly at all ) scored from 5 to 1 , respectively ( supplementary material ) . the total score can range between 10 and 50 , with higher scores corresponding to higher level of feeling of patient satisfaction from the inhaler . normality was assessed using dagostino pearson omnibus normality tests . to establish the homogeneity index of the fsi-10 questionnaire , the pearson s correlation coefficient regarding the scores on each question and the total score was calculated to assess the relationship between questions . reliability was assessed in terms of internal consistency according to the cronbach s , considering that values > 0.8 reflected good internal consistency . one - way analysis of variance followed by bonferroni post hoc correction or the pearson chi - square test with appropriate post hoc analysis were used to compare fsi-10 total scores and individual question frequencies between the tested inhaler devices . two - way analysis of variance was used for subgroup analysis with respect to age and disease severity . statistical software packages were used for data analysis and graph preparation ( spss version 20 ; spss inc . , chicago , il , usa , and prism version 5 ; graphpad software , inc . , la jolla , ca , usa , respectively ) . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were initially recruited in the study . eighty - two patients ( 38 asthma and 44 copd patients ) were lost to follow - up ( table 1 ) . the final patient groups consisted of 523 ( 332 women and 191 men ) asthmatic patients and 517 ( 120 women and 397 men ) copd patients , with a mean ( sd ) age of 50.3 ( 16.1 ) years and 66.1 ( 10.2 ) years , respectively . all patients used one of the three studied dpis for at least 2 months before enrollment , with a mean ( sd ) time of using the corresponding device of 12.3 ( 10.3 ) months and 11.3 ( 8.8 ) months for asthma and copd patients , respectively . there were no differences with respect to patient characteristics , dpi use , or disease severity between asthma and copd groups ( table 1 ) . the fsi-10 displayed adequate homogeneity with no redundancy , as no correlation > 0.8 was found between the questions ( table 2 ) . cronbach s test for the questionnaire as a whole showed very good internal consistency ( cronbach s = 0.898 ) . lower values ( 0.8790.890 ) were calculated , if any of the items was consecutively deleted . consistent and satisfactory results were obtained with all dpis tested ; however , certain statistically significant differences in the ratings between the devices were observed in both asthma and copd patients . specifically , regarding patients with asthma , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 41.4 ( 6.1 ) for the dk , 44.1 ( 5.3 ) for the eh , and 43 ( 5.3 ) for the th ( figure 1a ) . the eh was rated best in seven of the ten questions ( questions 1 , 37 , and 10 ; table 3 ) . it also had significantly higher mean total score compared to dk ( p<0.001 ; figure 1a ) , but not to th ( p=0.16 ; figure 1a ) . th was rated first in questions 7 and 8 of the fsi-10 questionnaire ( table 3 ) and presented a significantly higher mean total score compared to dk ( p<0.05 ; figure 1a ) . dk was rated first only in question 2 , but with no statistical difference from the other two devices ( table 3 ) . no significant differences were observed regarding age ( p=0.89 ; figure 1b ) or disease severity ( p=0.96 ; figure 1c ) , in any of the tested devices . with respect to copd patients , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 40.8 ( 6.9 ) for the dk , 44.7 ( 4.4 ) for the eh , and 41.5 ( 5.8 ) for the th ( figure 2a ) . eh was also rated first in eight of the ten questions ( questions 1 , 37 , and 910 ; table 3 ) having also a significantly higher mean total score compared to dk and th ( both p<0.001 ; figure 2a ) . th was rated first only in question 8 of the fsi-10 questionnaire ( table 3 ) , showing no statistically significant difference in mean total score compared to dk ( p=0.86 ; figure 2a ) . dk was rated first only in question 2 , but with no statistical difference compared to the other two devices ( table 3 ) . no significant differences in scores were observed regarding age ( p=0.12 ; figure 2b ) , in any of the tested devices . it should also be noted that patients suffering with severe copd tended to express a higher feeling of satisfaction with their devices compared to those with mild or moderate disease ( p<0.05 ; figure 2c ) . a total of 39 patients ( 13 with asthma and 26 with copd ) experienced an exacerbation during the study . no differences in exacerbation rates were observed between the three studied groups in either asthma or copd ( dk 3% and 4.2% , eh 1.3% and 5% , and th 2.9% and 5.6% , in asthma and copd patients , respectively ; p=0.76 ) . no differences were observed with respect to forced expiratory volume in 1 second measurements during the 1-month study period between all inhaler groups , in both asthma and copd patients . for the specific inhaled corticosteroid / long - acting 2-agonist fixed combinations treatments , the mean ( sd ) difference in forced expiratory volume in 1 second between visits 1 and 2 was 0.08 l ( 0.37 ) and 0.16 0.18 l ( 0.50 ) and 0.12 l ( 0.37 ) for th , and 0.20 l ( 0.49 ) and 0.18 l ( 0.45 ) for eh , respectively , for the asthma and copd groups ( p=0.08 and p=0.37 , respectively ) . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were initially recruited in the study . eighty - two patients ( 38 asthma and 44 copd patients ) were lost to follow - up ( table 1 ) . the final patient groups consisted of 523 ( 332 women and 191 men ) asthmatic patients and 517 ( 120 women and 397 men ) copd patients , with a mean ( sd ) age of 50.3 ( 16.1 ) years and 66.1 ( 10.2 ) years , respectively . all patients used one of the three studied dpis for at least 2 months before enrollment , with a mean ( sd ) time of using the corresponding device of 12.3 ( 10.3 ) months and 11.3 ( 8.8 ) months for asthma and copd patients , respectively . there were no differences with respect to patient characteristics , dpi use , or disease severity between asthma and copd groups ( table 1 ) . the fsi-10 displayed adequate homogeneity with no redundancy , as no correlation > 0.8 was found between the questions ( table 2 ) . cronbach s test for the questionnaire as a whole showed very good internal consistency ( cronbach s = 0.898 ) . lower values ( 0.8790.890 ) were calculated , if any of the items was consecutively deleted . consistent and satisfactory results were obtained with all dpis tested ; however , certain statistically significant differences in the ratings between the devices were observed in both asthma and copd patients . specifically , regarding patients with asthma , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 41.4 ( 6.1 ) for the dk , 44.1 ( 5.3 ) for the eh , and 43 ( 5.3 ) for the th ( figure 1a ) . the eh was rated best in seven of the ten questions ( questions 1 , 37 , and 10 ; table 3 ) . it also had significantly higher mean total score compared to dk ( p<0.001 ; figure 1a ) , but not to th ( p=0.16 ; figure 1a ) . th was rated first in questions 7 and 8 of the fsi-10 questionnaire ( table 3 ) and presented a significantly higher mean total score compared to dk ( p<0.05 ; figure 1a ) . dk was rated first only in question 2 , but with no statistical difference from the other two devices ( table 3 ) . no significant differences were observed regarding age ( p=0.89 ; figure 1b ) or disease severity ( p=0.96 ; figure 1c ) , in any of the tested devices . with respect to copd patients , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 40.8 ( 6.9 ) for the dk , 44.7 ( 4.4 ) for the eh , and 41.5 ( 5.8 ) for the th ( figure 2a ) . eh was also rated first in eight of the ten questions ( questions 1 , 37 , and 910 ; table 3 ) having also a significantly higher mean total score compared to dk and th ( both p<0.001 ; figure 2a ) . th was rated first only in question 8 of the fsi-10 questionnaire ( table 3 ) , showing no statistically significant difference in mean total score compared to dk ( p=0.86 ; figure 2a ) . dk was rated first only in question 2 , but with no statistical difference compared to the other two devices ( table 3 ) . no significant differences in scores were observed regarding age ( p=0.12 ; figure 2b ) , in any of the tested devices . it should also be noted that patients suffering with severe copd tended to express a higher feeling of satisfaction with their devices compared to those with mild or moderate disease ( p<0.05 ; figure 2c ) . a total of 39 patients ( 13 with asthma and 26 with copd ) experienced an exacerbation during the study . no differences in exacerbation rates were observed between the three studied groups in either asthma or copd ( dk 3% and 4.2% , eh 1.3% and 5% , and th 2.9% and 5.6% , in asthma and copd patients , respectively ; p=0.76 ) . no differences were observed with respect to forced expiratory volume in 1 second measurements during the 1-month study period between all inhaler groups , in both asthma and copd patients . for the specific inhaled corticosteroid / long - acting 2-agonist fixed combinations treatments , the mean ( sd ) difference in forced expiratory volume in 1 second between visits 1 and 2 was 0.08 l ( 0.37 ) and 0.16 0.18 l ( 0.50 ) and 0.12 l ( 0.37 ) for th , and 0.20 l ( 0.49 ) and 0.18 l ( 0.45 ) for eh , respectively , for the asthma and copd groups ( p=0.08 and p=0.37 , respectively ) . in this study , we investigated the ease of use and satisfaction of patients with asthma and copd in a cohort of greek patients regarding three different marketed dpi devices , which they had already been using . the assessment of patient satisfaction was performed by a specifically designed self - completed questionnaire , the fsi-10 , which was easily understood and completed by the participants . all dpis tested received satisfactory results ; however , eh consistently received higher fsi-10 scores in both copd and asthma patients . moreover , severe copd patients tended to express higher feeling of satisfaction than those with moderate or mild disease , irrespective of the device used . to the best of our knowledge , no other study has ever examined in a standardized fashion patient satisfaction with their inhaler devices in the greek population . several types of devices for delivery of inhaled drugs are currently available ; however , no inhaler currently fits the profile of the ideal inhaler device.18,19 many inhaler - related factors , such as size , portability , and ease of use can affect inhaler preference and patient satisfaction.11,1921 the issue of patient satisfaction with their inhaler devices has been increasingly addressed over the last few years , mostly because of the evidence linking inhaler satisfaction with patient adherence and improved clinical outcomes.10,2227 patients using their preferred inhaler are more likely to obtain a greater degree of satisfaction with therapy . this is of outmost importance as lack of satisfaction with treatment is associated with low compliance and , consequently , poor control and worse disease course.10,11,14 there are no standardized questionnaires assessing patient satisfaction with inhaler devices available for use in the greek language other than the fsi-10 , which was only recently translated and validated for use in greece.16 other types of instruments used in similar inhaler satisfaction studies range from simple preference questions to psychometrically developed and validated questionnaires.28,29 response scales of these instruments range from open - ended questions , through unclear response scales , to visual analog and likert scales.30,31 comparison between the visual analog scale and the likert measuring scale used in the fsi-10 questionnaire showed the latter to have better predictive performance.30 the fsi-10 questionnaire quantifies the various aspects that are important to patients who use inhaler devices , such as ease of use and portability . the fsi-10 questionnaire has been used in order to compare satisfaction and preferences in 112 stable asthmatic patients regarding three different devices th , accuhaler ( ie , dk ) , and novolizer ( nv ) , a multidose reservoir dpi ( meda pharmaceuticals , somerset , nj , usa ) , in a multi - center , prospective , observational study in spain.17 in this study , the fsi-10 was found to be a useful instrument for assessing the degree of satisfaction of adult and pediatric asthmatic patients . the highest scoring and most often preferred inhaler was the nv , while , in accordance to the findings of this study among the greek patients , the spanish asthma patients reported higher score for th compared to dk . this preference was , partly , attributed by the authors to th having been the device that was commonly used by the patients prior to the study , even though an overall younger age effect was evident in favor of nv.17 the fsi-10 questionnaire has also been used to evaluate patient satisfaction in turkey , in 442 patients with copd , in real - life clinical practice . data concerning the ease of use , the portability , and the usability of the device were collected on the basis of a single cross - sectional patient visit , for copd patients who had already been on budesonide / formoterol th therapy , for 3 months at least , prior to study enrollment . the results revealed that the majority of patients were able to use th , whereas older age was associated with higher rate of errors in inhalation maneuvers , while the majority of patients were satisfied with the overall use of the device.25 to the best of our knowledge , this study is the first one performed in a real - life clinical settings , evaluating patient satisfaction regarding three different widely used dpis , in a large cohort of both copd and asthma patients . the validation data from our study indicate that the fsi-10 questionnaire is comprehensible , easy to use , and has satisfactory measurement properties . it shows a good association between questions and a positive contribution of the score of each question to the total score . moreover , the questionnaire as a whole demonstrates very good internal consistency and no redundancies . however , no data are available regarding the sensitivity or the minimum clinically significant difference of the fsi-10 , which accounts for the interim and exploratory nature of the interpretation of its results . it is a judgment that a well - designed and conducted study , showing prospectively defined important statistical significance , has an effect size that is clinically meaningful , in the context of the clinical relevance of the end point used in a study . nonetheless , the statistically significant differences we observed , regarding the consistently higher fsi-10 scores for the eh in both copd and asthmatic patients , could , possibly , be important in the clinical setting . in a recent study , dk and th were compared to nexthaler , a multidose reservoir dpi ( chiesi , parma , italy ) , in terms of usability and asthma patient satisfaction.23 similar to the results obtained in our study concerning asthma patients who already use their devices , th presented the worst score corresponding to verification of dose delivery , in 66 dpi naive adult patients compared to the other devices , including dk.23 however , th was superior to dk in its easiness to keep the inhaler clean and in good working condition , its efficacy to continue normal activities with the use of the inhaler , its easiness in terms of size and weight , and its easiness to be carried , only in patients with asthma . in contrast , dk was found to be superior compared to th in another study of 169 powder - naive patients with asthma or copd , in terms of the presence of a dose counter , ease of learning to use , design , attached cover , and comfortable mouthpiece.26 similar to the findings of a comparative study in device - naive copd patients27 and our study as well , th was favored over dk regarding factors related to size and ease of holding . eh is a newly developed single - dose dpi , designed to administer fixed combinations of fluticasone propionate / salmeterol and budesonide / formoterol , similarly to dk and th , respectively.32,33 in a recent cross - sectional randomized cross - over multicentre study involving dpi - naive patients with copd and asthma , van der palen et al22 compared dk to eh with regard to dpi patient preference and satisfaction , along with inhaler technique critical errors . contrary to our findings , their study concluded that patients were more satisfied , in general , with dk and that more patients preferred dk over eh.22 various differences concerning the design of both studies could be accounted for this discrepancy . initially , our study intended to assess neither inhalation errors nor patient preference ; hence , it was not designed to be a cross - over study . second , patients in our study were already properly instructed and familiar with their inhalers . as van der palen et al22 have also indicated , their findings referred to the patients opinion at a first glance and their limited experience with the devices , as they were instructed only once , on how to use their assigned device . inhaler satisfaction may shift when patients become more accustomed with one device after more extended use , as was the case in our study . this could also explain why in question 2 of fsi-10 questionnaire ( was it easy to prepare the inhaler for use ? ) , despite significant differences in device preparation steps ( dk four and th five vs eh ten steps ) , although dk was rated first by both asthma and copd patients , there was no statistical difference from the other two devices . moreover , several issues regarding the design and methodology of the study could possibly be further responsible for the differences observed between the conclusions of the two studies.34 our study also revealed some interesting differences between tested dpis in specific components of the fsi-10 questionnaire . similar to the findings of other studies in asthma23 and copd,27 th was favored over dk and in our study over also eh regarding factors related to size and ease of holding , with significant higher scores in questions 7 ( was using the inhaler easy in terms of size and weight ? ) and 8 ( was it easy to carry the inhaler with you ? ) of the fsi-10 . on the other hand , one of the major advantages of eh seemed to be the verification of dose delivery , as > 60% of both asthma and copd patients scored 5 ( very satisfied ) in question 9 of fsi-10 ( after you ve used the inhaler , do you have the feeling that you used it correctly ? ) compared with < 30% for both dk and th . nevertheless , these differences must be interpreted with caution , as they are not derived from a direct comparison of the three dpi inhalers in a cross - sectional designed study and rather reflect patients general feeling and familiarity with their device . previous dpi patient preference and satisfaction clinical studies that involve a variety of devices have also lead to divergent conclusion.9,35 contradictive interpretations of the results may derive from the lack of a robust and widely used instrument for measuring preference and satisfaction and the exclusion of patients with unstable disease or unable to use inhalers , along with the involvement of the pharmaceutical industries in the study design . indeed , in the vast majority of the studies sponsored by a pharmaceutical industry , with dk and th featuring prominently , the sponsor s device was widely preferred.9 this study presents certain limitations . as already stated , we did not assess patients preference or inhaler technique , therefore our study was not designed to be a cross - over study , which is important when comparing devices that administer the same drug.9 however , we believe that the longitudinal design of our study was essential in addressing the main aim of our study . another limitation , as with other preference studies , was the fact that patients with unstable disease or those unable to use inhalers correctly were excluded from our study . in addition , the study included patients > 18 years of age , so further studies to investigate the patient satisfaction in pediatric and adolescent asthma patients could be warranted . nevertheless , evaluations across patients with asthma and copd , deriving from diverse ethnic and cultural background,17,25 led us to the conclusion that the fsi-10 is a reliable , practical , and responsive instrument for measuring inhalation device satisfaction . inhaler satisfaction is a useful patient - reported outcome , which should be considered in clinical studies assessing inhaler devices in copd and asthma . considering patients preference and satisfaction when choosing an inhaler device could possibly be associated with better adherence and enhanced clinical outcomes . future research should focus on the effect of patient - reported device preference and degree of satisfaction on adherence , quality of life , disease control , and clinical outcome .

backgroundpoor adherence to inhaled therapy is common in patients with asthma and copd . an inhaler selection based on patients preference could be beneficial to adherence and treatment effectiveness . properly designed questionnaires can assess patients satisfaction with their medication devices . the aim of this study was to estimate , using the feeling of satisfaction with inhaler ( fsi-10 ) questionnaire , the ease of use and satisfaction of patients regarding three different marketed dry powder inhalers ( dpis ) : diskus ( dk ) , elpenhaler ( eh ) , and turbuhaler ( th ) . the fsi-10 is a self - completed questionnaire to assess patients opinions regarding ease of use , portability , and usability of devices , irrespective of the drug used.patients and methodswe performed a 4-week , open , noninterventional , multicenter , parallel clinical study in 560 asthmatic and 561 copd patients . during the first visit , patients were classified into three groups according to the dpi they were already using . patients were regularly receiving their treatments ( seretide dk , rolenium eh , and symbicort th ) and agreed to complete the fsi-10 questionnaire in the second visit.resultsa total of 517 copd and 523 asthma patients completed the study . all dpis tested received satisfactory results , while the eh obtained consistently higher scores in the fsi-10 in both copd and asthma patients ( 44.7 and 44.1 vs 41.5 and 43 for th , 40.8 and 41.4 for dk , p<0.001 and p<0.01 , respectively ) . th was rated better than dk by asthma patients . patients suffering with severe copd tended to express higher feeling of satisfaction than those with moderate or mild disease , irrespective of the device used.conclusionall dpis tested were highly acceptable by asthma and copd patients of different ages ; nevertheless , eh received significantly higher ratings in most of the questionnaire domains . copd patients in advanced stages of the disease generally expressed higher level of satisfaction with their devices .

Introduction Patients and methods Study design and population FSI-10 questionnaire Statistical analysis Results Patient characteristics FSI-10 questionnaire validation data FSI-10 questionnaire scores Efficacy and safety Discussion Conclusion Supplementary material

asthma and copd are chronic obstructive lung diseases that affect millions of patients and add a huge burden to health care systems worldwide.13 based on current treatment guidelines , both diseases are treated with maintenance medications delivered daily by inhaler devices ( usually a combination of bronchodilators and inhaled corticosteroids).4,5 however , it is common for patients under prolonged treatment to have poor adherence to medication.6,7 patients adherence to treatment can be influenced by their overall state of being , as well as by the frequency of dosing , their expectations regarding treatment of their disease and its outcomes , and to a great extent by the quality and ease of use of the device . a wide choice of inhaler devices with different drug combinations exist nowadays that offer excellent therapeutic results , when used efficiently.8 however , there is still space for improvement regarding device - related attributes that are considered important to the patients , such as ease of use , size , and portability . it has been suggested that allowing the patient to choose the inhalation device might help to increase acceptance of therapy and adherence.912 the idea is that involving the patient in the decision - making process will result in enhancing patient satisfaction with the inhaler device , which in turn will lead to increased adherence , better clinical outcomes , and reduced health care expenditures.10 recent evidence - based guidelines for device selection and outcomes of aerosol therapy have pointed out the importance of tailoring the device to the patient.8 the global initiative for asthma ( gina ) guidelines recognize that individual patient preference , convenience , and ease of use may influence not only the efficiency of drug delivery but also patients adherence to treatment and long - term control . patients who use their preferred inhaler device may obtain a greater degree of satisfaction with therapy , while lack of satisfaction with treatment is associated with a worst disease course and poorer disease control in patients with asthma.14,15 moreover , both asthma and copd patients are more likely to be compliant and experience better outcomes when they are satisfied with their inhalers.10,11 this study was an open , noninterventional , multicenter , parallel , 4-week clinical trial designed to compare patient satisfaction with three different commercially available dry powder inhalers ( dpis ) delivering inhaled corticosteroid / long - acting 2-agonist fixed combinations : seretide diskus ( dk ) , a multidose blister dpi ( glaxosmithkline plc , london , uk ) ; rolenium elpenhaler ( eh ) , a single - dose blister dpi ( elpen pharmaceuticals co. inc . this study was conducted in a real - world clinical setting in a large pool of patients with both asthma and copd already treated with any of the three studied dpis for a rather long period of time , using a recently validated greek version of the fsi-10 questionnaire.16 this study was a prospective , observational , 4-week , open , noninterventional , multicenter , parallel clinical study performed in 79 private practice and hospital centers across greece between january and september 2011 . the primary outcome of the study was patient satisfaction with the inhaled device used and the secondary outcomes were treatment efficacy ( according to lung function measurements ) and safety . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were recruited in the study . the study s primary outcome was to assess patients satisfaction with the dpi devices they were already familiarized within a real - world clinical setting . disease severity for asthma and copd was defined according to current gina and global initiative for chronic obstructive lung disease guidelines , respectively.4,5 all patients were informed regarding the study procedures and objectives and signed an informed consent form before study enrollment . during the first visit ( day 0 ) , patients were informed about the study and a written consent was obtained . during the second visit ( week 4 ) , patients were interviewed regarding possible exacerbations about the use of rescue medication or the addition of oral corticosteroids , as well as the occurrence of any adverse events . patient satisfaction with their corresponding dpi was evaluated by means of fsi-10 questionnaire , which was adapted in linguistic and cultural terms for greek patients.16 the fsi-10 is a self - completed questionnaire designed to assess patients opinion regarding the satisfaction and usability of the inhalers , irrespective of the medication or formulation used.17 it consists of ten questions , each with five possible responses on a five - point likert scale ( very , fairly , somewhat , not very , and hardly at all ) scored from 5 to 1 , respectively ( supplementary material ) . to establish the homogeneity index of the fsi-10 questionnaire , the pearson s correlation coefficient regarding the scores on each question and the total score was calculated to assess the relationship between questions . this study was a prospective , observational , 4-week , open , noninterventional , multicenter , parallel clinical study performed in 79 private practice and hospital centers across greece between january and september 2011 . the primary outcome of the study was patient satisfaction with the inhaled device used and the secondary outcomes were treatment efficacy ( according to lung function measurements ) and safety . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were recruited in the study . demographic characteristics and distribution of patients with asthma and copd stratified by disease severity and inhaler device are presented in table 1 . the study s primary outcome was to assess patients satisfaction with the dpi devices they were already familiarized within a real - world clinical setting . disease severity for asthma and copd was defined according to current gina and global initiative for chronic obstructive lung disease guidelines , respectively.4,5 all patients were informed regarding the study procedures and objectives and signed an informed consent form before study enrollment . during the first visit ( day 0 ) , patients were informed about the study and a written consent was obtained . during the second visit ( week 4 ) , patients were interviewed regarding possible exacerbations about the use of rescue medication or the addition of oral corticosteroids , as well as the occurrence of any adverse events . patient satisfaction with their corresponding dpi was evaluated by means of fsi-10 questionnaire , which was adapted in linguistic and cultural terms for greek patients.16 the fsi-10 is a self - completed questionnaire designed to assess patients opinion regarding the satisfaction and usability of the inhalers , irrespective of the medication or formulation used.17 it consists of ten questions , each with five possible responses on a five - point likert scale ( very , fairly , somewhat , not very , and hardly at all ) scored from 5 to 1 , respectively ( supplementary material ) . to establish the homogeneity index of the fsi-10 questionnaire , the pearson s correlation coefficient regarding the scores on each question and the total score was calculated to assess the relationship between questions . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were initially recruited in the study . all patients used one of the three studied dpis for at least 2 months before enrollment , with a mean ( sd ) time of using the corresponding device of 12.3 ( 10.3 ) months and 11.3 ( 8.8 ) months for asthma and copd patients , respectively . consistent and satisfactory results were obtained with all dpis tested ; however , certain statistically significant differences in the ratings between the devices were observed in both asthma and copd patients . specifically , regarding patients with asthma , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 41.4 ( 6.1 ) for the dk , 44.1 ( 5.3 ) for the eh , and 43 ( 5.3 ) for the th ( figure 1a ) . th was rated first in questions 7 and 8 of the fsi-10 questionnaire ( table 3 ) and presented a significantly higher mean total score compared to dk ( p<0.05 ; figure 1a ) . with respect to copd patients , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 40.8 ( 6.9 ) for the dk , 44.7 ( 4.4 ) for the eh , and 41.5 ( 5.8 ) for the th ( figure 2a ) . th was rated first only in question 8 of the fsi-10 questionnaire ( table 3 ) , showing no statistically significant difference in mean total score compared to dk ( p=0.86 ; figure 2a ) . it should also be noted that patients suffering with severe copd tended to express a higher feeling of satisfaction with their devices compared to those with mild or moderate disease ( p<0.05 ; figure 2c ) . a total of 39 patients ( 13 with asthma and 26 with copd ) experienced an exacerbation during the study . no differences in exacerbation rates were observed between the three studied groups in either asthma or copd ( dk 3% and 4.2% , eh 1.3% and 5% , and th 2.9% and 5.6% , in asthma and copd patients , respectively ; p=0.76 ) . for the specific inhaled corticosteroid / long - acting 2-agonist fixed combinations treatments , the mean ( sd ) difference in forced expiratory volume in 1 second between visits 1 and 2 was 0.08 l ( 0.37 ) and 0.16 0.18 l ( 0.50 ) and 0.12 l ( 0.37 ) for th , and 0.20 l ( 0.49 ) and 0.18 l ( 0.45 ) for eh , respectively , for the asthma and copd groups ( p=0.08 and p=0.37 , respectively ) . a total of 1,121 patients ( 561 copd and 560 asthma patients ) were initially recruited in the study . all patients used one of the three studied dpis for at least 2 months before enrollment , with a mean ( sd ) time of using the corresponding device of 12.3 ( 10.3 ) months and 11.3 ( 8.8 ) months for asthma and copd patients , respectively . consistent and satisfactory results were obtained with all dpis tested ; however , certain statistically significant differences in the ratings between the devices were observed in both asthma and copd patients . specifically , regarding patients with asthma , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 41.4 ( 6.1 ) for the dk , 44.1 ( 5.3 ) for the eh , and 43 ( 5.3 ) for the th ( figure 1a ) . th was rated first in questions 7 and 8 of the fsi-10 questionnaire ( table 3 ) and presented a significantly higher mean total score compared to dk ( p<0.05 ; figure 1a ) . with respect to copd patients , the total score ( mean [ sd ] ) on the fsi-10 for each of the inhalers was 40.8 ( 6.9 ) for the dk , 44.7 ( 4.4 ) for the eh , and 41.5 ( 5.8 ) for the th ( figure 2a ) . th was rated first only in question 8 of the fsi-10 questionnaire ( table 3 ) , showing no statistically significant difference in mean total score compared to dk ( p=0.86 ; figure 2a ) . it should also be noted that patients suffering with severe copd tended to express a higher feeling of satisfaction with their devices compared to those with mild or moderate disease ( p<0.05 ; figure 2c ) . a total of 39 patients ( 13 with asthma and 26 with copd ) experienced an exacerbation during the study . no differences in exacerbation rates were observed between the three studied groups in either asthma or copd ( dk 3% and 4.2% , eh 1.3% and 5% , and th 2.9% and 5.6% , in asthma and copd patients , respectively ; p=0.76 ) . for the specific inhaled corticosteroid / long - acting 2-agonist fixed combinations treatments , the mean ( sd ) difference in forced expiratory volume in 1 second between visits 1 and 2 was 0.08 l ( 0.37 ) and 0.16 0.18 l ( 0.50 ) and 0.12 l ( 0.37 ) for th , and 0.20 l ( 0.49 ) and 0.18 l ( 0.45 ) for eh , respectively , for the asthma and copd groups ( p=0.08 and p=0.37 , respectively ) . in this study , we investigated the ease of use and satisfaction of patients with asthma and copd in a cohort of greek patients regarding three different marketed dpi devices , which they had already been using . the assessment of patient satisfaction was performed by a specifically designed self - completed questionnaire , the fsi-10 , which was easily understood and completed by the participants . all dpis tested received satisfactory results ; however , eh consistently received higher fsi-10 scores in both copd and asthma patients . moreover , severe copd patients tended to express higher feeling of satisfaction than those with moderate or mild disease , irrespective of the device used . several types of devices for delivery of inhaled drugs are currently available ; however , no inhaler currently fits the profile of the ideal inhaler device.18,19 many inhaler - related factors , such as size , portability , and ease of use can affect inhaler preference and patient satisfaction.11,1921 the issue of patient satisfaction with their inhaler devices has been increasingly addressed over the last few years , mostly because of the evidence linking inhaler satisfaction with patient adherence and improved clinical outcomes.10,2227 patients using their preferred inhaler are more likely to obtain a greater degree of satisfaction with therapy . this is of outmost importance as lack of satisfaction with treatment is associated with low compliance and , consequently , poor control and worse disease course.10,11,14 there are no standardized questionnaires assessing patient satisfaction with inhaler devices available for use in the greek language other than the fsi-10 , which was only recently translated and validated for use in greece.16 other types of instruments used in similar inhaler satisfaction studies range from simple preference questions to psychometrically developed and validated questionnaires.28,29 response scales of these instruments range from open - ended questions , through unclear response scales , to visual analog and likert scales.30,31 comparison between the visual analog scale and the likert measuring scale used in the fsi-10 questionnaire showed the latter to have better predictive performance.30 the fsi-10 questionnaire quantifies the various aspects that are important to patients who use inhaler devices , such as ease of use and portability . the fsi-10 questionnaire has been used in order to compare satisfaction and preferences in 112 stable asthmatic patients regarding three different devices th , accuhaler ( ie , dk ) , and novolizer ( nv ) , a multidose reservoir dpi ( meda pharmaceuticals , somerset , nj , usa ) , in a multi - center , prospective , observational study in spain.17 in this study , the fsi-10 was found to be a useful instrument for assessing the degree of satisfaction of adult and pediatric asthmatic patients . the highest scoring and most often preferred inhaler was the nv , while , in accordance to the findings of this study among the greek patients , the spanish asthma patients reported higher score for th compared to dk . this preference was , partly , attributed by the authors to th having been the device that was commonly used by the patients prior to the study , even though an overall younger age effect was evident in favor of nv.17 the fsi-10 questionnaire has also been used to evaluate patient satisfaction in turkey , in 442 patients with copd , in real - life clinical practice . data concerning the ease of use , the portability , and the usability of the device were collected on the basis of a single cross - sectional patient visit , for copd patients who had already been on budesonide / formoterol th therapy , for 3 months at least , prior to study enrollment . the results revealed that the majority of patients were able to use th , whereas older age was associated with higher rate of errors in inhalation maneuvers , while the majority of patients were satisfied with the overall use of the device.25 to the best of our knowledge , this study is the first one performed in a real - life clinical settings , evaluating patient satisfaction regarding three different widely used dpis , in a large cohort of both copd and asthma patients . nonetheless , the statistically significant differences we observed , regarding the consistently higher fsi-10 scores for the eh in both copd and asthmatic patients , could , possibly , be important in the clinical setting . in a recent study , dk and th were compared to nexthaler , a multidose reservoir dpi ( chiesi , parma , italy ) , in terms of usability and asthma patient satisfaction.23 similar to the results obtained in our study concerning asthma patients who already use their devices , th presented the worst score corresponding to verification of dose delivery , in 66 dpi naive adult patients compared to the other devices , including dk.23 however , th was superior to dk in its easiness to keep the inhaler clean and in good working condition , its efficacy to continue normal activities with the use of the inhaler , its easiness in terms of size and weight , and its easiness to be carried , only in patients with asthma . in contrast , dk was found to be superior compared to th in another study of 169 powder - naive patients with asthma or copd , in terms of the presence of a dose counter , ease of learning to use , design , attached cover , and comfortable mouthpiece.26 similar to the findings of a comparative study in device - naive copd patients27 and our study as well , th was favored over dk regarding factors related to size and ease of holding . eh is a newly developed single - dose dpi , designed to administer fixed combinations of fluticasone propionate / salmeterol and budesonide / formoterol , similarly to dk and th , respectively.32,33 in a recent cross - sectional randomized cross - over multicentre study involving dpi - naive patients with copd and asthma , van der palen et al22 compared dk to eh with regard to dpi patient preference and satisfaction , along with inhaler technique critical errors . , despite significant differences in device preparation steps ( dk four and th five vs eh ten steps ) , although dk was rated first by both asthma and copd patients , there was no statistical difference from the other two devices . similar to the findings of other studies in asthma23 and copd,27 th was favored over dk and in our study over also eh regarding factors related to size and ease of holding , with significant higher scores in questions 7 ( was using the inhaler easy in terms of size and weight ? ) previous dpi patient preference and satisfaction clinical studies that involve a variety of devices have also lead to divergent conclusion.9,35 contradictive interpretations of the results may derive from the lack of a robust and widely used instrument for measuring preference and satisfaction and the exclusion of patients with unstable disease or unable to use inhalers , along with the involvement of the pharmaceutical industries in the study design . nevertheless , evaluations across patients with asthma and copd , deriving from diverse ethnic and cultural background,17,25 led us to the conclusion that the fsi-10 is a reliable , practical , and responsive instrument for measuring inhalation device satisfaction .

advances in health policy , research , and information technology have converged to increase the electronic collection and use of patient - reported outcomes ( pros ) to support clinical care and research . through the affordable care act and other legislation , the united states government has invested in research and care delivery that focus on evidence - based treatments and reflect patients preferences and values.1,2 to support these initiatives , and concomitant with increasing electronic health record ( ehr ) adoption,35 researchers and clinicians are demanding information technology and data infrastructures that support routine collection and use of pros.68 pros allow patients to directly report on their health status and health care , such as quality of life , function , symptoms , or care experiences.9 furthermore , systematic pro collection and use in research supports the learning health care system goal of increasing the efficiency and scope of scientific discovery related to health care delivery.10 one approach to meeting the clinical and research demand for pros is to incorporate electronic data collection into everyday clinical processes.1113 this approach is appealing because it utilizes existing infrastructure and interactions with patients , which may allow data to be captured more efficiently and to be acquired for larger , more representative populations . also , by collecting data during care delivery , pros may be more easily integrated with complementary data , such as demographics , diagnoses , laboratory data , and medications . indeed , systems for collecting and sharing pros with clinicians have been shown to be usable,1416 to improve patient - clinician communication,1719 and to be supportive of both clinical care and research,20,21 such as the feasibility of linking pros with a cancer registry.22 furthermore , sharing pro data with clinicians at the point of care may improve patients health - related quality of life.23 however , broader analyses of the literature fail to reveal a consistent positive impact of pro collection and use on care processes , clinical decision - making , and health outcomes.2428 this lack of consistent impact may relate to inconsistencies in how electronic pro systems are implemented , including how burden on clinical workflows is managed and how pros are communicated to clinicians.2932 therefore , to more effectively meet the demand for pro data , there is a need to describe lessons learned in implementing pro systems . describing lessons learned with pro systems may help future implementers and researchers enhance the scale and sustainable use of such systems . the purpose of this case study is to describe a novel information system for electronic pros and our lessons learned in implementing the system , which supports clinical care and research , in an academic health center . uniquely , our lessons learned stem from a system that starts with point - of - care electronic pro collection and ends with institutionwide access to pros linked to other clinical data for research . we describe how pros are collected in primary care practices , integrated with other clinical data in an ehr , and then loaded into an institutional data warehouse . the system utilizes interfaces between freely available and commercial software and involves clinical and research workflows that support the collection , transformation , and research use of pro data . researchers in the institution can use web - based software33 to query for patients of interest based on pros and other clinical data before requesting detailed data for in - depth analysis . our evaluation of the system focused on assessing the feasibility of collecting , integrating , and then reusing pro data for research . therefore , we used qualitative data gleaned from informal and formal interactions with stakeholders in varied roles , including practice staff , clinicians , patients , system designers , researchers , and administrative leadership . these data were collected during the system s design , implementation , and the first six months after implementation . to supplement these qualitative data , we also collected quantitative data that describes the frequency with which pros were collected in clinical settings and reused by researchers . the technology and lessons learned described here are based on our initial implementation of pro data being collected in two family medicine practices affiliated with the university s family medicine department . the first goal was to improve care for chronic pain by incorporating patient - reported data into clinical processes . this goal was set by pain medicine physicians and family medicine physicians who were interested in improving chronic pain care . the second goal was to create a process through which pros collected during routine clinical care could also be reused by many researchers across the institution . this second goal was set by researchers and administrators in the institution who wanted to build infrastructure that supports the regular reuse of pros by many researchers . therefore , the family medicine practices began collecting pro data related to pain , physical function , sleep , and mental health . at the same time , the first and third authors of this case study led a related pragmatic clinical trial to evaluate the effect of integrating pros in an ehr on clinician and patient satisfaction with care for chronic pain.34 the system s design and implementation were led by a team that included two physician researchers , an information science researcher , system developers , and the health system s chief data officer . the team received support from administrative leadership , including the community health and family medicine department chairperson , the college of medicine s assistant dean for clinical informatics , and the academic health center information technology organization . while this article primarily describes the electronic data collection and workflows for research use of pros , details of clinician and patient use of the pro system the software and processes that comprise the system serve three main functions : ( 1 ) collecting electronic pros in clinical care , ( 2 ) integrating pro data with other clinical data not reported by the patient , and ( 3 ) disseminating data to researchers ( figures 1 and 2 ) . to accomplish these tasks , the system encompasses multiple software tools and interfaces between them . to collect pros at the point of care , we adopted an existing web - based system that administers pro assessments called the collaborative health outcomes information registry choir was first developed and implemented at stanford university to support specialty pain care and clinical research.35,36 our university received a no - cost license to use and modify the software within our health system . under a similar license , choir is also in various stages of design and implementation at other academic health centers.35 choir allows patients to complete pro assessments via web - enabled devices . choir also contains administrative functions that allow clinicians or practice staff to register patients , assign surveys , and review pro results at a point in time or longitudinally . through a computer - adaptive testing engine , choir administers pro measures , including patient - reported outcome measurement information system ( promis ) measures.6,37 computer - adaptive and static instruments can be combined to create different patient assessments . new instruments can also be added to those already available in choir . in the current system , patients complete an assessment that includes nine computer - adaptive promis measures ( pain interference , pain behavior , fatigue , physical function , sleep disturbance , sleep - related impairment , anger , depression , and anxiety)38,39 as well as static measures of pain location , pain intensity ( 010 scale ) , pain catastrophizing,40 and risk of opioid - related aberrant behavior.41 while these measures have specific relevance to patients with chronic pain , the domains are also generally relevant to primary care patients and clinical management of other chronic diseases . patients complete assessments using a tablet computer prior to seeing their provider , either in a waiting room or exam room . because the data are initially collected for regular clinical care , patients do not consent to research reuse when reporting their outcomes . however , as we describe later in the case , researchers who wish to reuse the pros must obtain institutional review board ( irb ) approval . once an assessment is complete for the promis measures , the standardized scores include a t - score ( mean 50 , standard deviation 10 ) and a percentage score ( 0100 percent ) , which are based on calibration samples that are representative of the united states population.42 also , choir produces a comprehensive pro result set in portable document format ( pdf ) that can be viewed , saved , or printed by clinicians . all data are stored as discrete entities in an oracle database within the health system s secure clinical data environment . to integrate pro results with other clinical data in support of clinical care the choir system sends the standardized pro scores to our health system s epic ehr . choir creates hl7 version 2.1 messages and sends discrete pro results through epic s laboratory and results interface . each message contains unique patient and encounter identifiers that link the pro results to patients other health record information . we are in the process of mapping the pro measures to logical observation identifiers names and codes ( loinc ) . loinc is a universal system for coding laboratory results and clinical observations , including patient assessments.43 also , within united states government initiatives to increase ehr use and health data exchange , loinc is the preferred vocabulary standard for results.44 therefore , by mapping the pro results to loinc , we expect to increase the ability to share results both internally and with external organizations over time . finally , in addition to the discrete numeric results , choir sends the comprehensive results set pdf document in a separate hl7 version 2.6 message . this pdf document contains the same pro results scores that are sent as discrete numeric results through the ehr s laboratory and results interface . however , the pdf also contains patients responses to the individual pro assessment items in case clinicians are interested in obtaining the full details of their patients responses . both the discrete pro scores and the pdf , which includes individual item responses , are available to clinicians in the ehr . using synopsis , which provides graphical trending functionality , the pro scores can be plotted against other clinical data , such as medications that have been prescribed . the pro scores can also be accessed through results review , which is the function through which laboratory results are typically viewed . the pro scores can also be accessed using shortcut phrases , which allow them to be pasted directly into clinicians notes . finally , the pdf , which contains pro scores and individual item responses , is available to clinicians through the ehr s document storage functionality called media tab . the health system s research enterprise makes pro data , which is integrated with other clinical data as described above , available to researchers through two mechanisms . first , university - affiliated researchers can obtain count - based information through the institution s i2b2 ( informatics for integrating biology and the bedside ) system by simply requesting access to the i2b2 web client.33,45 i2b2 provides count - based information such as the number of unique patients seen within the health system who meet certain clinical criteria and their breakdown by age , race , and sex . for example , a researcher could query for the number and age ranges of women with diabetes who have documented promis depression and anxiety scores in the 75 percentile or above . such information may be useful for determining feasibility of recruitment for prospective studies or availability of existing data for retrospective studies . second , researchers can obtain detailed data through the institution s integrated data repository ( idr).45 the idr encompasses an enterprise clinical data warehouse , supporting personnel , and information management and governance processes . the idr provides a single source of administrative and clinical information that supports research , clinical care , and operations . the idr is funded by the health system and the university s clinical and translational science institute ( ctsi ) . the idr is continuously growing in terms of the amount and types of clinical data that it contains . with irb approval , university - affiliated researchers can obtain detailed pro and other clinical data from the idr using a standardized data request process . thus , their studies may be deemed nonhuman by the irb ( if using de - identified data ) or may be approved with a waiver of patient informed consent . furthermore , the university , through the ctsi , proactively promotes and educates researchers on i2b2 and idr data access . this occurs through regular tutorials to students and faculty groups , an idr studio in which researchers bring specific research questions for review and consultation , and monthly email blasts that describe new data elements when they become available for research use . two export , transform , and load ( etl ) processes move data from the ehr to the idr and i2b2 , as described below . first , on a daily basis , select clinical data elements that were chosen to support operational and research needs are copied from the ehr s database to the idr s clinical data warehouse . both databases use sql server 2012 databases and reside within the health system s secure clinical data environment . second , on a monthly basis , the i2b2 database is refreshed with data from the clinical data warehouse . the i2b2 database , which is also implemented in sql server 2012 , contains a health insurance portability and accountability act ( hipaa ) limited data set of patient information and resides outside of the clinical enterprise s privacy wall so that researchers can query it using i2b2 . a separate mapping table that contains unique patient identifiers is maintained inside the enterprise s privacy wall and supports fulfillment of research data requests that are based on the results of previously executed i2b2 queries . to collect pros at the point of care , we adopted an existing web - based system that administers pro assessments called the collaborative health outcomes information registry ( choir ) . choir was first developed and implemented at stanford university to support specialty pain care and clinical research.35,36 our university received a no - cost license to use and modify the software within our health system . under a similar license , choir is also in various stages of design and implementation at other academic health centers.35 choir allows patients to complete pro assessments via web - enabled devices . choir also contains administrative functions that allow clinicians or practice staff to register patients , assign surveys , and review pro results at a point in time or longitudinally . through a computer - adaptive testing engine , choir administers pro measures , including patient - reported outcome measurement information system ( promis ) measures.6,37 computer - adaptive and static instruments can be combined to create different patient assessments . new instruments can also be added to those already available in choir . in the current system , patients complete an assessment that includes nine computer - adaptive promis measures ( pain interference , pain behavior , fatigue , physical function , sleep disturbance , sleep - related impairment , anger , depression , and anxiety)38,39 as well as static measures of pain location , pain intensity ( 010 scale ) , pain catastrophizing,40 and risk of opioid - related aberrant behavior.41 while these measures have specific relevance to patients with chronic pain , the domains are also generally relevant to primary care patients and clinical management of other chronic diseases . patients complete assessments using a tablet computer prior to seeing their provider , either in a waiting room or exam room . because the data are initially collected for regular clinical care , patients do not consent to research reuse when reporting their outcomes . however , as we describe later in the case , researchers who wish to reuse the pros must obtain institutional review board ( irb ) approval . once an assessment is complete for the promis measures , the standardized scores include a t - score ( mean 50 , standard deviation 10 ) and a percentage score ( 0100 percent ) , which are based on calibration samples that are representative of the united states population.42 also , choir produces a comprehensive pro result set in portable document format ( pdf ) that can be viewed , saved , or printed by clinicians . all data are stored as discrete entities in an oracle database within the health system s secure clinical data environment . to integrate pro results with other clinical data in support of clinical care the choir system sends the standardized pro scores to our health system s epic ehr . choir creates hl7 version 2.1 messages and sends discrete pro results through epic s laboratory and results interface . each message contains unique patient and encounter identifiers that link the pro results to patients other health record information . we are in the process of mapping the pro measures to logical observation identifiers names and codes ( loinc ) . loinc is a universal system for coding laboratory results and clinical observations , including patient assessments.43 also , within united states government initiatives to increase ehr use and health data exchange , loinc is the preferred vocabulary standard for results.44 therefore , by mapping the pro results to loinc , we expect to increase the ability to share results both internally and with external organizations over time . finally , in addition to the discrete numeric results , choir sends the comprehensive results set pdf document in a separate hl7 version 2.6 message . this pdf document contains the same pro results scores that are sent as discrete numeric results through the ehr s laboratory and results interface . however , the pdf also contains patients responses to the individual pro assessment items in case clinicians are interested in obtaining the full details of their patients responses . both the discrete pro scores and the pdf , which includes individual item responses , are available to clinicians in the ehr . using synopsis , which provides graphical trending functionality , the pro scores can be plotted against other clinical data , such as medications that have been prescribed . the pro scores can also be accessed through results review , which is the function through which laboratory results are typically viewed . the pro scores can also be accessed using shortcut phrases , which allow them to be pasted directly into clinicians notes . finally , the pdf , which contains pro scores and individual item responses , is available to clinicians through the ehr s document storage functionality called media tab . the health system s research enterprise makes pro data , which is integrated with other clinical data as described above , available to researchers through two mechanisms . first , university - affiliated researchers can obtain count - based information through the institution s i2b2 ( informatics for integrating biology and the bedside ) system by simply requesting access to the i2b2 web client.33,45 i2b2 provides count - based information such as the number of unique patients seen within the health system who meet certain clinical criteria and their breakdown by age , race , and sex . for example , a researcher could query for the number and age ranges of women with diabetes who have documented promis depression and anxiety scores in the 75 percentile or above . such information may be useful for determining feasibility of recruitment for prospective studies or availability of existing data for retrospective studies . second , researchers can obtain detailed data through the institution s integrated data repository ( idr).45 the idr encompasses an enterprise clinical data warehouse , supporting personnel , and information management and governance processes . the idr provides a single source of administrative and clinical information that supports research , clinical care , and operations . the idr is funded by the health system and the university s clinical and translational science institute ( ctsi ) . the idr is continuously growing in terms of the amount and types of clinical data that it contains . with irb approval , university - affiliated researchers can obtain detailed pro and other clinical data from the idr using a standardized data request process . thus , their studies may be deemed nonhuman by the irb ( if using de - identified data ) or may be approved with a waiver of patient informed consent . furthermore , the university , through the ctsi , proactively promotes and educates researchers on i2b2 and idr data access . this occurs through regular tutorials to students and faculty groups , an idr studio in which researchers bring specific research questions for review and consultation , and monthly email blasts that describe new data elements when they become available for research use . two export , transform , and load ( etl ) processes move data from the ehr to the idr and i2b2 , as described below . first , on a daily basis , select clinical data elements that were chosen to support operational and research needs are copied from the ehr s database to the idr s clinical data warehouse . both databases use sql server 2012 databases and reside within the health system s secure clinical data environment . second , on a monthly basis , the i2b2 database is refreshed with data from the clinical data warehouse . the i2b2 database , which is also implemented in sql server 2012 , contains a health insurance portability and accountability act ( hipaa ) limited data set of patient information and resides outside of the clinical enterprise s privacy wall so that researchers can query it using i2b2 . a separate mapping table that contains unique patient identifiers is maintained inside the enterprise s privacy wall and supports fulfillment of research data requests that are based on the results of previously executed i2b2 queries . pro data collection , integration with other clinical data , and dissemination to researchers is currently operating in the institution . for each , we qualitatively summarize barriers and facilitators to clinic operation , which we identified through regular check - ins with practice staff ; our own workflow observations ; and discussions with patients , clinicians , system developers , and administrators . when possible , we also quantitatively describe the extent to which each function was used in the first six months following implementation ( table 1 ) . in terms of pro data collection , clinicians in two family - medicine practice locations have been using the choir system to collect electronic pros from select patients on a daily basis . also , two other departments are currently reviewing the system for adoption in their clinics . in the two family - medicine practice locations , initial barriers to data collection included clinician uncertainty about the clinical benefit of the data and concerns about slowing down workflow . to help overcome the barriers , we developed a process that leveraged ehr data to pre - identify patients with chronic pain who were then targeted to complete the pro assessments . in collaboration with practice staff and providers , we also found existing patient wait times within workflows during which patients could reliably complete assessments before seeing their provider . in our testing , patients typically completed the questionnaires in less than 10 minutes . also , patients typically expressed willingness to use the tablet computers to report outcomes . while we did not precisely measure the number of patients who refused the pro assessment in practices , in our related study on the same types of patients with chronic pain,34 84 percent of patients responded feel comfortable and able to use a tablet computer to answer questions about your health at your doctor s office . finally , after implementation , clinicians and staff did not express concerns that the data collection slowed down patient throughput . therefore , the practice processes tolerated regular data collection without significant changes to existing workflows . we further discuss elsewhere barriers to pro data collection in clinics and how we overcame them.30 while a majority of patients with chronic pain who were approached were willing and able to complete the pro assessment prior to seeing a provider , patients with known chronic pain conditions represented a minority of all patients in the practices . still , in the first six months , a total of 309 complete pro assessments , which include 13 distinct pro measures , were recorded by 203 unique patients . these pro data were integrated with clinical data in the ehr and became part of the regular data loads in the clinical data warehouse and i2b2 ( figure 2 ) . this represents a moderate - size and growing data set on which future research analyses can be conducted . we discovered no problems in integrating the pro data with patients electronic records once the system went live in the practices . pro results consistently loaded into the ehr after patients reported them . furthermore , because the integration of pros into the clinical data warehouse and i2b2 followed established institutional processes for curating and integrating data , the pros quickly became a standard part of regular data loads from the clinical - information system infrastructure to the research - information system infrastructure . with respect to facilitating research use of pros , we have two examples of research studies that were actively supported by the system in the first six months after implementation . our previously mentioned study on the effect of introducing pros into the ehr during patient visits has been supported by all three systems.34 that study obtains regular data extracts , including pros and other clinical data , through the idr s data request process . second , a future study that aims to qualitatively examine patient reporting of pros during office visits recently used i2b2 to identify a cohort of patients with high pain interference who can be contacted about potential research participation . overall , in the first six months after implementation , researchers ran 39 i2b2 queries in support of these 2 exemplar studies . with that said , one of the goals of the system is to support pro data reuse by a larger and more diverse group of researchers across the institution . as an early understanding of success in that regard , other researchers in the institution ran i2b2 queries involving pro data 18 times , or an average of 3 queries per month . over the same period , i2b2 received an average of 519 total queries per month , which indicates overall system feasibility . therefore , while it is still in the early stages of use , we expect the system will increasingly be used for point - of - care pro data collection and subsequent reuse of pro data for research . our successful design and implementation of an information system that supports pros in research was driven by three overarching strategies ( table 2 ) . first , we selected and implemented multiple interfaced technologies to support pro collection , management , and research use . in other words , rather than using one system only ( e.g. , epic ) to collect , integrate , and disseminate pros to researchers , we chose to leverage different systems with unique strengths in these functions . second , we aimed to use standardized approaches to measuring pros , sending pros between systems , and disseminating pros . finally , we focused on using technologies and processes that aligned with existing clinical - research information - management strategies within our organization . together , these strategies helped drive a successful system that is currently collecting pros from patients on a daily basis and feeding those data into the research enterprise for widespread use . furthermore , we believe these strategies will increase the long - term growth and sustainability of the system locally and extensibility of our general approach to other institutions . below , we expand on these strategies and describe specific challenges and lessons learned from our experiences . we implemented technologies from multiple sources , including an open - source tool in i2b2 and another freely licensable tool in choir . we chose this approach so that we could take advantage of innovative software and functionality that were unavailable in the ehr . using epic to collect pros this would have increased patient response burden and required patients to be registered with epic s personal health record tool . instead , choir provides an ehr - independent platform into which we can easily incorporate and modify static and computer adaptive assessments . our technology choices also increased the availability of pro data for researchers in our institution and the potential for our process to be replicated by and allow data sharing with other health systems . rather than keeping pro data in choir , we made it available through our institution s enterprisewide idr and i2b2 . this process makes pro data collected by one clinician or collected for a particular research study readily available to other researchers . also , because choir and i2b2 software are freely available and ehr independent , they offer low - cost options for other institutions to acquire . i2b2 has already been adopted by more than 100 institutions worldwide and provides a platform for increasing cross - institutional sharing of pro and other clinical data.46 at the same time , because they are not mature vendor - based products , choir and i2b2 require additional overhead in terms of development and maintenance . another lesson learned and ongoing challenge relates to the ongoing cost of maintaining multiple systems from different sources , including maintaining the interfaces between these systems . currently , the interfaces require minimal maintenance to remain operational because they use standard approaches to data exchange that are also used to share data between other systems within our institution . however , as systems are updated or organizational policies change , the interfaces for the pro system may also need updating . for example , if organizational policies were changed to no longer support sending pro data through the ehr s laboratory and results interface , an alternate approach such as using epic s device interface would be needed . this would require continued administrative support for the pro system so that relevant development , testing , and implementation resources could be allocated . furthermore , we are hopeful that the increasing informatics and policy emphasis on systems and data interoperability will help minimize the cost of ongoing interface maintenance between our ehr and other systems.47,48 our success thus far has required champions in key clinical and research leadership positions and dedicated funding to support system development . on one hand , the choir software implementation was supported by a single - study research grant . on the other hand , i2b2 and related idr resources are supported by the health system and the university s research enterprise as part of general investments in research informatics infrastructure . such funding will likely need to come from research infrastructure funds and clinical departments interested in collecting pros to support their clinical services . in designing our system , we also aimed for processes that use widely available data , technology , and messaging standards for measuring pros , communicating pros between systems , and disseminating pros to researchers . for measuring pros , we focused on promis measures , whose development , validation , and use has grown dramatically in recent years . promis provides an increasingly popular approach to measuring pros , computer - adaptive assessments , and measures that span a broad range of domains , which we can add to our system in the future . for sending pros from choir to the ehr hl7 messaging standards are widely understood and would allow our interface process to be replicated by other institutions and in other ehr systems . in disseminating pros to researchers , our system reports pro results using standardized quantitative scores , including t - scores and population percentiles . each of these choices increases the potential scalability of our processes , in part or in whole , within and beyond our institution . in particular , the relative novelty of promis has made it difficult to interpret the clinical significance of promis results . similarly , the newness of promis has prevented us from linking each promis result to a standard coding system , such as loinc . therefore , we are currently in the process of mapping our pro measures to existing loinc codes , and we will likely request the creation of new loinc codes for some measures . finally , our design and implementation strategy required us to align our decisions and technologies with institutional strategies for collecting , managing , and disseminating clinical data for research . this alignment between individual systems and overarching strategies was necessary for both short- and long - term support for the growth and sustainability of the system . however , we learned that alignment with organizational strategies can be a moving target . for example , the goal of mapping promis results to loinc was prompted by not only the general value of standardization for communicating pro data but also by our institution s shift to using loinc , generally , for results . similarly , our institution has increasingly fielded requests from researchers and clinicians to interface its ehr system with other systems . because of this , administrators have reconsidered how many different interfaces and interface processes should be supported . therefore , in the future , maintaining the pro system may require reworking to adapt to changing institutionwide strategies . overall , as we prospectively devised and retrospectively reflected on our approach to collecting , integrating , and disseminating pros , we identified many specific challenges and lessons learned . beyond those above , we also continue to be challenged by the need to make pro data collection as easy as possible and clinically relevant in practice settings . while collecting pros at the point of care is critical because it allows data capture from essentially all patients , the value of pro data for research is not generally a compelling rationale for busy clinicians and office staff with a full schedule of patients . therefore , the cost of collecting pros must be negligible and the benefits must be clearly articulated . we implemented technologies from multiple sources , including an open - source tool in i2b2 and another freely licensable tool in choir . we chose this approach so that we could take advantage of innovative software and functionality that were unavailable in the ehr . using epic to collect pros this would have increased patient response burden and required patients to be registered with epic s personal health record tool . instead , choir provides an ehr - independent platform into which we can easily incorporate and modify static and computer adaptive assessments . our technology choices also increased the availability of pro data for researchers in our institution and the potential for our process to be replicated by and allow data sharing with other health systems . rather than keeping pro data in choir , we made it available through our institution s enterprisewide idr and i2b2 . this process makes pro data collected by one clinician or collected for a particular research study readily available to other researchers . also , because choir and i2b2 software are freely available and ehr independent , they offer low - cost options for other institutions to acquire . i2b2 has already been adopted by more than 100 institutions worldwide and provides a platform for increasing cross - institutional sharing of pro and other clinical data.46 at the same time , because they are not mature vendor - based products , choir and i2b2 require additional overhead in terms of development and maintenance . another lesson learned and ongoing challenge relates to the ongoing cost of maintaining multiple systems from different sources , including maintaining the interfaces between these systems . currently , the interfaces require minimal maintenance to remain operational because they use standard approaches to data exchange that are also used to share data between other systems within our institution . however , as systems are updated or organizational policies change , the interfaces for the pro system may also need updating . for example , if organizational policies were changed to no longer support sending pro data through the ehr s laboratory and results interface , an alternate approach such as using epic s device interface would be needed . this would require continued administrative support for the pro system so that relevant development , testing , and implementation resources could be allocated . furthermore , we are hopeful that the increasing informatics and policy emphasis on systems and data interoperability will help minimize the cost of ongoing interface maintenance between our ehr and other systems.47,48 our success thus far has required champions in key clinical and research leadership positions and dedicated funding to support system development . on one hand , the choir software implementation was supported by a single - study research grant . on the other hand , i2b2 and related idr resources are supported by the health system and the university s research enterprise as part of general investments in research informatics infrastructure . such funding will likely need to come from research infrastructure funds and clinical departments interested in collecting pros to support their clinical services . in designing our system , we also aimed for processes that use widely available data , technology , and messaging standards for measuring pros , communicating pros between systems , and disseminating pros to researchers . for measuring pros , we focused on promis measures , whose development , validation , and use has grown dramatically in recent years . promis provides an increasingly popular approach to measuring pros , computer - adaptive assessments , and measures that span a broad range of domains , which we can add to our system in the future . for sending pros from choir to the ehr hl7 messaging standards are widely understood and would allow our interface process to be replicated by other institutions and in other ehr systems . in disseminating pros to researchers , our system reports pro results using standardized quantitative scores , including t - scores and population percentiles . each of these choices increases the potential scalability of our processes , in part or in whole , within and beyond our institution . our choices of standards have also introduced some challenges . in particular , the relative novelty of promis has made it difficult to interpret the clinical significance of promis results . similarly , the newness of promis has prevented us from linking each promis result to a standard coding system , such as loinc . therefore , we are currently in the process of mapping our pro measures to existing loinc codes , and we will likely request the creation of new loinc codes for some measures . finally , our design and implementation strategy required us to align our decisions and technologies with institutional strategies for collecting , managing , and disseminating clinical data for research . this alignment between individual systems and overarching strategies was necessary for both short- and long - term support for the growth and sustainability of the system . for example , the goal of mapping promis results to loinc was prompted by not only the general value of standardization for communicating pro data but also by our institution s shift to using loinc , generally , for results . similarly , our institution has increasingly fielded requests from researchers and clinicians to interface its ehr system with other systems . because of this , administrators have reconsidered how many different interfaces and interface processes should be supported . therefore , in the future , maintaining the pro system may require reworking to adapt to changing institutionwide strategies . overall , as we prospectively devised and retrospectively reflected on our approach to collecting , integrating , and disseminating pros , we identified many specific challenges and lessons learned . beyond those above , we also continue to be challenged by the need to make pro data collection as easy as possible and clinically relevant in practice settings . while collecting pros at the point of care is critical because it allows data capture from essentially all patients , the value of pro data for research is not generally a compelling rationale for busy clinicians and office staff with a full schedule of patients . therefore , the cost of collecting pros must be negligible and the benefits must be clearly articulated . this case study described the context and lessons learned in implementing an information system that supports collecting , integrating , and using pros for research in a learning health care system . our description and lessons learned can help others as they think about how to implement useful research information systems while adhering to principles that allow their systems to scale and persist over time . with this case study , we also hope to spur dialogue in the research community on others successes ( and failures ) in designing , implementing , and managing systems for collecting and managing electronic pros in support of research . as other academic health centers grapple with the challenge of managing electronic clinical data and technology for research , the community would benefit from a more robust and diverse body of literature describing experiences . these descriptions may come from experiences of consortiums , such as those funded by the patient - centered outcomes research institute49 and others that manage cross - institutional data research networks . also , these descriptions could come from individual institutions that developed effective systems to support individual clinical studies that grew to be part of broader organizational solutions and can thus provide more general informatics - oriented contributions . initially , growth in published literature on such systems may offer qualitative syntheses of lessons learned across organizations . as the knowledge base matures , it may be possible to conduct cross - organizational quantitative studies that examine relationships between pro and other data management strategies and outcomes such as research productivity and quality . as with other case studies , our experiences and lessons the complete set of processes has been in place for only six months and pro data collection is not yet in operation throughout the health system . thus , it remains to be seen whether the strategies we employed will scale up within our institution or be adopted by others . in addition to more qualitative evaluations via stakeholder feedback , quantitative evaluation should include ongoing tracking of the volume of electronic pro data collection as it expands to other care settings and new types of patients . also , the institution logs i2b2 query volume and detailed research data requests . therefore , to assess ongoing research use of pros , we should track the volume and types of queries and data requests involving pro data . an academic health center that affiliates with multiple health systems may face additional challenges in using a system like ours . in such a setting , system designers may face challenges in developing consensus on a common pro data set and challenges in developing standards for integrating and disseminating data that come from multiple organizations . however , our general approach of using multiple systems that interface using standard data and messaging approaches may also be beneficial in settings with multiple health systems . our approach does not require each health system to use the same ehr or even to use choir for collecting pros . this flexibility may also be helpful if a health system decided to withdraw support for a given software or vendor and switch to another . also , using choir required significant start - up and moderate ongoing maintenance effort given that it is not a mature , vendor - based product . therefore , another institution that aimed to adopt the same technologies would need in - house software development expertise . we also note that the pro data are initially generated in a clinical setting without a researcher present . thus , the quality of the data may not be as high as data generated in a controlled setting where a researcher directly oversees the process . this concern is mitigated by the fact that the pro assessments were designed to be completed independently by patients . therefore , we believe the pro data are of sufficient quality for most observational studies and for cohort discovery purposes . we also believe that the pro data are likely of similar or better quality as other clinical data commonly used for these research purposes . new challenges will certainly arise as the corpus of pro data in the institution grows and researcher demand for the data grows with it . that said , we are actively improving our system and working to scale up the implementation in our health system . we are also partnering with other institutions that are planning to adopt our approaches to regularly capturing and using electronic pros in research . in conclusion , if learning health care systems across the united states and worldwide are to incorporate efficient solutions for generating pro data for research on large , diverse populations , it is important to continue sharing and learning from these experiences and advancing research on the management of these systems .

introduction : advances in health policy , research , and information technology have converged to increase the electronic collection and use of patient - reported outcomes ( pros ) . therefore , it is important to share lessons learned in implementing pros in research information systems.case description : the purpose of this case study is to describe a novel information system for electronic pros and lessons learned in implementing that system to support research in an academic health center . the system incorporates freely available and commercial software and involves clinical and research workflows that support the collection , transformation , and research use of pro data . the software and processes that comprise the system serve three main functions , ( i ) collecting electronic pros in clinical care , ( ii ) integrating pro data with non - patient generated clinical data , and ( iii ) disseminating data to researchers through the institution s research informatics infrastructure , including the i2b2 ( informatics for integrating biology and the bedside ) system.strategies:our successful design and implementation was driven by three overarching strategies . first , we selected and implemented multiple interfaced technologies to support pro collection , management , and research use . second , we aimed to use standardized approaches to measuring pros , sending pros between systems , and disseminating pros . finally , we focused on using technologies and processes that aligned with existing clinical research information management strategies within our organization.conclusion:these experiences and lessons may help future implementers and researchers enhance the scale and sustainable use of systems for research use of pros .

Introduction Context Case Description Electronic PRO Data Collection in Clinical Care Integrating PRO Data with Other Clinical Data Disseminating Data to Researchers Findings Strategies Strategic Selection and Implementation of Multiple Technologies to Support PROs Using Standardized Approaches to Measure, Exchange, and Disseminate PROs Aligning Technologies and Processes with Existing Clinical Research Information Management Strategies Discussion and Conclusion

advances in health policy , research , and information technology have converged to increase the electronic collection and use of patient - reported outcomes ( pros ) to support clinical care and research . through the affordable care act and other legislation , the united states government has invested in research and care delivery that focus on evidence - based treatments and reflect patients preferences and values.1,2 to support these initiatives , and concomitant with increasing electronic health record ( ehr ) adoption,35 researchers and clinicians are demanding information technology and data infrastructures that support routine collection and use of pros.68 pros allow patients to directly report on their health status and health care , such as quality of life , function , symptoms , or care experiences.9 furthermore , systematic pro collection and use in research supports the learning health care system goal of increasing the efficiency and scope of scientific discovery related to health care delivery.10 one approach to meeting the clinical and research demand for pros is to incorporate electronic data collection into everyday clinical processes.1113 this approach is appealing because it utilizes existing infrastructure and interactions with patients , which may allow data to be captured more efficiently and to be acquired for larger , more representative populations . also , by collecting data during care delivery , pros may be more easily integrated with complementary data , such as demographics , diagnoses , laboratory data , and medications . indeed , systems for collecting and sharing pros with clinicians have been shown to be usable,1416 to improve patient - clinician communication,1719 and to be supportive of both clinical care and research,20,21 such as the feasibility of linking pros with a cancer registry.22 furthermore , sharing pro data with clinicians at the point of care may improve patients health - related quality of life.23 however , broader analyses of the literature fail to reveal a consistent positive impact of pro collection and use on care processes , clinical decision - making , and health outcomes.2428 this lack of consistent impact may relate to inconsistencies in how electronic pro systems are implemented , including how burden on clinical workflows is managed and how pros are communicated to clinicians.2932 therefore , to more effectively meet the demand for pro data , there is a need to describe lessons learned in implementing pro systems . describing lessons learned with pro systems may help future implementers and researchers enhance the scale and sustainable use of such systems . the purpose of this case study is to describe a novel information system for electronic pros and our lessons learned in implementing the system , which supports clinical care and research , in an academic health center . uniquely , our lessons learned stem from a system that starts with point - of - care electronic pro collection and ends with institutionwide access to pros linked to other clinical data for research . we describe how pros are collected in primary care practices , integrated with other clinical data in an ehr , and then loaded into an institutional data warehouse . the system utilizes interfaces between freely available and commercial software and involves clinical and research workflows that support the collection , transformation , and research use of pro data . researchers in the institution can use web - based software33 to query for patients of interest based on pros and other clinical data before requesting detailed data for in - depth analysis . our evaluation of the system focused on assessing the feasibility of collecting , integrating , and then reusing pro data for research . therefore , we used qualitative data gleaned from informal and formal interactions with stakeholders in varied roles , including practice staff , clinicians , patients , system designers , researchers , and administrative leadership . these data were collected during the system s design , implementation , and the first six months after implementation . to supplement these qualitative data , we also collected quantitative data that describes the frequency with which pros were collected in clinical settings and reused by researchers . the technology and lessons learned described here are based on our initial implementation of pro data being collected in two family medicine practices affiliated with the university s family medicine department . therefore , the family medicine practices began collecting pro data related to pain , physical function , sleep , and mental health . at the same time , the first and third authors of this case study led a related pragmatic clinical trial to evaluate the effect of integrating pros in an ehr on clinician and patient satisfaction with care for chronic pain.34 the system s design and implementation were led by a team that included two physician researchers , an information science researcher , system developers , and the health system s chief data officer . the team received support from administrative leadership , including the community health and family medicine department chairperson , the college of medicine s assistant dean for clinical informatics , and the academic health center information technology organization . while this article primarily describes the electronic data collection and workflows for research use of pros , details of clinician and patient use of the pro system the software and processes that comprise the system serve three main functions : ( 1 ) collecting electronic pros in clinical care , ( 2 ) integrating pro data with other clinical data not reported by the patient , and ( 3 ) disseminating data to researchers ( figures 1 and 2 ) . to collect pros at the point of care , we adopted an existing web - based system that administers pro assessments called the collaborative health outcomes information registry choir was first developed and implemented at stanford university to support specialty pain care and clinical research.35,36 our university received a no - cost license to use and modify the software within our health system . under a similar license , choir is also in various stages of design and implementation at other academic health centers.35 choir allows patients to complete pro assessments via web - enabled devices . through a computer - adaptive testing engine , choir administers pro measures , including patient - reported outcome measurement information system ( promis ) measures.6,37 computer - adaptive and static instruments can be combined to create different patient assessments . because the data are initially collected for regular clinical care , patients do not consent to research reuse when reporting their outcomes . loinc is a universal system for coding laboratory results and clinical observations , including patient assessments.43 also , within united states government initiatives to increase ehr use and health data exchange , loinc is the preferred vocabulary standard for results.44 therefore , by mapping the pro results to loinc , we expect to increase the ability to share results both internally and with external organizations over time . the health system s research enterprise makes pro data , which is integrated with other clinical data as described above , available to researchers through two mechanisms . first , university - affiliated researchers can obtain count - based information through the institution s i2b2 ( informatics for integrating biology and the bedside ) system by simply requesting access to the i2b2 web client.33,45 i2b2 provides count - based information such as the number of unique patients seen within the health system who meet certain clinical criteria and their breakdown by age , race , and sex . second , researchers can obtain detailed data through the institution s integrated data repository ( idr).45 the idr encompasses an enterprise clinical data warehouse , supporting personnel , and information management and governance processes . the idr provides a single source of administrative and clinical information that supports research , clinical care , and operations . this occurs through regular tutorials to students and faculty groups , an idr studio in which researchers bring specific research questions for review and consultation , and monthly email blasts that describe new data elements when they become available for research use . first , on a daily basis , select clinical data elements that were chosen to support operational and research needs are copied from the ehr s database to the idr s clinical data warehouse . second , on a monthly basis , the i2b2 database is refreshed with data from the clinical data warehouse . choir was first developed and implemented at stanford university to support specialty pain care and clinical research.35,36 our university received a no - cost license to use and modify the software within our health system . under a similar license , choir is also in various stages of design and implementation at other academic health centers.35 choir allows patients to complete pro assessments via web - enabled devices . through a computer - adaptive testing engine , choir administers pro measures , including patient - reported outcome measurement information system ( promis ) measures.6,37 computer - adaptive and static instruments can be combined to create different patient assessments . because the data are initially collected for regular clinical care , patients do not consent to research reuse when reporting their outcomes . to integrate pro results with other clinical data in support of clinical care the choir system sends the standardized pro scores to our health system s epic ehr . loinc is a universal system for coding laboratory results and clinical observations , including patient assessments.43 also , within united states government initiatives to increase ehr use and health data exchange , loinc is the preferred vocabulary standard for results.44 therefore , by mapping the pro results to loinc , we expect to increase the ability to share results both internally and with external organizations over time . the health system s research enterprise makes pro data , which is integrated with other clinical data as described above , available to researchers through two mechanisms . first , university - affiliated researchers can obtain count - based information through the institution s i2b2 ( informatics for integrating biology and the bedside ) system by simply requesting access to the i2b2 web client.33,45 i2b2 provides count - based information such as the number of unique patients seen within the health system who meet certain clinical criteria and their breakdown by age , race , and sex . second , researchers can obtain detailed data through the institution s integrated data repository ( idr).45 the idr encompasses an enterprise clinical data warehouse , supporting personnel , and information management and governance processes . the idr provides a single source of administrative and clinical information that supports research , clinical care , and operations . the idr is funded by the health system and the university s clinical and translational science institute ( ctsi ) . this occurs through regular tutorials to students and faculty groups , an idr studio in which researchers bring specific research questions for review and consultation , and monthly email blasts that describe new data elements when they become available for research use . first , on a daily basis , select clinical data elements that were chosen to support operational and research needs are copied from the ehr s database to the idr s clinical data warehouse . second , on a monthly basis , the i2b2 database is refreshed with data from the clinical data warehouse . pro data collection , integration with other clinical data , and dissemination to researchers is currently operating in the institution . for each , we qualitatively summarize barriers and facilitators to clinic operation , which we identified through regular check - ins with practice staff ; our own workflow observations ; and discussions with patients , clinicians , system developers , and administrators . in terms of pro data collection , clinicians in two family - medicine practice locations have been using the choir system to collect electronic pros from select patients on a daily basis . these pro data were integrated with clinical data in the ehr and became part of the regular data loads in the clinical data warehouse and i2b2 ( figure 2 ) . we discovered no problems in integrating the pro data with patients electronic records once the system went live in the practices . furthermore , because the integration of pros into the clinical data warehouse and i2b2 followed established institutional processes for curating and integrating data , the pros quickly became a standard part of regular data loads from the clinical - information system infrastructure to the research - information system infrastructure . with respect to facilitating research use of pros , we have two examples of research studies that were actively supported by the system in the first six months after implementation . our previously mentioned study on the effect of introducing pros into the ehr during patient visits has been supported by all three systems.34 that study obtains regular data extracts , including pros and other clinical data , through the idr s data request process . with that said , one of the goals of the system is to support pro data reuse by a larger and more diverse group of researchers across the institution . therefore , while it is still in the early stages of use , we expect the system will increasingly be used for point - of - care pro data collection and subsequent reuse of pro data for research . our successful design and implementation of an information system that supports pros in research was driven by three overarching strategies ( table 2 ) . first , we selected and implemented multiple interfaced technologies to support pro collection , management , and research use . , epic ) to collect , integrate , and disseminate pros to researchers , we chose to leverage different systems with unique strengths in these functions . second , we aimed to use standardized approaches to measuring pros , sending pros between systems , and disseminating pros . finally , we focused on using technologies and processes that aligned with existing clinical - research information - management strategies within our organization . furthermore , we believe these strategies will increase the long - term growth and sustainability of the system locally and extensibility of our general approach to other institutions . below , we expand on these strategies and describe specific challenges and lessons learned from our experiences . our technology choices also increased the availability of pro data for researchers in our institution and the potential for our process to be replicated by and allow data sharing with other health systems . rather than keeping pro data in choir , we made it available through our institution s enterprisewide idr and i2b2 . currently , the interfaces require minimal maintenance to remain operational because they use standard approaches to data exchange that are also used to share data between other systems within our institution . this would require continued administrative support for the pro system so that relevant development , testing , and implementation resources could be allocated . furthermore , we are hopeful that the increasing informatics and policy emphasis on systems and data interoperability will help minimize the cost of ongoing interface maintenance between our ehr and other systems.47,48 our success thus far has required champions in key clinical and research leadership positions and dedicated funding to support system development . on the other hand , i2b2 and related idr resources are supported by the health system and the university s research enterprise as part of general investments in research informatics infrastructure . in designing our system , we also aimed for processes that use widely available data , technology , and messaging standards for measuring pros , communicating pros between systems , and disseminating pros to researchers . for measuring pros , we focused on promis measures , whose development , validation , and use has grown dramatically in recent years . promis provides an increasingly popular approach to measuring pros , computer - adaptive assessments , and measures that span a broad range of domains , which we can add to our system in the future . in disseminating pros to researchers , our system reports pro results using standardized quantitative scores , including t - scores and population percentiles . therefore , we are currently in the process of mapping our pro measures to existing loinc codes , and we will likely request the creation of new loinc codes for some measures . finally , our design and implementation strategy required us to align our decisions and technologies with institutional strategies for collecting , managing , and disseminating clinical data for research . overall , as we prospectively devised and retrospectively reflected on our approach to collecting , integrating , and disseminating pros , we identified many specific challenges and lessons learned . beyond those above , we also continue to be challenged by the need to make pro data collection as easy as possible and clinically relevant in practice settings . while collecting pros at the point of care is critical because it allows data capture from essentially all patients , the value of pro data for research is not generally a compelling rationale for busy clinicians and office staff with a full schedule of patients . our technology choices also increased the availability of pro data for researchers in our institution and the potential for our process to be replicated by and allow data sharing with other health systems . rather than keeping pro data in choir , we made it available through our institution s enterprisewide idr and i2b2 . i2b2 has already been adopted by more than 100 institutions worldwide and provides a platform for increasing cross - institutional sharing of pro and other clinical data.46 at the same time , because they are not mature vendor - based products , choir and i2b2 require additional overhead in terms of development and maintenance . currently , the interfaces require minimal maintenance to remain operational because they use standard approaches to data exchange that are also used to share data between other systems within our institution . furthermore , we are hopeful that the increasing informatics and policy emphasis on systems and data interoperability will help minimize the cost of ongoing interface maintenance between our ehr and other systems.47,48 our success thus far has required champions in key clinical and research leadership positions and dedicated funding to support system development . on the other hand , i2b2 and related idr resources are supported by the health system and the university s research enterprise as part of general investments in research informatics infrastructure . in designing our system , we also aimed for processes that use widely available data , technology , and messaging standards for measuring pros , communicating pros between systems , and disseminating pros to researchers . for measuring pros , we focused on promis measures , whose development , validation , and use has grown dramatically in recent years . promis provides an increasingly popular approach to measuring pros , computer - adaptive assessments , and measures that span a broad range of domains , which we can add to our system in the future . in disseminating pros to researchers , our system reports pro results using standardized quantitative scores , including t - scores and population percentiles . therefore , we are currently in the process of mapping our pro measures to existing loinc codes , and we will likely request the creation of new loinc codes for some measures . finally , our design and implementation strategy required us to align our decisions and technologies with institutional strategies for collecting , managing , and disseminating clinical data for research . this alignment between individual systems and overarching strategies was necessary for both short- and long - term support for the growth and sustainability of the system . overall , as we prospectively devised and retrospectively reflected on our approach to collecting , integrating , and disseminating pros , we identified many specific challenges and lessons learned . beyond those above , we also continue to be challenged by the need to make pro data collection as easy as possible and clinically relevant in practice settings . while collecting pros at the point of care is critical because it allows data capture from essentially all patients , the value of pro data for research is not generally a compelling rationale for busy clinicians and office staff with a full schedule of patients . this case study described the context and lessons learned in implementing an information system that supports collecting , integrating , and using pros for research in a learning health care system . our description and lessons learned can help others as they think about how to implement useful research information systems while adhering to principles that allow their systems to scale and persist over time . with this case study , we also hope to spur dialogue in the research community on others successes ( and failures ) in designing , implementing , and managing systems for collecting and managing electronic pros in support of research . as other academic health centers grapple with the challenge of managing electronic clinical data and technology for research , the community would benefit from a more robust and diverse body of literature describing experiences . as with other case studies , our experiences and lessons the complete set of processes has been in place for only six months and pro data collection is not yet in operation throughout the health system . therefore , to assess ongoing research use of pros , we should track the volume and types of queries and data requests involving pro data . an academic health center that affiliates with multiple health systems may face additional challenges in using a system like ours . in such a setting , system designers may face challenges in developing consensus on a common pro data set and challenges in developing standards for integrating and disseminating data that come from multiple organizations . therefore , we believe the pro data are of sufficient quality for most observational studies and for cohort discovery purposes . new challenges will certainly arise as the corpus of pro data in the institution grows and researcher demand for the data grows with it . we are also partnering with other institutions that are planning to adopt our approaches to regularly capturing and using electronic pros in research . in conclusion , if learning health care systems across the united states and worldwide are to incorporate efficient solutions for generating pro data for research on large , diverse populations , it is important to continue sharing and learning from these experiences and advancing research on the management of these systems .

the polymorphism of the antimicrobial drug 4-amino - n-(thiazol-2-ylidene)-benzene sulfonamide 1 ( trivial name sulfathiazole ) has been extensively studied and is now a classic example of this phenomenon . to date , five crystalline polymorphs of unsolvated 1 are fully characterized by single - crystal x - ray diffraction and over 100 crystalline solvates . although compound 1 can exist as imino ( 1a ) and amido tautomers ( 1b ) , in the crystal phase it is exclusively found as the imino tautomer 1a . the literature on the polymorphism of 1 can be confusing and contradictory , especially as different authors have used different polymorph numbering schemes . herein we adopt the csd enumeration scheme , and to avoid any possible confusion , this scheme is detailed in table s1 ( supporting information ) , together with the standard unit cell and reduced cell constants . a detailed overview of the preparation and characterization of the polymorphs of 1 has been recently been given by nagy and co - workers , in which they emphasize the difficulties of reproducibly obtaining the pure polymorphic forms by crystallization techniques . all forms crystallize in the monoclinic space group p21/c , although for convenience the alternative setting of p21/n is used in the literature for forms iv and v. forms ii and iv crystallize with one molecule in the asymmetric unit , and the remaining forms have two molecules . a large number of wide - ranging studies on the polymorphism of 1 have been undertaken . even answering the vexed , if important , question of the relative thermodynamic stabilities of the polymorphs of 1 has proved a challenge , due in part to the interconversion between the phases , but also because of their closely similar energies . it is now clear that the order of thermodynamic stability is quite temperature - dependent , and the inconsistencies in the literature regarding this matter have been pointed out recently by croker and co - workers . their own solubility and differential scanning calorimetric measurements suggest that in the temperature range 283323 k the stability order is i < v < iv < ii < iii . above 373 k , the relative order changes and form i becomes progressively more stable . close to the melting points , the relative order is ii < iii < iv < v < i , that is , almost a complete reversal of the room temperature order . in this study , we report high - resolution single crystal x - ray studies on forms i iv and single crystal neutron diffraction studies on forms ii iv at 100 k. we were unable to obtain single crystals of form v of sufficient quality to merit similar investigations on this polymorphic form . multipole refinements and topological analysis of the resulting density models were undertaken using the experimental x - ray structure factors and theoretical static structure factors obtained from periodic density functional theory ( dft ) calculations , and lattice energies were derived from the multipole populations . these results agree reasonably well with theoretical calculations on the lattice energies and intermolecular interaction energies but are insufficiently accurate to determine the relative polymorph stabilities . compound 1 was obtained from commercial sources and recrystallized according to literature procedures to afford the various polymorphs . as discussed previously , these procedures do not consistently afford the pure polymorphs , and in our hands we also found this to be the case . in some cases , we observed concomitant crystallization , and crystals had to be separated by hand and their polymorphic form confirmed by x - ray diffraction . single crystal x - ray diffraction data were collected near 100 k on a bruker - nonius kappaccd ( forms i , ii , and iv ) or bruker axs apex - ii ( form iii ) diffractometer , running under nonius collect or apex-2 software . after collection of the low - order reflection data , the same scan - sets were repeated with 1/10th of the exposure time . these rapid images were used to record the intense low - order data more accurately , including all reflections that were overexposed in the first set of images . the unit cell dimensions used for refinement purposes were determined by postrefinement of the setting angles of a significant portion of the data set , using the scalepack or saint software . the kappaccd frame images were integrated using denzo(smn ) with a sufficiently large spot size ( < 0.85 ) to account for the k1-2 splitting , which becomes quite significant at 50. the resultant raw intensity files from denzo(smn ) were processed using a locally modified version of denzox . an absorption correction by gaussian quadrature was then applied to the reflection data ( except for form iii ) . a second semiempirical correction ( without a theta - dependent correction ) was then applied to remove any residual absorption anisotropy due to the mounting medium and account for other errors such as machine instabilities . the data were scaled and merged using sortav to provide a set of unique reflections without systematic absences . all data sets were essentially complete and highly redundant . a spherical atom refinement using shelxl-2013 was initially undertaken , with full - matrix least - squares on f and using all the unique data . all non - h atoms were allowed anisotropic thermal motion . details of these refinements are given in table 1 . rw = { (w(fo fc))/(w(fo))}. rw = { (w(fo fc))/(w(fo))}. r = [ (fo)]/ [ fo ] . fo ( mean)|/fo ( summation carried out when more than one symmetry equivalent is averaged ) . neutron diffraction data were collected for forms ii , iii , and iv at 100 k on the sxd instrument at the isis spallation neutron source , using the time - of - flight ( tof ) laue diffraction method . reflection intensities were reduced to structure factors ( shelx style hklf 2 ) using standard sxd procedures , as implemented in the computer program sxd2001 . the unit cells used for refinements with shelxl-2013 were taken from the x - ray determinations . anisotropic displacement parameters were used for all atoms , including the h atoms . in the final cycles , the merged and extinction - corrected reflection data obtained from a converged shelxl refinement was used as the input reflection file . rw = { (w(fo fc))/(w(fo))}. rw = { (w(fo fc))/(w(fo))}. r = [(fo)]/ [ fo ] . rint = {n/(n 1}|fo fo(mean)|/fo ( summation is carried out only where more than one symmetry equivalent is averaged ) . the multipole formalism of hansen and coppens as implemented in the xd-2006 program suite was used . the function minimized in the least - squares procedure was w(|fo| the multipole expansion was truncated at the hexadecapole level for the s atoms , the octupole level for the o , n , and c atoms , and the quadrupole level for the h atoms . the importance of employing anisotropic displacement parameters ( adp s ) for h atoms in multipole refinements has been emphasized recently by several authors . the method of madsen ( shade2 program ) is known to provide an excellent approximation to h - atom adp s . for those forms of 1 for which neutron diffraction data were available , models using h atom adp s from the shade2 calculated or the scaled experimental neutron values were carefully compared , and little difference was found . in the final refinements for forms iv , the scaled experimental neutron adp s were used with fixed contributions , while for form i , the shade determined parameters were used in the same way . each pseudoatom was assigned a core and spherical - valence scattering factor derived from the relativistic dirac fock wave functions of su and coppens expanded in terms of the single- functions of bunge , barrientos and bunge . the radial fit of these functions was optimized by refinement of the expansion - contraction parameter . the valence deformation functions for the c , o , and h atoms used a single- slater - type radial function multiplied by the density - normalized spherical harmonics . the radial fits for the chemically distinct atoms were optimized by refinement of their expansion contraction parameters , . the radial functions used for the s atoms were those recommended by dominiak and coppens . for all forms , anharmonic motion for the sulfur atoms examination of the resultant pdf s using the xdpdf module in xd2006 indicated that this modeling was physically reasonable . the successful deconvolution of thermal motion was judged by the hirshfeld rigid bond criterion . scatter plots of the scale factor fobs / fcalc against sin()/ were flat across almost the entire resolution range , while difference fourier maps and residual density analysis showed that , for forms iii and iv , essentially no unmodeled features remained in the data ( figures s1s3 , supporting information ) . for forms i and ii however , some positive residuals were observed , which could be attributed to minor disorder or possibly minor twinning . for form i , there is a single residual peak close to the inversion center at ( 0.5 , 0.5 , 0 ) with an integrated electron density of 0.15 e , while for form ii there are two peaks that could be attributable to the s atoms from a second orientation . there are no previous reports of disorder in any polymorph of 1 , but if it is present for forms i and ii , then it is certainly present at no greater than the 12% level . there is no evidence in the neutron diffraction study for any disorder in ii , and no disorder model was used in the final refinements of the x - ray data . a careful comparison of the resultant multipole models and topological properties shows no evidence of discrepancies between forms i and ii and forms iii and iv which could be attributed to any putative disorder ( see below ) . gas - phase dft calculations on 1 were undertaken with the program gaussian09 at both the experimental and optimized geometries , using several functionals and basis sets . we quote here the results from the optimized geometry with the m02 - 2x functional and using the def2-tzvp basis for all atoms . fully periodic b3lyp calculations based on the experimental crystallographic parameters were also undertaken with crystal09 , using standard 6 - 31 g * * basis sets for all centers . lattice energies were calculated from the multipole models using the xd2006 suite and also using the gavezzotti atom atom coulomb london topological analysis on the gas - phase wave function was undertaken using the aimall software package , while hirshfeld surface analysis was conducted using the crystalexplorer program . to obtain a benchmark interaction energy , an mp2 complete basis set ( cbs ) limit was estimated using results from aug - cc - pvtz and aug - cc - pvqz basis sets . the hf component of the energy was extrapolated using the formula of karton , and the correlation energies were extrapolated using the formula of halkier . correlation was also performed at the df - lccsd(t0)/aug - cc - pvdz level , comparing the correlation energy with that from df - lmp2/aug - cc - pvdz . the difference in correlation energy was then added to the mp2/cbs total energies , and the resultant energies are referred to as cbs(t ) . counterpoise corrections were not used , as the local correlation methods are bsse free . the molecular structure , crystal packing , and h - bonding intermolecular interactions in polymorphs i iv of 1 are very well established , and only salient features will be discussed here . the two independent molecules for forms i and iii are denoted here as ia / b and iiia / b respectively . thermal ellipsoid plots for ia and ib are shown in figure 1 , and plots for the other forms are in figures s3s7 ( supporting information ) . are essentially identical , while ia and ib have a different conformation , as is clearly visible from the overlay plot shown in figure 2 and the characterizing torsion angles given in table 3 . these differences arise from differing torsions within the two rings and also a 180 rotation of the nh2 group . the differing molecular conformations in form i result in a distinct h - bonding arrangement and crystal packing compared with the other forms , as was previously discussed in detail by blagden et al . and , however , the sulfur atom of the thiazole ring lies close to one of the oxygen atoms of the sulfone group , with an s12o11 distance in the range 2.8734(4)2.9848(5 ) , significantly shorter than the sum ( 3.3 ) of their van der waals radii . the gas - phase structure ( optimized from the experimental geometry , figure s8 , supporting information ) has the same short so = s interaction ( 2.74 ) . this intramolecular interaction is clearly detectable in the topological analysis of the electron density in all forms , and its importance in locking the molecular conformation in 1 is discussed in further detail below . ortep plots of molecules ia ( top ) and ib ( bottom ) showing the atomic labeling scheme , with thermal ellipsoids drawn at the 50% probability level . anisotropic thermal parameters for h atoms were calculated using the shade procedure . molecular best - fit overlay plot ( a ) forms green , mol . 2 yellow ; form ii , red ; form iii mol . 1 , purple , mol . conformation 10 ( see text ) . in principle , neutron diffraction studies should provide more accurate anisotropic displacement parameters ( adp s ) than conventional x - ray studies , though in practice this is not always the case , for well - known reasons . on the basis of the hirshfeld rigid bond criterion , the x - ray derived adp s ( after multipole refinement ) appear slightly more reliable . for form ii , the average mean - square displacement amplitudes ( -msda s ) are 0.0019 ( 0.0026 ) for x - ray ( neutron ) , and for form iv they are 0.0017 ( 0.0020 ) for the atoms with anisotropic thermal parameters . several features are consistently observed for all polymorphs , in both the x - ray and neutron refinements . the adp of the thiazole sulfur atom is consistently larger and more anisotropic than that of the sulfone sulfur atom . the elongation perpendicular to the ring is clearly visible in figure 1 and figures s4s7 ( supporting information ) [ mean ueq - thiazole / ueq - sulfone = 1.73 , mean 1/3 for thiazole and sulfone are 3.30 , 1.64 respectively ] . this feature of the thiazole s atom is also observed in some other structures containing thiazole rings , for which adp s are available ( see figure s9 , supporting information ) . to ascertain whether this was due to genuine thermal motion , rather than a disguised disorder ( and nonplanarity ) in the thiazole ring , variable temperature x - ray diffraction data on form ii in the temperature range 100200 k were obtained . the same crystal was used for all data sets and the resolution ( sin / = 0.7 ) and data processing protocols were identical . analysis of the uij tensors of both sulfur atoms showed a linear dependence of components between 200 and 100 k ( figure s10 , supporting information ) . this is the expected behavior for a quantum oscillator and suggests that the adp for the thiazole s atom is representative of true thermal motion rather than a convolution of static or dynamic disorder . in our case , the msda s are not zero when extrapolated to 0 k , presumably due to systematic errors in the adp s ( possibly due to some anharmonicity ) . while the thiazole ring in the imino tautomer 1a is not formally aromatic , the sp hybridization of all three c atoms imposes planarity on the ring , and the contributions to the atomic displacement parameters from the computed frequencies of the internal modes are shown in figure s11 ( supporting information ) . they are consistent with an extended thermal motion of the s atom perpendicular to the ring , but it should be stressed that for heavy atoms such as sulfur , the internal modes make quite minor contributions to the observed adp , which is dominated by the low frequency modes s for all non - h atoms in the x - ray structures is their general similarity in all the polymorphic forms , suggesting the observed adp s reflect molecular vibrations that are hardly differentiated by the potential field due to the crystal packing . whitten and spackman have compared adp s using a quantitative similarity index s12 = 100(1 r12 ) , where r12 measures the overlap between the probability densities functions described by two displacement tensors u1 and u2 . this procedure has been coded into the simadp routine of wingx , in which the two molecules or fragments to be compared are rotated to minimize the discrepancy in their positional coordinates , and the orthogonalized uij tensors are then compared . since s12 does not provide a direct measure of the relative orientation of the eigenvectors of the tensors ( particularly if the tensors are close to isotropic ) , a combined figure of merit ( fom ) based on the similarity index s12 , reigval ( an r value based on the magnitudes of corresponding eigenvalues ) and rmseigvec ( the rms angles between corresponding eigenvectors ) is also computed . a perfect fit gives a fom of 0.0 and a value less than 0.05 indicates a very close fit . these results provide quantitative confirmation that corresponding atomic adp s in forms ii , iii , and iv are extremely similar the noticeably worse fits associated with comparisons involving form i may be due in part to its different conformation . the hirshfeld surface , defined as the surface where wa(r ) = promolecule(r)/procrystal(r ) = 0.5 , has been proposed as a useful graphical tool for examining the differing intermolecular interactions in polymorphic systems . one such example of its use in characterizing the differences in polymorphs is the case of carbamazepine . this surface is , of course , just one of many possible molecular surfaces , but it has the unique property of encoding the intermolecular contacts from its very definition . the hirshfeld surfaces for all individual molecules of 1 are shown in figure 3 , where the normalized contact distance ( dnorm ) is color - mapped onto the surface . red points mark intermolecular atomic contacts shorter than , while blue points mark contacts longer than the van der waals contact . it is immediately visually obvious that the sulfathiazole molecules in forms ii iv have similar intermolecular contacts , while those in form i are different from each other and also from forms ii iv . the so - called fingerprint plots , where the di and de distribution of surface points is shown as a frequency - coded scatter plot , are capable of providing useful quantitative information about the relative proportions of differing types of intermolecular contact . the fingerprint plot for form iv is shown in figure 4 , specifically broken down into contributions from the ch , hh , nh , oh , and sh contacts which together make up 91.2% of the surface . the fingerprint plots for all forms are shown in figure s12 ( supporting information ) . the differing types of h - bonding intermolecular interactions are indicated as types 18 and average distances given in table s12 , supporting information . iv compared with form i are clearly obvious from this graphical representation . in particular , although the n ho and n hn hydrogen bonding networks in the various polymorphs of 1 have been discussed in detail with reference to the different packing arrangements , it is clear from these fingerprint plots that the ch and hh contacts also make up a significant portion of the hirshfeld surface and so may make a considerable contribution to the intermolecular interaction energies and lattice energies . fingerprint plots of form iv showing the contributions from ( a ) all intermolecular contacts , ( b ) ch contacts ( 22.4% ) , ( c ) hh contacts ( 26.2% ) , ( d ) nh contacts ( 11.2% ) , ( e ) oh ( 22.2% ) , and ( f ) sh contacts ( 9.2% ) . de and di represent the distances from the surface to nearest external and internal atoms , respectively . the color coding gray - blue - cyan represents increasing numbers of surface contributors at individual de / di points . the electron density in the six independent experimental molecules ( ia , ib , ii , iiia , iiib , and iv ) and the theoretical optimized structures were subjected to a quantum theory of atoms in molecules ( qtaim ) analysis . the topological properties at the bond critical points ( bcp s ) for a few selected bonds are given in table 5 , and a full list is given in table s13 ( supporting information ) . since in this study we have effectively six different experimental determinations of these properties , the sample mean and standard deviation were computed and are also given in the tables . the experimental molecular graphs are all homeomorphic , and a representative plot for form iv is shown in figure 5a . bond paths and associated bcp s were detected for all the conventional covalent bonds . the experimental electron densities at the bcp s for all forms agree surprisingly well , and the theoretical value is within three standard deviations of the experimental sample mean in virtually all cases . as is commonly observed , the laplacian values , especially for the polar covalent bonds , show slightly greater discrepancies between theory and experiment . this is primarily due to differences in 3 , arising from the slightly differing positions of the bcp along the bond path . in particular for the s(11)n(10 ) bond , the magnitude of the laplacian is very sensitive to the position of the bcp , and there is a linear dependence of the laplacian on the percentage displacement of the bcp along the bond vector ( see figure s13 , supporting information ) . molecular graphs of ( a ) experimental form iv , ( b ) theoretical conformer 10 , ( c ) theoretical conformer 1180 . atomic color coding : h pale blue , c gray , n blue , o red , s orange ; bond critical points are shown as small red spheres , and ring critical points are shown as small yellow spheres . first six lines correspond to experimental forms ia , ib , ii , iiia , iiib , iv , respectively ; the next line ( italic ) corresponds to reference density from wave function of optimized geometry , and the final two lines ( bold ) are the sample mean and sample standard deviation for the experimental data . perhaps the most interesting feature in the molecular graphs is the bond path associated with the s12o11 intramolecular interaction . this feature was observed in all molecules and suggests it may be responsible for the observed molecular conformation . a representative plot of the laplacian in the plane of this interaction is shown in figure 6 , while a complete set of plots is given in the supporting information ( figure s14 ) . a local charge concentration at the o atom is linked , via the bond path , to an area of relative charge depletion on the s atom , suggesting that this interaction is primarily electrostatic in nature , that is , so(=s ) . this view is confirmed by the electrostatic potential shown in figure 7 and s15 ( supporting information ) where a relatively positive zone on the s atom ( in green ) is close to the negatively charged o atom ( in red ) . the source function at the so bcp as a reference point is also consistent with this view , as almost all atomic basins have a noticeable influence on the electron density ; that is , the source is highly delocalized ( see figure s16a , supporting information ) . plot of the laplacian function in form iiia through the s12n1o11 plane , showing the bond paths and bond ( red circles ) and ring ( yellow circles ) critical points associated with the s12o11 intramolecular interaction . electrostatic potential ( e ) for form iv mapped onto the 0.5 e electron density isosurface . the potential at + 1.495 e is shown in purple and 0.044 e in red . in a charge density study by guru row and co - workers on the closely related molecule 2-(4-amino - benzosulfonimido)-5-methyl-3h-1,3,4-thiadiazole ( trivial name sulfamethizole ) and its salts , a closely similar intramolecular so(=s ) interaction has been observed . recently , jackson et al . have discussed the importance of so ( and similar ) interactions in locking the molecular configuration in a number of thiophene based systems . they conclude that in general this so interaction is not the dominating factor in determining the molecular conformation , and x ho interactions were shown , in certain cases , to be energetically more favorable . in view of these results , we have examined the conformation of 1 in more detail , specifically the effect of driving the s11n10c11s12 torsion angle . two low energy conformations were observed ( figure s8 , tables s14 and s15 , supporting information ) , one where is close to zero ( as found in the experimental structures , conformation 10 ) , and one where is 180 , conformation 1180 engendering a significant n ho interaction . the molecular graphs for these two conformations are shown in figure 5b , c and demonstrate the presence of bond paths for the respective weak intramolecular interactions . the source function for conformation 1180 ( figure s16b , supporting information ) is consistent with the interpretation of the n the evolution of the total energy and the adjacent c111s11n10c11 torsion angle as a function of is shown in figure s17 ( supporting information ) and indicates a barrier of 50 kj mol to interconversion between conformers . this barrier presumably arises because of partial loss of character in the iminium c11n10 bond ( mean experimental distance 1.327(1 ) , optimized theoretical distance 1.292 ) during the rotation . the c11n10 internuclear distance increases by less than 0.01 during the rotation , but the delocalization index (c11,n10 ) decreases from 1.325 to 1.275 , consistent with a small loss of character . interaction is marginally more stable by 6 kj mol in the gas phase but has never been observed in an experimental structure . it is interesting to speculate why this is the case , but the most likely reason is that the various intermolecular interactions involving this h atom in the crystal structures [ n hn(imine ) in form i , n n ho in form v ] provide a greater stabilization than can be achieved from the involvement of this group in an intramolecular interaction . this is certainly borne out by the analysis of the h - bond contributions to the lattice energies discussed below . with regard to the idea that the intramolecular so ( and n ho ) interactions have a locking effect on the molecular conformation , some clear evidence for this is provided by the evolution of the c s n - c torsion and the associated so and ho distances , shown in figure s17 ( supporting information ) . this evolution is not smooth , but significant jumps in the torsion are observed , which can be traced to the energetic favorability of these intramolecular interactions . as soon as the driving torsion approaches a value whereby the so ( or n ho ) interactions become sterically feasible , the torsion immediately adjusts to accommodate the interaction . as a result , the so ( or ho ) distances remain quite short , 2.7 ( 1.9 ) , over a significant range of . the calculated barrier indicates that both conformers of the imine tautomer ought to be detectable and their interchange observable by nmr spectroscopy . finally , some mention should be made of the effect of minor disorder on derived topological parameters . as intimated in the experimental section , there are residual density features present for forms i and ii , which could be attributed to some minor disorder . the accepted wisdom is that any disorder in a data set renders that data very suspect , at least in terms of a charge density analysis . in our case , the level of disorder was so low that it could not be satisfactorily modeled by a secondary site and was effectively ignored . one way of quantifying the global effect that any such disorder has on a derived parameter p is through an r(par ) value : where p(model ) is the refined parameter value from the experimental data and p(theor ) is that obtained from theory and the summation is over all experimental measurements of the parameter . table 6 lists the r(par ) values for the derived topological parameters prho = bcp and pdelrho = (bcp ) , where the wave function density is used as the reference theoretical density . as expected , the global agreements between experiment and theoretical parameters are much better for bcp than for (bcp ) , but there is no obvious worsening for forms i and ii ( with minor disorder ) , compared with forms iii and iv ( with no detectable disorder ) . similar results are obtained for other derived topological parameters . in this study at least , we find no evidence that the proposed minor disorder has any detectable deleterious effect on the derived static density . the multipole populations obtained from the least - squares process seem relatively insensitive to errors in the structure factors from the minor disorder . the difficulties in predicting whether a molecule will exhibit polymorphism ( and if so , which form is the thermodynamically most stable one ) are well - known and have been recently summarized by price . since the energy differences between polymorphs may only be on the order of a few kj mol , any prediction of rank - order stabilities is very challenging indeed . the unit cell volumes we obtain for forms i iii at 100 k agree extremely well ( < 0.15% discrepancy ) with the previously reported 100 k cell volumes . solely on the basis of the crystal densities at 100 k ( 150 k for form v ) the expected relative order of thermodynamic stability is v < i < ii < iii < iv , but the presence of strong h - bonding in 1 is very likely to change this ordering . as stated in the introduction , the lattice energy calculation ( laten option in xd2006 ) relies on the estimation of the total intermolecular interaction energies eint , which is decomposed into several terms : the electrostatic term is estimated using the ep / mm method and is considered to be essentially accurate . the induction term is included in the experimentally determined multipole populations ( since these are determined in the crystal environment ) , while the exchange - repulsion and dispersion terms are approximated by atom atom potentials of williams and cox . initial publications by volkov and coppens were very encouraging regarding the comparability of this method with theoretical estimates of the interaction energy , and we were interested to use this approach to determine the relative lattice energies of the polymorphs of sulfathiazole from the charge density analysis . to reassess the accuracy of interaction energies determined from experimental multipole models , we have compared the interaction energies between the two independent molecules in the asymmetric unit of form iii using the interen option in xd2006 and computational methods , including a complete basis set limit calculation ( see experimental section ) , which may be taken as providing a bench mark value . the results are listed in table 7 and demonstrate the importance of including dispersion effects , since the dispersion - corrected functional b97-d gives a value much closer to the bench mark than the standard b3lyp functional . the multipole - derived value is reasonably close to the bench mark value but is clearly not as good as the dispersion - corrected dft method . the lattice energies computed from the experimental multipoles are highly dependent on the exact refinement model used ( table s16 , supporting information ) , in particular on the treatment of the h atom thermal motion . in table 8 , values obtained from the best multipole model are compared with several theoretical estimates , including gavezotti s pixel methodology . all these methods provide estimates of the lattice energy at 0 k , that is , only the enthalpic component . it is clear that they do not even agree on the relative order of stabilities , except that most methods indicate that form i is the least stable . although error estimates for the derived lattice energies are not available in the xd2006 program suite , a study by destro et al . suggests a typical error in the region of 10 kj mol . the discrepancies between experimental lattice energies from our multipole models and several theoretical approaches are in line with this estimate , but it is equally clear that no method is sufficiently accurate to allow a definitive stability order to be determined , at least for sulfathiazole . other methods of using information on the electron density topology to obtain a relative ranking of lattice energies in polymorphs are even more approximate and hence ( generally ) less useful . one such method involves identifying the intermolecular interactions through their qtaim signature the bond path . these bond paths are generally related to well established bonding situations , such as strong and weak h - bonds , and the individual h - bond bonding energies may be estimated using the approximation of espinosa et al . recently , abramov has employed this method to estimate the stability ranking of several polymorphic compounds , including sulfathiazole , for which the relative ranking was ii iii > i > v. given the approximate nature of this method , it is not surprising that the stability ranking differs from other estimations . to investigate this methodology in more detail , the intermolecular interactions that contribute to the total lattice energy calculations provided by the pixel method were analyzed in more detail . the results are summarized in tables s17 and s18 ( supporting information ) . only the intermolecular interactions of the first shell , that is , the closest neighbor h - bond interactions , were taken into consideration . the total intermolecular interaction energies of the first shell are greatest in form i , followed by iii , iv , and ii . however , the total interaction energy within the crystal lattice shows a different ranking stability of ii > iii > iv > i. in the first shell , the intermolecular interactions were limited only to the h - bonds ; however , there are also repulsive interactions that contribute to the total lattice energy . in the case of form i , for example , there is a considerable repulsive interaction of 15.4 kj mol between two iaia molecules ; this is also observed in forms iii and iv , where values of 17 kj mol and 19.0 kj mol , respectively , are found . in form ii , the intermolecular repulsive interaction energies are much smaller , at 3.9 kj mol . these are of comparable magnitudes to the h - bond energies , and we conclude that it is not possible to estimate the relative stability of the polymorphic forms of sulfathiazole only on the basis of the h - bond interactions . the topological analysis of the electron density for the polymorphs of sulfathiazole shows an essentially identical set of properties for all forms , indicating that the potential fields due to the differing crystal packings have little effect on the examined molecular properties . an intramolecular so(=s ) interaction between the thiazole sulfur atom and an oxygen in the sulfone group is consistently observed in all polymorphs . two low energy conformers in the gas phase have been identified , one being very similar to that found in the crystalline phase , while the other exhibiting an n ho interaction of the ring nh proton with a sulfone oxygen atom . the lattice energies obtained from the experimental multipole populations , although in reasonable agreement with those obtained theoretically , do not have the precision and accuracy required to rank the polymorph stabilities of sulfathiazole .

high resolution x - ray diffraction data on forms i iv of sulfathiazole and neutron diffraction data on forms ii iv have been collected at 100 k and analyzed using the atoms in molecules topological approach . the molecular thermal motion as judged by the anisotropic displacement parameters ( adp s ) is very similar in all four forms . the adp of the thiazole sulfur atom had the greatest amplitude perpendicular to the five - membered ring , and analysis of the temperature dependence of the adps indicates that this is due to genuine thermal motion rather than a concealed disorder . a minor disorder ( 12% ) is evident for forms i and ii , but a statistical analysis reveals no deleterious effect on the derived multipole populations . the topological analysis reveals an intramolecular s os interaction , which is consistently present in all experimental topologies . analysis of the gas - phase conformation of the molecule indicates two low - energy theoretical conformers , one of which possesses the same intramolecular s os interaction observed in the experimental studies and the other an s oh n intermolecular interaction . these two interactions appear responsible for locking the molecular conformation . the lattice energies of the various polymorphs computed from the experimental multipole populations are highly dependent on the exact refinement model . they are similar in magnitude to theoretically derived lattice energies , but the relatively high estimated errors mean that this method is insufficiently accurate to allow a definitive stability order for the sulfathiazole polymorphs at 0 k to be determined .

Introduction Experimental Section Results and Discussion Conclusions

to date , five crystalline polymorphs of unsolvated 1 are fully characterized by single - crystal x - ray diffraction and over 100 crystalline solvates . all forms crystallize in the monoclinic space group p21/c , although for convenience the alternative setting of p21/n is used in the literature for forms iv and v. forms ii and iv crystallize with one molecule in the asymmetric unit , and the remaining forms have two molecules . it is now clear that the order of thermodynamic stability is quite temperature - dependent , and the inconsistencies in the literature regarding this matter have been pointed out recently by croker and co - workers . their own solubility and differential scanning calorimetric measurements suggest that in the temperature range 283323 k the stability order is i < v < iv < ii < iii . in this study , we report high - resolution single crystal x - ray studies on forms i iv and single crystal neutron diffraction studies on forms ii iv at 100 k. we were unable to obtain single crystals of form v of sufficient quality to merit similar investigations on this polymorphic form . multipole refinements and topological analysis of the resulting density models were undertaken using the experimental x - ray structure factors and theoretical static structure factors obtained from periodic density functional theory ( dft ) calculations , and lattice energies were derived from the multipole populations . these results agree reasonably well with theoretical calculations on the lattice energies and intermolecular interaction energies but are insufficiently accurate to determine the relative polymorph stabilities . in some cases , we observed concomitant crystallization , and crystals had to be separated by hand and their polymorphic form confirmed by x - ray diffraction . single crystal x - ray diffraction data were collected near 100 k on a bruker - nonius kappaccd ( forms i , ii , and iv ) or bruker axs apex - ii ( form iii ) diffractometer , running under nonius collect or apex-2 software . neutron diffraction data were collected for forms ii , iii , and iv at 100 k on the sxd instrument at the isis spallation neutron source , using the time - of - flight ( tof ) laue diffraction method . the function minimized in the least - squares procedure was w(|fo| the multipole expansion was truncated at the hexadecapole level for the s atoms , the octupole level for the o , n , and c atoms , and the quadrupole level for the h atoms . the importance of employing anisotropic displacement parameters ( adp s ) for h atoms in multipole refinements has been emphasized recently by several authors . for those forms of 1 for which neutron diffraction data were available , models using h atom adp s from the shade2 calculated or the scaled experimental neutron values were carefully compared , and little difference was found . in the final refinements for forms iv , the scaled experimental neutron adp s were used with fixed contributions , while for form i , the shade determined parameters were used in the same way . the valence deformation functions for the c , o , and h atoms used a single- slater - type radial function multiplied by the density - normalized spherical harmonics . for all forms , anharmonic motion for the sulfur atoms examination of the resultant pdf s using the xdpdf module in xd2006 indicated that this modeling was physically reasonable . the successful deconvolution of thermal motion was judged by the hirshfeld rigid bond criterion . scatter plots of the scale factor fobs / fcalc against sin()/ were flat across almost the entire resolution range , while difference fourier maps and residual density analysis showed that , for forms iii and iv , essentially no unmodeled features remained in the data ( figures s1s3 , supporting information ) . for forms i and ii however , some positive residuals were observed , which could be attributed to minor disorder or possibly minor twinning . there are no previous reports of disorder in any polymorph of 1 , but if it is present for forms i and ii , then it is certainly present at no greater than the 12% level . there is no evidence in the neutron diffraction study for any disorder in ii , and no disorder model was used in the final refinements of the x - ray data . a careful comparison of the resultant multipole models and topological properties shows no evidence of discrepancies between forms i and ii and forms iii and iv which could be attributed to any putative disorder ( see below ) . gas - phase dft calculations on 1 were undertaken with the program gaussian09 at both the experimental and optimized geometries , using several functionals and basis sets . lattice energies were calculated from the multipole models using the xd2006 suite and also using the gavezzotti atom atom coulomb london topological analysis on the gas - phase wave function was undertaken using the aimall software package , while hirshfeld surface analysis was conducted using the crystalexplorer program . the hf component of the energy was extrapolated using the formula of karton , and the correlation energies were extrapolated using the formula of halkier . the difference in correlation energy was then added to the mp2/cbs total energies , and the resultant energies are referred to as cbs(t ) . the molecular structure , crystal packing , and h - bonding intermolecular interactions in polymorphs i iv of 1 are very well established , and only salient features will be discussed here . thermal ellipsoid plots for ia and ib are shown in figure 1 , and plots for the other forms are in figures s3s7 ( supporting information ) . and , however , the sulfur atom of the thiazole ring lies close to one of the oxygen atoms of the sulfone group , with an s12o11 distance in the range 2.8734(4)2.9848(5 ) , significantly shorter than the sum ( 3.3 ) of their van der waals radii . the gas - phase structure ( optimized from the experimental geometry , figure s8 , supporting information ) has the same short so = s interaction ( 2.74 ) . this intramolecular interaction is clearly detectable in the topological analysis of the electron density in all forms , and its importance in locking the molecular conformation in 1 is discussed in further detail below . in principle , neutron diffraction studies should provide more accurate anisotropic displacement parameters ( adp s ) than conventional x - ray studies , though in practice this is not always the case , for well - known reasons . on the basis of the hirshfeld rigid bond criterion , the x - ray derived adp s ( after multipole refinement ) appear slightly more reliable . for form ii , the average mean - square displacement amplitudes ( -msda s ) are 0.0019 ( 0.0026 ) for x - ray ( neutron ) , and for form iv they are 0.0017 ( 0.0020 ) for the atoms with anisotropic thermal parameters . several features are consistently observed for all polymorphs , in both the x - ray and neutron refinements . the adp of the thiazole sulfur atom is consistently larger and more anisotropic than that of the sulfone sulfur atom . this feature of the thiazole s atom is also observed in some other structures containing thiazole rings , for which adp s are available ( see figure s9 , supporting information ) . to ascertain whether this was due to genuine thermal motion , rather than a disguised disorder ( and nonplanarity ) in the thiazole ring , variable temperature x - ray diffraction data on form ii in the temperature range 100200 k were obtained . analysis of the uij tensors of both sulfur atoms showed a linear dependence of components between 200 and 100 k ( figure s10 , supporting information ) . this is the expected behavior for a quantum oscillator and suggests that the adp for the thiazole s atom is representative of true thermal motion rather than a convolution of static or dynamic disorder . in our case , the msda s are not zero when extrapolated to 0 k , presumably due to systematic errors in the adp s ( possibly due to some anharmonicity ) . while the thiazole ring in the imino tautomer 1a is not formally aromatic , the sp hybridization of all three c atoms imposes planarity on the ring , and the contributions to the atomic displacement parameters from the computed frequencies of the internal modes are shown in figure s11 ( supporting information ) . they are consistent with an extended thermal motion of the s atom perpendicular to the ring , but it should be stressed that for heavy atoms such as sulfur , the internal modes make quite minor contributions to the observed adp , which is dominated by the low frequency modes s for all non - h atoms in the x - ray structures is their general similarity in all the polymorphic forms , suggesting the observed adp s reflect molecular vibrations that are hardly differentiated by the potential field due to the crystal packing . these results provide quantitative confirmation that corresponding atomic adp s in forms ii , iii , and iv are extremely similar the noticeably worse fits associated with comparisons involving form i may be due in part to its different conformation . it is immediately visually obvious that the sulfathiazole molecules in forms ii iv have similar intermolecular contacts , while those in form i are different from each other and also from forms ii iv . the fingerprint plot for form iv is shown in figure 4 , specifically broken down into contributions from the ch , hh , nh , oh , and sh contacts which together make up 91.2% of the surface . in particular , although the n ho and n hn hydrogen bonding networks in the various polymorphs of 1 have been discussed in detail with reference to the different packing arrangements , it is clear from these fingerprint plots that the ch and hh contacts also make up a significant portion of the hirshfeld surface and so may make a considerable contribution to the intermolecular interaction energies and lattice energies . the electron density in the six independent experimental molecules ( ia , ib , ii , iiia , iiib , and iv ) and the theoretical optimized structures were subjected to a quantum theory of atoms in molecules ( qtaim ) analysis . the topological properties at the bond critical points ( bcp s ) for a few selected bonds are given in table 5 , and a full list is given in table s13 ( supporting information ) . the experimental electron densities at the bcp s for all forms agree surprisingly well , and the theoretical value is within three standard deviations of the experimental sample mean in virtually all cases . this is primarily due to differences in 3 , arising from the slightly differing positions of the bcp along the bond path . in particular for the s(11)n(10 ) bond , the magnitude of the laplacian is very sensitive to the position of the bcp , and there is a linear dependence of the laplacian on the percentage displacement of the bcp along the bond vector ( see figure s13 , supporting information ) . first six lines correspond to experimental forms ia , ib , ii , iiia , iiib , iv , respectively ; the next line ( italic ) corresponds to reference density from wave function of optimized geometry , and the final two lines ( bold ) are the sample mean and sample standard deviation for the experimental data . this feature was observed in all molecules and suggests it may be responsible for the observed molecular conformation . this view is confirmed by the electrostatic potential shown in figure 7 and s15 ( supporting information ) where a relatively positive zone on the s atom ( in green ) is close to the negatively charged o atom ( in red ) . they conclude that in general this so interaction is not the dominating factor in determining the molecular conformation , and x ho interactions were shown , in certain cases , to be energetically more favorable . two low energy conformations were observed ( figure s8 , tables s14 and s15 , supporting information ) , one where is close to zero ( as found in the experimental structures , conformation 10 ) , and one where is 180 , conformation 1180 engendering a significant n ho interaction . it is interesting to speculate why this is the case , but the most likely reason is that the various intermolecular interactions involving this h atom in the crystal structures [ n hn(imine ) in form i , n n ho in form v ] provide a greater stabilization than can be achieved from the involvement of this group in an intramolecular interaction . this is certainly borne out by the analysis of the h - bond contributions to the lattice energies discussed below . with regard to the idea that the intramolecular so ( and n ho ) interactions have a locking effect on the molecular conformation , some clear evidence for this is provided by the evolution of the c s n - c torsion and the associated so and ho distances , shown in figure s17 ( supporting information ) . this evolution is not smooth , but significant jumps in the torsion are observed , which can be traced to the energetic favorability of these intramolecular interactions . as intimated in the experimental section , there are residual density features present for forms i and ii , which could be attributed to some minor disorder . one way of quantifying the global effect that any such disorder has on a derived parameter p is through an r(par ) value : where p(model ) is the refined parameter value from the experimental data and p(theor ) is that obtained from theory and the summation is over all experimental measurements of the parameter . as expected , the global agreements between experiment and theoretical parameters are much better for bcp than for (bcp ) , but there is no obvious worsening for forms i and ii ( with minor disorder ) , compared with forms iii and iv ( with no detectable disorder ) . in this study at least , we find no evidence that the proposed minor disorder has any detectable deleterious effect on the derived static density . the multipole populations obtained from the least - squares process seem relatively insensitive to errors in the structure factors from the minor disorder . the unit cell volumes we obtain for forms i iii at 100 k agree extremely well ( < 0.15% discrepancy ) with the previously reported 100 k cell volumes . solely on the basis of the crystal densities at 100 k ( 150 k for form v ) the expected relative order of thermodynamic stability is v < i < ii < iii < iv , but the presence of strong h - bonding in 1 is very likely to change this ordering . as stated in the introduction , the lattice energy calculation ( laten option in xd2006 ) relies on the estimation of the total intermolecular interaction energies eint , which is decomposed into several terms : the electrostatic term is estimated using the ep / mm method and is considered to be essentially accurate . initial publications by volkov and coppens were very encouraging regarding the comparability of this method with theoretical estimates of the interaction energy , and we were interested to use this approach to determine the relative lattice energies of the polymorphs of sulfathiazole from the charge density analysis . to reassess the accuracy of interaction energies determined from experimental multipole models , we have compared the interaction energies between the two independent molecules in the asymmetric unit of form iii using the interen option in xd2006 and computational methods , including a complete basis set limit calculation ( see experimental section ) , which may be taken as providing a bench mark value . the lattice energies computed from the experimental multipoles are highly dependent on the exact refinement model used ( table s16 , supporting information ) , in particular on the treatment of the h atom thermal motion . all these methods provide estimates of the lattice energy at 0 k , that is , only the enthalpic component . although error estimates for the derived lattice energies are not available in the xd2006 program suite , a study by destro et al . the discrepancies between experimental lattice energies from our multipole models and several theoretical approaches are in line with this estimate , but it is equally clear that no method is sufficiently accurate to allow a definitive stability order to be determined , at least for sulfathiazole . the total intermolecular interaction energies of the first shell are greatest in form i , followed by iii , iv , and ii . these are of comparable magnitudes to the h - bond energies , and we conclude that it is not possible to estimate the relative stability of the polymorphic forms of sulfathiazole only on the basis of the h - bond interactions . the topological analysis of the electron density for the polymorphs of sulfathiazole shows an essentially identical set of properties for all forms , indicating that the potential fields due to the differing crystal packings have little effect on the examined molecular properties . an intramolecular so(=s ) interaction between the thiazole sulfur atom and an oxygen in the sulfone group is consistently observed in all polymorphs . two low energy conformers in the gas phase have been identified , one being very similar to that found in the crystalline phase , while the other exhibiting an n ho interaction of the ring nh proton with a sulfone oxygen atom . the lattice energies obtained from the experimental multipole populations , although in reasonable agreement with those obtained theoretically , do not have the precision and accuracy required to rank the polymorph stabilities of sulfathiazole .

resistance exercise has been shown to intensify the resting energy expenditure ( ee ) ( 25 , 26 ) and increase post - exercise lipid oxidation ( 20 ) , which would be desirable for weight loss ( 15 , 18 ) . the respiratory exchange ratio ( rer ) is a surrogate of substrate utilization , and can be used to identify the rate of lipid oxidation ( 2 , 11 , 15 , 22 , 25 ) . however , its response to resistance exercise sessions has not been extensively investigated , and data from a few available studies are varied ( 2 , 20 , 21 , 29 , 33 ) . prior researchers investigating the influence of resistance training variables upon ee , lipid oxidation , or rer have focused on the intensity ( 33 ) , volume ( 11 , 12 , 22 ) , rest intervals ( 29 ) , type of exercise ( 15 ) , or session design ( 25 ) . some studies tested the influence of complete sessions of resistance exercises ( 2 , 15 , 21 , 25 ) or compared resistance and aerobic exercise ( 3 , 9 , 20 ) . a previous study by our group ( 8) demonstrated that ee estimated from oxygen uptake net ( vo2net ) during and after exercise was higher after exercises performed with larger than smaller muscle mass . on the other hand , differences of substrate utilization between exercises with different muscle mass have not been yet addressed . this information would be valuable , since it is acknowledged that the amount of muscle mass influences ee and substrate utilization during and after exercise sessions ( 7 ) . the assessment of rer during and after resistance exercise sessions would be useful to address this issue , as demonstrated by several previous studies ( 1 , 2 , 12 , 21 ) . carbohydrates are used for glycogen resynthesis following exercise , which helps to explain the increased lipid oxidation during the excess post - exercise consumption ( epoc ) ( 13 ) . it can be speculated that ee and the need for posterior glycogenesis would be higher if a greater amount of muscle mass is exercised . thus , it is feasible to think that the amount of muscle mass used for exercises may determine the substrate utilization during post - exercise recovery and that the rer would reflect differences in this regard . this study compared the impact of multiple sets of acute resistance exercises performed with similar intensity but different muscle mass upon ee and substrate utilization , as reflected by rer during and after exercise . the hypothesis was that the exercised muscle mass would influence the total vo2 and rate of oxidation of nutrients estimated by means of the rer , especially lipid oxidation . ten healthy men with at least one year of experience with resistance training ( 1.60.4 yrs . ) volunteered for the study ( 263 yrs . ; 1796 cm ; 787 kg ) . the institutional ethical committee approved the study and participants signed written informed consent prior to enrolling in the study . data were assessed on four non - consecutive days , interspersed with intervals of 4872 hours , using a within - group study design . on the first day researchers determined the resting vo2 , rer , and load corresponding to 15 repetition - maximum ( 15rm ) for the horizontal leg press ( lp ) and the chest fly ( cf ) . on the second day , resting measures and 15rm determination protocols were repeated to check for reliability . subjects performed two exercise sessions in a counterbalanced design on the third and fourth days . upon arrival at the laboratory , subjects were positioned to perform the exercise , installing the vo2 mask and equipment before the standardized warm - up ( 12 repetitions with 30% 15rm ) . the exercise protocols began five minutes after the warm - up ( 5 sets of 10 repetitions with 15rm workloads and 1-min interval between sets ) . all subjects performed lp ( larger muscle mass ) and cf ( smaller muscle mass ) protocols on different days . prior to the 15rm tests , a warm - up of 12 repetitions with 30% of pre - determined 15rm was performed . after five minutes , three to four maximal trials with 5-minute rest intervals were allowed to determine 15rm either for lp or cf ( technogym , gambettola , fc , italy ) . similar procedures were adopted for determining the load corresponding to 15 rm in lp and cf , being repeated after 4872 hours to assess the load reliability . test - retest intraclass correlation ( icc ) and coefficient of variation were considered satisfactory ( lp : icc=0.96 , p<0.001 and cv=4.2% ; cf : icc=0.97 , p<0.001 and cv=4.8% ) . weight was measured to the nearest 1gram and height was measured in millimeters using a wall mounted stadiometer ( harpenden , cambridge , uk ) . body surface ( m ) was determined using classic standard protocol ( 6 ) , and body composition was calculated based on chest , abdomen and thigh skinfolds ( 14 , 32 ) . in order to avoid bias due to the thermic effect of food , the proportion of macronutrients in total energetic value ( tev ) recommended by the world health organization ( 24 ) and daily ee of approximately 2400 kcal / day were adopted as references ( 15 , 23 ) . on the day before the exercise sessions , participants were instructed to have dinner at 89 pm to minimize possible metabolic effects of previous nutritional intake . all tests were performed in the morning , and 2 hours prior to the experiment , a standard food intake was provided ( 16 ) : a glass of fruit juice ( 200 ml ) and 6 salt crackers . energy values of fruit juice and crackers were 88 kcal ( 22 g of carbohydrate ) and 240 kcal ( 38 g of carbohydrate , 8 g of fat , and 4 g of protein ) , respectively . prior to respiratory measurements at rest , subjects abstained from physical exercise , alcohol , soft drinks , and caffeine for 48 hours , fasted for 810 hours , and made minimum effort when travelling to the laboratory . upon arrival , they laid in a calm environment for 20 minutes . the vo2 and ventilation ( ve ) were measured using a vo2000 analyzer ( medical graphics , saint louis , mo , usa ) with subjects at the supine position . a facemask ( hans rudolph v mask ; hans rudolph inc . , shawnee , ks , usa ) covering the mouth and nose was attached to a bidirectional digital flow valve and fastened using a mesh hairnet and velcro straps . standard conditions were respected during the assessment , including length of fasting and resting period before measurement to allow subjects to achieve a steady state . the rer under typical metabolic conditions with stable respiratory function ranges from 0.7 to 1.0 . if rer is lower than 0.7 or higher than 1.0 , prolonged starvation or excessive recent energy consumption should be suspected , both events representing protocol violation of resting metabolic rate ( 4 ) . the rer at rest was assessed using a low - flow pneumotachometer ( 230 l / min ) and data output frequency of three respiratory cycles . the vo2 ( ml / kg / min ) was measured for 40 minutes , and analyses were performed using five minutes of steady state data after the 10-minute stabilization period . all resting measurements were made in a temperature and humidity controlled environment ( 2022 c and 6070% respectively ) . intraclass correlations and coefficients of variation were calculated ( icc=0.89 , p=0.023 and cv=5.2% ) . before the exercise protocols , subjects remained at rest in a quiet environment for 15 minutes or until rer matched the previously assessed resting values ( variation could not exceed 5% ) . during exercise , vo2 , vco2 , and ve were assessed using a medium - flow pneumotachometer ( 10120 l / min ) . upon completion of the exercise sequences , the same procedures adopted for rer assessment at rest were applied to measurements during post - exercise recovery . however , it was critical to change the pneumotachometer from medium- to low - flow . the change was made within five to ten minutes of recovery and approximately three minutes were required to reprogram the software , recalibrate the system , and resume assessment . data from the first minute of measurement after the pneumotachometer replacement were discarded . in brief , post - exercise breath - by - breath vo2 data were averaged immediately following the last set of a given exercise , and at 1 , 2 , 3 , 4 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , and 90 minutes of recovery . individual breath - by - breath data points for vo2 ( ml / kg / min ) were averaged for the entire set and each minute of resting intervals . the total vo2 ( l ) for a given exercise was calculated by adding the values obtained during the sets and rest intervals . the absolute vo2 ( l / min ) and rer during each set , between set intervals , and post - exercise recovery were used to calculate the total vo2 ( defined as the sum of vo2 obtained during the exercises , recovery intervals , and 90-minute epoc ) . the net vo2 ( total vo2 resting vo2 ) was adopted as a surrogate of total ee . the occurrence of lipid oxidation was related to rer < 0.71 , whereas a value > 1.00 was considered to reflect carbohydrate oxidation . considering a rer > 0.85 as an index of fat to carbohydrate oxidation , total fat and carbohydrate ( cho ) oxidation rates were calculated according to the non - protein respiratory quotient ( 28 ) : cho oxidation rate = 4.585 vco2 3.226 vo2 ; fat oxidation rate = 1.695 vo2 1.701 vco2 , with vo2 and vco2 expressed in liters per minute ( l / min ) and oxidation rates in grams per minute ( g / min ) . it assumes that the amount of protein oxidized is negligible , and that other metabolic processes involving the production or utilization of o2 or co2 are also quantitatively negligible compared to glucose and fatty acid oxidation ( 28 ) . the post - exercise cho and fat consumption were averaged at each five minutes of recovery , starting with 1115 until 8690 minutes . the first 10 minutes of recovery were discarded to avoid influence of the pneumotachometer change , as well as bias due to peak vo2 attained during the exercise sessions . therefore total vo2 , cho and fat consumption during and after exercises were compared using student t - tests for paired samples . the rer measured during exercise was compared by 2-way repeated measures anova ( exercises and sets as main factors ) . differences between rer , cho , and fat consumption at baseline and during recovery were tested by repeated measures anova . in all cases , fisher post hoc tests were applied in the event of significant f ratios . a probability level of p the same statistical software was used for all calculations ( stata / se 8.2 , college station , tx , usa ) . ten healthy men with at least one year of experience with resistance training ( 1.60.4 yrs . ) volunteered for the study ( 263 yrs . ; 1796 cm ; 787 kg ) . the institutional ethical committee approved the study and participants signed written informed consent prior to enrolling in the study . data were assessed on four non - consecutive days , interspersed with intervals of 4872 hours , using a within - group study design . on the first day researchers determined the resting vo2 , rer , and load corresponding to 15 repetition - maximum ( 15rm ) for the horizontal leg press ( lp ) and the chest fly ( cf ) . on the second day , resting measures and 15rm determination protocols were repeated to check for reliability . subjects performed two exercise sessions in a counterbalanced design on the third and fourth days . upon arrival at the laboratory , subjects were positioned to perform the exercise , installing the vo2 mask and equipment before the standardized warm - up ( 12 repetitions with 30% 15rm ) . the exercise protocols began five minutes after the warm - up ( 5 sets of 10 repetitions with 15rm workloads and 1-min interval between sets ) . all subjects performed lp ( larger muscle mass ) and cf ( smaller muscle mass ) protocols on different days . prior to the 15rm tests , a warm - up of 12 repetitions with 30% of pre - determined 15rm was performed . after five minutes , three to four maximal trials with 5-minute rest intervals were allowed to determine 15rm either for lp or cf ( technogym , gambettola , fc , italy ) . similar procedures were adopted for determining the load corresponding to 15 rm in lp and cf , being repeated after 4872 hours to assess the load reliability . test - retest intraclass correlation ( icc ) and coefficient of variation were considered satisfactory ( lp : icc=0.96 , p<0.001 and cv=4.2% ; cf : icc=0.97 , p<0.001 and cv=4.8% ) . weight was measured to the nearest 1gram and height was measured in millimeters using a wall mounted stadiometer ( harpenden , cambridge , uk ) . body surface ( m ) was determined using classic standard protocol ( 6 ) , and body composition was calculated based on chest , abdomen and thigh skinfolds ( 14 , 32 ) . in order to avoid bias due to the thermic effect of food , the proportion of macronutrients in total energetic value ( tev ) recommended by the world health organization ( 24 ) and daily ee of approximately 2400 kcal / day were adopted as references ( 15 , 23 ) . on the day before the exercise sessions , participants were instructed to have dinner at 89 pm to minimize possible metabolic effects of previous nutritional intake . all tests were performed in the morning , and 2 hours prior to the experiment , a standard food intake was provided ( 16 ) : a glass of fruit juice ( 200 ml ) and 6 salt crackers . energy values of fruit juice and crackers were 88 kcal ( 22 g of carbohydrate ) and 240 kcal ( 38 g of carbohydrate , 8 g of fat , and 4 g of protein ) , respectively . , subjects abstained from physical exercise , alcohol , soft drinks , and caffeine for 48 hours , fasted for 810 hours , and made minimum effort when travelling to the laboratory . upon arrival , they laid in a calm environment for 20 minutes . the vo2 and ventilation ( ve ) were measured using a vo2000 analyzer ( medical graphics , saint louis , mo , usa ) with subjects at the supine position . , shawnee , ks , usa ) covering the mouth and nose was attached to a bidirectional digital flow valve and fastened using a mesh hairnet and velcro straps . standard conditions were respected during the assessment , including length of fasting and resting period before measurement to allow subjects to achieve a steady state . the rer under typical metabolic conditions with stable respiratory function ranges from 0.7 to 1.0 . if rer is lower than 0.7 or higher than 1.0 , prolonged starvation or excessive recent energy consumption should be suspected , both events representing protocol violation of resting metabolic rate ( 4 ) . the rer at rest was assessed using a low - flow pneumotachometer ( 230 l / min ) and data output frequency of three respiratory cycles . the vo2 ( ml / kg / min ) was measured for 40 minutes , and analyses were performed using five minutes of steady state data after the 10-minute stabilization period . all resting measurements were made in a temperature and humidity controlled environment ( 2022 c and 6070% respectively ) . intraclass correlations and coefficients of variation were calculated ( icc=0.89 , p=0.023 and cv=5.2% ) . before the exercise protocols , subjects remained at rest in a quiet environment for 15 minutes or until rer matched the previously assessed resting values ( variation could not exceed 5% ) . during exercise , vo2 , vco2 , and ve were assessed using a medium - flow pneumotachometer ( 10120 l / min ) . upon completion of the exercise sequences , the same procedures adopted for rer assessment at rest were applied to measurements during post - exercise recovery . however , it was critical to change the pneumotachometer from medium- to low - flow . the change was made within five to ten minutes of recovery and approximately three minutes were required to reprogram the software , recalibrate the system , and resume assessment . data from the first minute of measurement after the pneumotachometer replacement were discarded . in brief , post - exercise breath - by - breath vo2 data were averaged immediately following the last set of a given exercise , and at 1 , 2 , 3 , 4 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , and 90 minutes of recovery . individual breath - by - breath data points for vo2 ( ml / kg / min ) were averaged for the entire set and each minute of resting intervals . the total vo2 ( l ) for a given exercise the absolute vo2 ( l / min ) and rer during each set , between set intervals , and post - exercise recovery were used to calculate the total vo2 ( defined as the sum of vo2 obtained during the exercises , recovery intervals , and 90-minute epoc ) . the net vo2 ( total vo2 resting vo2 ) was adopted as a surrogate of total ee . the occurrence of lipid oxidation was related to rer < 0.71 , whereas a value > 1.00 was considered to reflect carbohydrate oxidation . considering a rer > 0.85 as an index of fat to carbohydrate oxidation , total fat and carbohydrate ( cho ) oxidation rates were calculated according to the non - protein respiratory quotient ( 28 ) : cho oxidation rate = 4.585 vco2 3.226 vo2 ; fat oxidation rate = 1.695 vo2 1.701 vco2 , with vo2 and vco2 expressed in liters per minute ( l / min ) and oxidation rates in grams per minute ( g / min ) . the non - protein respiratory quotient approach has been extensively applied . it assumes that the amount of protein oxidized is negligible , and that other metabolic processes involving the production or utilization of o2 or co2 are also quantitatively negligible compared to glucose and fatty acid oxidation ( 28 ) . the post - exercise cho and fat consumption were averaged at each five minutes of recovery , starting with 1115 until 8690 minutes . the first 10 minutes of recovery were discarded to avoid influence of the pneumotachometer change , as well as bias due to peak vo2 attained during the exercise sessions . therefore total vo2 , cho and fat consumption during and after exercises were compared using student t - tests for paired samples . the rer measured during exercise was compared by 2-way repeated measures anova ( exercises and sets as main factors ) . differences between rer , cho , and fat consumption at baseline and during recovery were tested by repeated measures anova . in all cases , fisher post hoc tests were applied in the event of significant f ratios . a probability level of p the same statistical software was used for all calculations ( stata / se 8.2 , college station , tx , usa ) . table 1 . presents subjects characteristics concerning anthropometric data , diet recall , physiological measurements at rest , and loads corresponding to 15rm . the vo2 during exercise was higher in lp than in cf ( 6.541.26 l vs. 3.310.71 lp=0.006 ) . the epoc was also higher after lp than after cf until four minutes of recovery ( p<0.001 ) , producing greater epoc net in lp than in cf ( 7.361.10 l vs. 4.730.99 l , respectively ; p<0.001 l at 40 min ) . on the other hand , the type of exercise did not affect the epoc duration . the vo2 remained significantly higher compared to pre - exercise until 40 minutes of recovery after cf and lp ( p<0.0001 ) . in the two exercises , approximately 45% of epoc net was recorded until five minutes of recovery , whereas another 55% corresponded to measurements taken within 1090 minutes . the total vo2 net in lp was significantly higher than cf ( lp=17.573.63 l vs. cf=9.962.86 l at 40 min recovery ; p=0.003 ) . however , rer was in general higher in lp than in cf ( 1 set , p=0.005 ; 3 set , p=0.0003 ; 4 set , p=0.0003 ) . depicts rer during post - exercise recovery . in the first minutes of recovery , the rer was higher after lp than cf ( first four minutes , p=0.019 to p=0.009 ) , but data indicate that lipid oxidation occurred after both exercise protocols . three marked phases were identified during epoc : 1 ) in the first four minutes , the rer remained significantly higher compared to baseline ( p=0.02 to p<0.0001 ) ; 2 ) the rer decreased steadily within 1040 minutes and no difference was detected against pre - exercise ( p>0.05 ) ; 3 ) after 40 ( cf ) or 50 ( lp ) minutes until the end of recovery , the rer was significantly lower than pre - exercise ( rer=0.780.04 ) and values were compatible with lipid oxidation ( at 90 min : rer - cf=0.680.02 , p=0.03 ; rer - lp= 0.650.04 ; p=0.001 ) . macronutrient analyses during exercise from baseline to the last interval between sets ( s1 to s4 ) are presented in table 2 . as expected , the cho utilization increased from the first to fourth intervals in both exercises ( p<0.01 ) , with higher values being found for lp ( p<0.01 ) . the cho consumption reached zero at 35 minutes and 45 minutes following lp and cf , respectively . the fat consumption predominated until the last interval with slightly higher values being detected in lp than in cf ( mean total fat oxidation = 10.9 g vs. 8.4 g , after lp and cf , respectively ; p<0.01 ) . in both exercises , cho utilization in the first interval ( e.g. , 1115 minutes of recovery ) was lower and fat oxidation was higher compared to all intervals after 20 minutes of recovery ( p<0.001 ) . this study investigated the effects of resistance exercises performed with different muscle mass upon ee and substrate utilization , as reflected by rer . the main results were : a ) total vo2 during epoc ( and therefore overall ee ) was significantly higher in exercise performed with larger than smaller muscle mass ; b ) based on rer values , lipid oxidation occurred during epoc after both exercise protocols . however , macronutrient analyses suggested that fat oxidation would be slightly more pronounced after lp than cf . many factors may influence the comparison of respiratory responses during resistance exercises , such as subjects physical activity level or dietary behavior ( 19 ) . this study adopted a within - group design and subjects were homogeneous in regards to physical activity , body surface area , and caloric intake , minimizing the influence of these variables on the metabolic rate . the estimated daily caloric intake and proportion of macronutrients in relation to tev were consistent with current available recommendations ( 23 ) . moreover , the standardized breakfast provided in the study prior to data assessment was quite similar to the usual breakfast related in the diet recall . the utilization of indirect calorimetry during exercises with increased co2 production to assess the substrate balance has been questioned ( 17 ) . the difficulty in reaching an acid - base balance during resistance exercise could compromise an accurate determination of substrate utilization . moreover , when recovering from intense activities , the rer may be influenced by the replenishment of bicarbonate reserves , which requires the incorporation of co2 in their molecular structure ( 21 ) . this could result in lower vco2 and decreased rer , leading to the false idea that fat oxidation is increasing . however , this limitation mostly applies to recovery from exhaustive activities , which was not the case in the present protocols . moreover , comparisons between indirect calorimetry and stricter techniques have shown that absolute substrate oxidation could be well determined , regardless of the stable bicarbonate pool ( 31 ) . actually , many previous studies assessing the rer during and after resistance exercises have used similar techniques and approaches adopted in the present experiment ( 15 , 18 , 21 , 29 ) . the choice of lp and cf must be justified . first of all , resistance exercise sessions are mostly composed of exercises for lower and upper limbs . the cf was chosen instead of more common exercises , as the bench press for two reasons : a ) the agonistic action of the triceps would increase the amount of recruited muscle mass compared to cf , which could compromise the comparison between exercises . adopting a monoarticular exercise instead of a multiarticular exercise as the bench press seemed more appropriate to assure a substantial difference in the exercised muscle mass . secondly , in the context of weight management , weight training is hardly performed with maximum repetitions . instead , this kind of program is usually designed with submaximal load and repetitions , since subjects typically lack experience with resistance training ( 5 ) . a greater number of successive sets increased the ee to levels that made easier the detection of differences between exercises . our results concur with previous studies suggesting that acute resistance exercise may increase the epoc ( 7 , 8 , 11 , 28 ) . however , we failed to demonstrate differences in epoc duration related to exercised muscle mass . after both lp and cf , the vo2 returned to resting levels after approximately 40 minutes of recovery . on the other hand , the overall ee seemed to be influenced by the type of exercise , since total vo2 net in lp was almost twice the value obtained for cf . it is well accepted that exercise volume is a major determinant of ee during this type of exercise ( 7 ) . collectively , available studies have shown that total vo2 would increase in protocols with greater vs. lower exercise volume ( intensity x number of repetitions x number of sets ) ( 8 , 19 , 33 ) . it has been suggested that ee during resistance exercise would be proportional to the recruited muscle mass ( 8 , 15 , 30 ) . our findings ratify the premise that the amount of exercise muscle mass influence the ee elicited by resistance exercises , since the vo2 net in lp was significantly higher than cf . whether resistance exercise sessions are able to induce fat oxidation during recovery is an interesting issue , but its assessment is problematic . although rer might be used for evaluating potential lipid oxidation , its assessment should be performed concomitantly to substrate utilization analysis ( 10 ) to avoid misinterpretation of metabolic responses . the rer in resistance exercises has not been extensively investigated and little is known about energy substrate utilization in recovery ( 11 , 15 , 18 , 25 ) . it is acknowledged that during resistance exercise there would be a substantial contribution of high - energy phosphate and glucose breakdown to supply energy ( 27 ) , but during post - exercise , data are mixed and controversial . some studies have shown that lipids would be the main energy substrate during post - exercise recovery ( 11 , 18 , 22 , 25 ) , while others suggested that carbohydrate oxidation would be predominant ( 21 , 29 ) or that differences of substrate utilization would not exist ( 33 ) . it appears that the anaerobic nature of each set increases the co2 levels , resulting in high rer values . on the other hand , high levels of co2 may stimulate a central mediated increase in ve and subsequent reduction in rer during the rest intervals . lengthening the rest intervals increases the proportion of time the subject is not exercising and consequently the recovery from anaerobic stress , lowering the rer throughout the sets in comparison with shorter intervals . it was not surprising that multiple sets with 1-min intervals , as applied in our protocol , provoked a substantial increase in rer that could not be offset by this relatively short recovery period . ( 29 ) compared the rer during and after five sets of bench press performed with five or ten repetitions at 85% or 75% 1rm , respectively . the rer values during the exercise bouts reported in that study ( within 1.051.36 ) were compatible with our findings . the condition that approached the characteristics of our protocol ( 5 sets of 10 reps with 12 min intervals ) elicited the highest rer values ( between 1.211.36 ) . study ( 29 ) was detected up to 1015 minutes after the end of the exercise , but no lipid oxidation was observed until 30 minutes of recovery . the present study extended the period of observation and was able to detect lipid oxidation after 40 minutes of recovery , with fat utilization predominance until 90 minutes after both exercise protocols , but greater in lp than in cp . another issue that is not clear in the literature is the relative influence of training intensity and volume upon rer and substrate utilization . for instance , thornton and potteiger ( 33 ) failed to observe lipid oxidation during 120 minutes recovery after exercises performed with similar volume but different intensities ( 85% or 45% 1rm ) in young men . on the other hand , haddock and wilkin ( 11 ) and melby et al . ( 22 ) suggested that lipid oxidation would occur for several hours after resistance exercises performed with similar intensities , regardless of training volume . our findings are in line with the idea that lipid oxidation may indeed occur after multiple sets of resistance exercise and that exercise volume in the present case related to the exercised muscle mass would be a determinant of such response . an increase in fat utilization occurred after approximately 30 minutes of recovery , irrespective of the type of exercise , which has implications to exercise programs designed for health promotion . a limitation of this study relates to the potential application of our results to actual exercise prescription . the protocols involved just one exercise performed with moderate intensity , which is not the current practice in resistance training . the subjects participating in the study had normal weight and were young and experienced in resistance training , which limits the application of our findings to middle aged populations , which are frequently targets of adult weight management programs . future research is needed to ratify the possible effects of resistance exercise performed with different intensities and volumes upon fat oxidation in overweight populations . in brief , increases in post - exercise ee and fat oxidation were greater after resistance exercise performed with larger muscle mass . in a practical perspective , this means that lipid oxidation will occur during post - exercise recovery irrespective of the exercises included in the routine . however , larger muscle groups should be exercised if the purpose is to increase ee and perhaps optimize fat oxidation following resistance exercise sessions . the epoc duration was similar after protocols including multiple sets of lp and cf , but total vo2 and rer were influenced by the exercised muscle mass , being higher in lp ( larger muscle mass ) than in cf ( smaller muscle mass ) . the rer after both protocols indicated the occurrence of lipid oxidation during post - exercise recovery . additional macronutrient analyses showed that lipid oxidation after lp was greater over cf , suggesting that substrate utilization may be influenced by the amount of exercised muscle mass .

this study investigated the energy expenditure ( ee ) and substrate utilization reflected by the respiratory - exchange ratio ( rer ) during and after resistance exercises performed with different muscle mass . ten male volunteers ( meansd ; 264yr , 1796 cm , 778 kg ) performed multiple sets of the horizontal leg press ( lp ) and chest fly ( cf ) ( 5 sets of 10 repetitions with 15 repetition - maximum , 1-minute between - set intervals ) in a counterbalanced design . oxygen uptake and carbon dioxide production were measured during 40 minutes of resting ; resistance exercise protocols ( sets and intervals ) ; 90 minutes of post - exercise recovery . total fat and carbohydrate oxidation rates were calculated according to the non - protein respiratory quotient . both exercise conditions elicited net excess post - exercise oxygen consumption ( epoc ) of similar duration ( approximately 40min ) . the epoc magnitude at 40 minutes was greater after lp than after cf ( 7.361.10l vs. 4.730.99l ; p<0.001 ) . the rer was higher in lp ( 1.300.04 ) than cf ( 1.160.05 , p=0.0003 ) during exercise . during recovery the rer was similar in lp and cf ( p>0.05 ) and lower than pre - exercise ( pre - exercise=0.780.04 vs. cf40min=0.740.04 ; cf90min=0.680.02 and lp50min=0.730.06 ; lp90min=0.650.04 , p<0.05 ) . however , fat oxidation after lp was greater than cf between 3090 minutes of recovery ( mean total fat oxidation : lp=10.9 g vs. cf=8.4 g ; p<0.01 ) . the increases of ee and fat oxidation during post - exercise recovery were greater after multiple sets of resistance exercises performed with larger muscle mass than smaller muscle mass . this finding has practical implications for resistance training designed as part of weight management programs .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

resistance exercise has been shown to intensify the resting energy expenditure ( ee ) ( 25 , 26 ) and increase post - exercise lipid oxidation ( 20 ) , which would be desirable for weight loss ( 15 , 18 ) . the respiratory exchange ratio ( rer ) is a surrogate of substrate utilization , and can be used to identify the rate of lipid oxidation ( 2 , 11 , 15 , 22 , 25 ) . however , its response to resistance exercise sessions has not been extensively investigated , and data from a few available studies are varied ( 2 , 20 , 21 , 29 , 33 ) . prior researchers investigating the influence of resistance training variables upon ee , lipid oxidation , or rer have focused on the intensity ( 33 ) , volume ( 11 , 12 , 22 ) , rest intervals ( 29 ) , type of exercise ( 15 ) , or session design ( 25 ) . some studies tested the influence of complete sessions of resistance exercises ( 2 , 15 , 21 , 25 ) or compared resistance and aerobic exercise ( 3 , 9 , 20 ) . a previous study by our group ( 8) demonstrated that ee estimated from oxygen uptake net ( vo2net ) during and after exercise was higher after exercises performed with larger than smaller muscle mass . on the other hand , differences of substrate utilization between exercises with different muscle mass have not been yet addressed . this information would be valuable , since it is acknowledged that the amount of muscle mass influences ee and substrate utilization during and after exercise sessions ( 7 ) . the assessment of rer during and after resistance exercise sessions would be useful to address this issue , as demonstrated by several previous studies ( 1 , 2 , 12 , 21 ) . carbohydrates are used for glycogen resynthesis following exercise , which helps to explain the increased lipid oxidation during the excess post - exercise consumption ( epoc ) ( 13 ) . thus , it is feasible to think that the amount of muscle mass used for exercises may determine the substrate utilization during post - exercise recovery and that the rer would reflect differences in this regard . this study compared the impact of multiple sets of acute resistance exercises performed with similar intensity but different muscle mass upon ee and substrate utilization , as reflected by rer during and after exercise . the hypothesis was that the exercised muscle mass would influence the total vo2 and rate of oxidation of nutrients estimated by means of the rer , especially lipid oxidation . ; 1796 cm ; 787 kg ) . on the first day researchers determined the resting vo2 , rer , and load corresponding to 15 repetition - maximum ( 15rm ) for the horizontal leg press ( lp ) and the chest fly ( cf ) . subjects performed two exercise sessions in a counterbalanced design on the third and fourth days . the exercise protocols began five minutes after the warm - up ( 5 sets of 10 repetitions with 15rm workloads and 1-min interval between sets ) . all subjects performed lp ( larger muscle mass ) and cf ( smaller muscle mass ) protocols on different days . prior to the 15rm tests , a warm - up of 12 repetitions with 30% of pre - determined 15rm was performed . similar procedures were adopted for determining the load corresponding to 15 rm in lp and cf , being repeated after 4872 hours to assess the load reliability . test - retest intraclass correlation ( icc ) and coefficient of variation were considered satisfactory ( lp : icc=0.96 , p<0.001 and cv=4.2% ; cf : icc=0.97 , p<0.001 and cv=4.8% ) . in order to avoid bias due to the thermic effect of food , the proportion of macronutrients in total energetic value ( tev ) recommended by the world health organization ( 24 ) and daily ee of approximately 2400 kcal / day were adopted as references ( 15 , 23 ) . all tests were performed in the morning , and 2 hours prior to the experiment , a standard food intake was provided ( 16 ) : a glass of fruit juice ( 200 ml ) and 6 salt crackers . energy values of fruit juice and crackers were 88 kcal ( 22 g of carbohydrate ) and 240 kcal ( 38 g of carbohydrate , 8 g of fat , and 4 g of protein ) , respectively . if rer is lower than 0.7 or higher than 1.0 , prolonged starvation or excessive recent energy consumption should be suspected , both events representing protocol violation of resting metabolic rate ( 4 ) . intraclass correlations and coefficients of variation were calculated ( icc=0.89 , p=0.023 and cv=5.2% ) . before the exercise protocols , subjects remained at rest in a quiet environment for 15 minutes or until rer matched the previously assessed resting values ( variation could not exceed 5% ) . upon completion of the exercise sequences , the same procedures adopted for rer assessment at rest were applied to measurements during post - exercise recovery . in brief , post - exercise breath - by - breath vo2 data were averaged immediately following the last set of a given exercise , and at 1 , 2 , 3 , 4 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , and 90 minutes of recovery . the absolute vo2 ( l / min ) and rer during each set , between set intervals , and post - exercise recovery were used to calculate the total vo2 ( defined as the sum of vo2 obtained during the exercises , recovery intervals , and 90-minute epoc ) . considering a rer > 0.85 as an index of fat to carbohydrate oxidation , total fat and carbohydrate ( cho ) oxidation rates were calculated according to the non - protein respiratory quotient ( 28 ) : cho oxidation rate = 4.585 vco2 3.226 vo2 ; fat oxidation rate = 1.695 vo2 1.701 vco2 , with vo2 and vco2 expressed in liters per minute ( l / min ) and oxidation rates in grams per minute ( g / min ) . the post - exercise cho and fat consumption were averaged at each five minutes of recovery , starting with 1115 until 8690 minutes . the first 10 minutes of recovery were discarded to avoid influence of the pneumotachometer change , as well as bias due to peak vo2 attained during the exercise sessions . therefore total vo2 , cho and fat consumption during and after exercises were compared using student t - tests for paired samples . the rer measured during exercise was compared by 2-way repeated measures anova ( exercises and sets as main factors ) . differences between rer , cho , and fat consumption at baseline and during recovery were tested by repeated measures anova . on the first day researchers determined the resting vo2 , rer , and load corresponding to 15 repetition - maximum ( 15rm ) for the horizontal leg press ( lp ) and the chest fly ( cf ) . subjects performed two exercise sessions in a counterbalanced design on the third and fourth days . the exercise protocols began five minutes after the warm - up ( 5 sets of 10 repetitions with 15rm workloads and 1-min interval between sets ) . all subjects performed lp ( larger muscle mass ) and cf ( smaller muscle mass ) protocols on different days . prior to the 15rm tests , a warm - up of 12 repetitions with 30% of pre - determined 15rm was performed . similar procedures were adopted for determining the load corresponding to 15 rm in lp and cf , being repeated after 4872 hours to assess the load reliability . test - retest intraclass correlation ( icc ) and coefficient of variation were considered satisfactory ( lp : icc=0.96 , p<0.001 and cv=4.2% ; cf : icc=0.97 , p<0.001 and cv=4.8% ) . in order to avoid bias due to the thermic effect of food , the proportion of macronutrients in total energetic value ( tev ) recommended by the world health organization ( 24 ) and daily ee of approximately 2400 kcal / day were adopted as references ( 15 , 23 ) . all tests were performed in the morning , and 2 hours prior to the experiment , a standard food intake was provided ( 16 ) : a glass of fruit juice ( 200 ml ) and 6 salt crackers . energy values of fruit juice and crackers were 88 kcal ( 22 g of carbohydrate ) and 240 kcal ( 38 g of carbohydrate , 8 g of fat , and 4 g of protein ) , respectively . if rer is lower than 0.7 or higher than 1.0 , prolonged starvation or excessive recent energy consumption should be suspected , both events representing protocol violation of resting metabolic rate ( 4 ) . before the exercise protocols , subjects remained at rest in a quiet environment for 15 minutes or until rer matched the previously assessed resting values ( variation could not exceed 5% ) . upon completion of the exercise sequences , the same procedures adopted for rer assessment at rest were applied to measurements during post - exercise recovery . the change was made within five to ten minutes of recovery and approximately three minutes were required to reprogram the software , recalibrate the system , and resume assessment . in brief , post - exercise breath - by - breath vo2 data were averaged immediately following the last set of a given exercise , and at 1 , 2 , 3 , 4 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , and 90 minutes of recovery . the total vo2 ( l ) for a given exercise the absolute vo2 ( l / min ) and rer during each set , between set intervals , and post - exercise recovery were used to calculate the total vo2 ( defined as the sum of vo2 obtained during the exercises , recovery intervals , and 90-minute epoc ) . considering a rer > 0.85 as an index of fat to carbohydrate oxidation , total fat and carbohydrate ( cho ) oxidation rates were calculated according to the non - protein respiratory quotient ( 28 ) : cho oxidation rate = 4.585 vco2 3.226 vo2 ; fat oxidation rate = 1.695 vo2 1.701 vco2 , with vo2 and vco2 expressed in liters per minute ( l / min ) and oxidation rates in grams per minute ( g / min ) . the non - protein respiratory quotient approach has been extensively applied . the post - exercise cho and fat consumption were averaged at each five minutes of recovery , starting with 1115 until 8690 minutes . the first 10 minutes of recovery were discarded to avoid influence of the pneumotachometer change , as well as bias due to peak vo2 attained during the exercise sessions . therefore total vo2 , cho and fat consumption during and after exercises were compared using student t - tests for paired samples . the rer measured during exercise was compared by 2-way repeated measures anova ( exercises and sets as main factors ) . differences between rer , cho , and fat consumption at baseline and during recovery were tested by repeated measures anova . the vo2 during exercise was higher in lp than in cf ( 6.541.26 l vs. 3.310.71 lp=0.006 ) . the epoc was also higher after lp than after cf until four minutes of recovery ( p<0.001 ) , producing greater epoc net in lp than in cf ( 7.361.10 l vs. 4.730.99 l , respectively ; p<0.001 l at 40 min ) . the vo2 remained significantly higher compared to pre - exercise until 40 minutes of recovery after cf and lp ( p<0.0001 ) . in the two exercises , approximately 45% of epoc net was recorded until five minutes of recovery , whereas another 55% corresponded to measurements taken within 1090 minutes . the total vo2 net in lp was significantly higher than cf ( lp=17.573.63 l vs. cf=9.962.86 l at 40 min recovery ; p=0.003 ) . however , rer was in general higher in lp than in cf ( 1 set , p=0.005 ; 3 set , p=0.0003 ; 4 set , p=0.0003 ) . depicts rer during post - exercise recovery . in the first minutes of recovery , the rer was higher after lp than cf ( first four minutes , p=0.019 to p=0.009 ) , but data indicate that lipid oxidation occurred after both exercise protocols . three marked phases were identified during epoc : 1 ) in the first four minutes , the rer remained significantly higher compared to baseline ( p=0.02 to p<0.0001 ) ; 2 ) the rer decreased steadily within 1040 minutes and no difference was detected against pre - exercise ( p>0.05 ) ; 3 ) after 40 ( cf ) or 50 ( lp ) minutes until the end of recovery , the rer was significantly lower than pre - exercise ( rer=0.780.04 ) and values were compatible with lipid oxidation ( at 90 min : rer - cf=0.680.02 , p=0.03 ; rer - lp= 0.650.04 ; p=0.001 ) . as expected , the cho utilization increased from the first to fourth intervals in both exercises ( p<0.01 ) , with higher values being found for lp ( p<0.01 ) . the cho consumption reached zero at 35 minutes and 45 minutes following lp and cf , respectively . the fat consumption predominated until the last interval with slightly higher values being detected in lp than in cf ( mean total fat oxidation = 10.9 g vs. 8.4 g , after lp and cf , respectively ; p<0.01 ) . , 1115 minutes of recovery ) was lower and fat oxidation was higher compared to all intervals after 20 minutes of recovery ( p<0.001 ) . this study investigated the effects of resistance exercises performed with different muscle mass upon ee and substrate utilization , as reflected by rer . the main results were : a ) total vo2 during epoc ( and therefore overall ee ) was significantly higher in exercise performed with larger than smaller muscle mass ; b ) based on rer values , lipid oxidation occurred during epoc after both exercise protocols . however , macronutrient analyses suggested that fat oxidation would be slightly more pronounced after lp than cf . moreover , when recovering from intense activities , the rer may be influenced by the replenishment of bicarbonate reserves , which requires the incorporation of co2 in their molecular structure ( 21 ) . this could result in lower vco2 and decreased rer , leading to the false idea that fat oxidation is increasing . actually , many previous studies assessing the rer during and after resistance exercises have used similar techniques and approaches adopted in the present experiment ( 15 , 18 , 21 , 29 ) . the cf was chosen instead of more common exercises , as the bench press for two reasons : a ) the agonistic action of the triceps would increase the amount of recruited muscle mass compared to cf , which could compromise the comparison between exercises . secondly , in the context of weight management , weight training is hardly performed with maximum repetitions . instead , this kind of program is usually designed with submaximal load and repetitions , since subjects typically lack experience with resistance training ( 5 ) . our results concur with previous studies suggesting that acute resistance exercise may increase the epoc ( 7 , 8 , 11 , 28 ) . however , we failed to demonstrate differences in epoc duration related to exercised muscle mass . after both lp and cf , the vo2 returned to resting levels after approximately 40 minutes of recovery . on the other hand , the overall ee seemed to be influenced by the type of exercise , since total vo2 net in lp was almost twice the value obtained for cf . it is well accepted that exercise volume is a major determinant of ee during this type of exercise ( 7 ) . it has been suggested that ee during resistance exercise would be proportional to the recruited muscle mass ( 8 , 15 , 30 ) . our findings ratify the premise that the amount of exercise muscle mass influence the ee elicited by resistance exercises , since the vo2 net in lp was significantly higher than cf . whether resistance exercise sessions are able to induce fat oxidation during recovery is an interesting issue , but its assessment is problematic . the rer in resistance exercises has not been extensively investigated and little is known about energy substrate utilization in recovery ( 11 , 15 , 18 , 25 ) . it is acknowledged that during resistance exercise there would be a substantial contribution of high - energy phosphate and glucose breakdown to supply energy ( 27 ) , but during post - exercise , data are mixed and controversial . some studies have shown that lipids would be the main energy substrate during post - exercise recovery ( 11 , 18 , 22 , 25 ) , while others suggested that carbohydrate oxidation would be predominant ( 21 , 29 ) or that differences of substrate utilization would not exist ( 33 ) . ( 29 ) compared the rer during and after five sets of bench press performed with five or ten repetitions at 85% or 75% 1rm , respectively . the condition that approached the characteristics of our protocol ( 5 sets of 10 reps with 12 min intervals ) elicited the highest rer values ( between 1.211.36 ) . study ( 29 ) was detected up to 1015 minutes after the end of the exercise , but no lipid oxidation was observed until 30 minutes of recovery . the present study extended the period of observation and was able to detect lipid oxidation after 40 minutes of recovery , with fat utilization predominance until 90 minutes after both exercise protocols , but greater in lp than in cp . another issue that is not clear in the literature is the relative influence of training intensity and volume upon rer and substrate utilization . for instance , thornton and potteiger ( 33 ) failed to observe lipid oxidation during 120 minutes recovery after exercises performed with similar volume but different intensities ( 85% or 45% 1rm ) in young men . ( 22 ) suggested that lipid oxidation would occur for several hours after resistance exercises performed with similar intensities , regardless of training volume . our findings are in line with the idea that lipid oxidation may indeed occur after multiple sets of resistance exercise and that exercise volume in the present case related to the exercised muscle mass would be a determinant of such response . an increase in fat utilization occurred after approximately 30 minutes of recovery , irrespective of the type of exercise , which has implications to exercise programs designed for health promotion . the protocols involved just one exercise performed with moderate intensity , which is not the current practice in resistance training . the subjects participating in the study had normal weight and were young and experienced in resistance training , which limits the application of our findings to middle aged populations , which are frequently targets of adult weight management programs . future research is needed to ratify the possible effects of resistance exercise performed with different intensities and volumes upon fat oxidation in overweight populations . in brief , increases in post - exercise ee and fat oxidation were greater after resistance exercise performed with larger muscle mass . in a practical perspective , this means that lipid oxidation will occur during post - exercise recovery irrespective of the exercises included in the routine . however , larger muscle groups should be exercised if the purpose is to increase ee and perhaps optimize fat oxidation following resistance exercise sessions . the epoc duration was similar after protocols including multiple sets of lp and cf , but total vo2 and rer were influenced by the exercised muscle mass , being higher in lp ( larger muscle mass ) than in cf ( smaller muscle mass ) . the rer after both protocols indicated the occurrence of lipid oxidation during post - exercise recovery . additional macronutrient analyses showed that lipid oxidation after lp was greater over cf , suggesting that substrate utilization may be influenced by the amount of exercised muscle mass .

historically , wellbeing research has largely focused on adults in developed countries ( 1 ) . there has been little research on the self - reported ( i.e. , subjective ) wellbeing of children and or adolescents , particularly in developing countries ( 2 ) . similarly , there has been minuscule research , which focuses comparisons of subjective wellbeing among different adolescent groups ( e.g. , gender , age , ethnicity , parental education , economic status and physical activity etc . ) there are an estimated 1.2 billion young people aged 1019 in the world , comprising the largest generation of adolescents in history ( 3 ) . approximately 70 percent of these young people live in developing countries where complex economic , social , political and environmental contexts create a wide range of challenges for adolescents to surmount as they journey to adulthood . many of these disadvantaged adolescents have few personal resources and little social support to confront the conditions that propagate poverty , inequity , and gender discrimination . data indicate that more than half of all youth live in poverty worldwide ( 4 ) . who estimates that approximately one in five young people under the age of 18 experiences some form of developmental , emotional or behavioral problem , and one in eight experiences a mental disorder ( 5 ) , international studies show that youth physical activity has decreased over time ( 68 ) , it is responsible for 6% of deaths globally around 3.2 million deaths per year , including 2.6 million in low- and middle income countries ( 9 ) . consequently children and adolescents belonging to different socioeconomic , demographic groups are thought to be more vulnerable for disparities that will impact their health and well - being ( 10 ) . also mental illness has been estimated by 2020 to become a 15% of the global burden of disease ( 11 ) . some of the leading mental health problems are depression , anxiety and eating disorders especially among the young individuals ( 12 , 13 ) . from a broad perspective , the measurement and promotion of adolescent well - being is a desirable social and political objective ( 14 , 15 ) . psychological wellbeing of adolescents means being content with life and understanding an abundance of positive emotions , when joined with the absence of psychopathology , is linked with greatest academic function , social skills and support and physical health , being a stage that lays strong foundation for future personality , and a critical period during human development in which life goals , values , direction and purpose in life are created ( 16 , 17 ) , guaranteeing psychological wellbeing of adolescents is a socio psychological necessity . a growing number of longitudinal studies confirm the power of well - being scales to predict outcomes , for example , longevity , physical health , quality of life , criminality , drug and alcohol use , employment , earnings and pro - social behavior ( e.g. volunteering ) ( who , 2009 ) . moreover , given the ever evading nature of complexities typical to their phase of development , researches into factors contributing to adolescent psychological well - being was always intimidating task for scientific community . for any genuine approach for ensuring psychological well - being of a group , exploration into demographic correlates and predictors of psychological well - being by tracing the environmental , physiological or neurological underpinnings is not sufficient ( 18 ) . psychological distress is something strongly correlated with physical morbidity , reduced quality and duration of life and increased use of health service ( 19 ) . at the same time , there is no guarantee that both psychological well - being and psychological distress will not occur together in a personality . according to one study positive psychological factors may have such a strong relationship with health as negative ones and extend to which these psychological states are independent of each other may vary according to the external and internal environmental challenges people face and researches will need to make choices about the value of measuring both ( 18 ) . however , no particular study in pakistan has been noticed to assess the association between general levels of physical activity and ( who - five ) psychological wellbeing in adolescents . lifestyle , including physical activity is considered as one of the major determinants of health in a population ( 19 ) , although strategies to increase physical activity are being developed ( 20 , 21 ) , and regularly physical activity is well documented to increase physical ( 22 , 23 ) , as well as mental health ( 24 , 25 ) . the concept of adolescence as a distinct period of human development is still fairly new in pakistan . the pakistan population association reports that 65% of pakistani households contain one or more adolescents ( 26 ) . among adolescents , physical activity is associated with benefits in the prevention and control of emotional distress , and improvement of self - esteem ( 27 ) . the purpose of this research is to provide a broad picture of the psychological well - being of school adolescents and its relationship with general levels of physical activity and socio - demographic factors . the study analyses have evaluated the degree of co - existence of demographic factors , and to determine any gender , age , grade , physical activity specific differences in psychological wellbeing among school adolescents in gilgit , pakistan . a population - based cross - sectional quantitative correlational ( descriptive and analytic ) research design employed in this study to examine to understand if there is significant relationship between the psychological well - being , physical activity and socio - demographic factors carried out using a self - administered questionnaire . the study was approved by the research ethical committee of tehran university of medical sciences in 2013 . gilgit is the one of district and capital city of gilgit - baltistan province of pakistan comprising of mixture of all major ethnic groups ( sheena , burushaski , khuwar and wakhee ) . the subjects of sample size of three hundred and forty five [ n=345 ] was drawn for school adolescent ( aged 1218 ) , among them , one hundred and ninety one ( n = 191 ) were girls and one hundred and fifty four ( n = 154 ) boys , mean age was 14.64 year . the probability simple random sampling method was applied to select the five schools from the list of 47 schools . the participation adolescents was voluntary from the class 6th to 10th and participant were from a five computerized randomly selected school were given chance to participate in survey from the list of 47 schools . the response rate was 99% except those students who were absent ; completed the questionnaire in their classrooms at school timings in the presence of class teacher and guidance of researcher . a tool , compromising of 2 frequently used and reliable questionnaires , was redesigned by the conductor of this study . a pilot study was conducted in a school with in same city and the questionnaires were given to 25 adolescents that were similar to the study participants in terms of all characteristics . data used in the thesis is based on self - reported data from the questionnaire and consisted of three sections ( a ) socio - demographic part along with ( b ) well - being ( who five index ) and ( c ) physical activity questionnaire for adolescents ( paq - a ) . the ( who - five index ) captures emotional , psychological well - being and was developed from the world health organization - ten well - being index ( 28 ) . it was conceptualized as a one - dimensional measure that contains five positively worded items : i have felt cheerful and in good spirits ; i have felt calm and relaxed ; i have felt active and vigorous ; i woke up feeling fresh and rested ; and my daily life has been filled with things that interest me . the degree to which the aforementioned positive feelings were present in the last 2 weeks is scored on a 6-point likert scale ranging from 0 ( not present ) to 5 ( constantly present ) . the raw scores are transformed to a score from 0 ( worst thinkable well - being ) to 100 ( best thinkable well - being ) . a score < 50 suggests poor emotional well - being and is a sign for further testing . a score 28 is indicative of depression ( 29 ) . in the present study 28 was for likely depression , 50 was for low mood or low level of emotional well - being in categorical is , it comes between 50 and 75 percentile was show moderate / medium and > 75th was show high level of well - being . the paq - a was used to measure general levels of physical activity in adolescents . the paq - a was self - administer it provides last seven days summary of physical activity score derived from eight items , each scored on a 5-point scale ( 30 ) . a score of 1 indicates low physical activity , whereas a score of 5 indicates high physical activity . a population - based cross - sectional quantitative correlational ( descriptive and analytic ) research design employed in this study to examine to understand if there is significant relationship between the psychological well - being , physical activity and socio - demographic factors carried out using a self - administered questionnaire . the study was approved by the research ethical committee of tehran university of medical sciences in 2013 . gilgit is the one of district and capital city of gilgit - baltistan province of pakistan comprising of mixture of all major ethnic groups ( sheena , burushaski , khuwar and wakhee ) . the subjects of sample size of three hundred and forty five [ n=345 ] was drawn for school adolescent ( aged 1218 ) , among them , one hundred and ninety one ( n = 191 ) were girls and one hundred and fifty four ( n = 154 ) boys , mean age was 14.64 year . the probability simple random sampling method was applied to select the five schools from the list of 47 schools . the participation adolescents was voluntary from the class 6th to 10th and participant were from a five computerized randomly selected school were given chance to participate in survey from the list of 47 schools . the response rate was 99% except those students who were absent ; completed the questionnaire in their classrooms at school timings in the presence of class teacher and guidance of researcher . a tool , compromising of 2 frequently used and reliable questionnaires , was redesigned by the conductor of this study . a pilot study was conducted in a school with in same city and the questionnaires were given to 25 adolescents that were similar to the study participants in terms of all characteristics . data used in the thesis is based on self - reported data from the questionnaire and consisted of three sections ( a ) socio - demographic part along with ( b ) well - being ( who five index ) and ( c ) physical activity questionnaire for adolescents ( paq - a ) . the ( who - five index ) captures emotional , psychological well - being and was developed from the world health organization - ten well - being index ( 28 ) . it was conceptualized as a one - dimensional measure that contains five positively worded items : i have felt cheerful and in good spirits ; i have felt calm and relaxed ; i have felt active and vigorous ; i woke up feeling fresh and rested ; and my daily life has been filled with things that interest me . the degree to which the aforementioned positive feelings were present in the last 2 weeks is scored on a 6-point likert scale ranging from 0 ( not present ) to 5 ( constantly present ) . the raw scores are transformed to a score from 0 ( worst thinkable well - being ) to 100 ( best thinkable well - being ) . a score < 50 suggests poor emotional well - being and is a sign for further testing . a score 28 is indicative of depression ( 29 ) . in the present study 28 was for likely depression , 50 was for low mood or low level of emotional well - being in categorical is , it comes between 50 and 75 percentile was show moderate / medium and > 75th was show high level of well - being . the paq - a was used to measure general levels of physical activity in adolescents . the paq - a was self - administer it provides last seven days summary of physical activity score derived from eight items , each scored on a 5-point scale ( 30 ) . a score of 1 indicates low physical activity , whereas a score of 5 indicates high physical activity . the data were analyzed using statistical package for social sciences ( spss ) 21 for windows ( spss , inc . , , all forms were checked for completeness and consistency as well as coding of open ended responses . descriptive statistics ( including means and standard deviations , ) were calculated for all scales and subscales . associations between well - being and socio - demographic variables were investigated in regression models and linear regression , with well - being as the dependent variable and socio - demographic variables as independents . the bivariate analysis mann - whitney is nonparametric test for two groups , kruskal - wallis test was used to compare three or more samples and spearman s rank correlation coefficients ( rho ) were calculated for correlations of pwb ( score - a ) and pa ( paq - score ) . while multi and bivariate regression model was used to estimate correlation with other predictors . multivariate analysis were used after adjusted ( r2= 0.15 ) to control the effect of the other confounders and multiple comparisons were performed to compare participants pwb with different classes using bonferroni method , while this opportunity is not possible in bivariate analysis . the data were analyzed using statistical package for social sciences ( spss ) 21 for windows ( spss , inc . , , all forms were checked for completeness and consistency as well as coding of open ended responses . descriptive statistics ( including means and standard deviations , ) were calculated for all scales and subscales . associations between well - being and socio - demographic variables were investigated in regression models and linear regression , with well - being as the dependent variable and socio - demographic variables as independents . the bivariate analysis mann - whitney is nonparametric test for two groups , kruskal - wallis test was used to compare three or more samples and spearman s rank correlation coefficients ( rho ) were calculated for correlations of pwb ( score - a ) and pa ( paq - score ) . while multi and bivariate regression model was used to estimate correlation with other predictors . multivariate analysis were used after adjusted ( r2= 0.15 ) to control the effect of the other confounders and multiple comparisons were performed to compare participants pwb with different classes using bonferroni method , while this opportunity is not possible in bivariate analysis . a total of 345 adolescents were participated , the mean age of adolescents was 14.64 ( sd=1.275 ) years ; 55.4% were female and without gender differences that the majority ( 43.4% ) of adolescents were shown moderate level of pwb , ( 33.3% ) high while ( 23.2% ) participants were revealed low level of pwb and were likely to have depression ( table 1 ) . descriptive statistics for participants pa score and standardized scores of ( who-5 ) pwb this study estimated the mean score of pwb ( m=69.96 ) for adolescents , was observed moderate with standard deviation ( sd=14.914 ) but the maximum score was 100 complete high and minimum pwb was 28.00 found in some of the adolescents , it indicates depression . while the mean general level of pa was found ( m=2.087 ) with standard deviation ( sd=1.041 ) socio - demographics variables examined for group differences on the pwb measures as well as for the general levels of pa ( paq - a ) among voluntarily participated school adolescents . the mean of high pwb ( m=85.1765 ) , among class 6th was higher than other classes with standard deviation ( sd=11.38110 ) and ( ci=95% ) confidence interval , although the mean pwb ( m=66.7755 ) of class 6th was moderate with ( sd= 15.36265 ) . almost for all the demographic variables the mean pwb was remained moderate but shows distribution of sample according to pa score in relation to the gender , is showing that the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m=2.0666 ) with ( sd=1.05998 ) among females in the present investigation . the below table 2 in present study found that pwb score in categories ( 1-low , 2-moderate and 3-high ) a pwb was low in ( n=80 ) participants and the mean pa ( m=2.108 ) with standard deviation ( sd= 1.00758 ) was moderate among subjects with low pwb its shows depression or low mood , while participants ( n=115 ) were have high categorical pwb found with moderate level of mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) . this study finds that pa did nt have any big difference in participants with high and low pwb . descriptive for pwb in categorical and general level of pa as a continuous variable by spearman s , pwb ( score a ) of adolescents and general levels of pa ( paq _ score ) the correlation coefficient = 0.36 was attainable value ( b / w 01 ) but the association was not significant ( p = 0.511 ) figures are mentioned in the table 3 . but according to literature many studies reported the strong relation between individuals pwb and pa however for this study it did nt showed . secondly as presented in table 4 it s also demonstrated the associations between measured covariates and standardized scores of pwb along with their corresponding p - value in the bivariate a total score was significantly correlated with gender and class as stated school adolescent boys aged 12 to 18 ( 14.1% ) were have high mean pwb ( m=71.74 ) than the mean of pwb ( m=68.5 ) among girls ( 15.4% ) . the spearman s rho correlation between paq - score and score - a the bivariate analysis of the association between some measured covariates and standardized scores of ( who - five ) pwb in school adolescents there was positive correlation between two factors pwb and gender it was statistically significant at ( p = 0.046 ) . in the kruskal - wallis test , was found strong indirect important association between pwb and classes of the participant with ( p = 0.0001 ) . but it was nt found any major association among last grade , language with pwb ( p = 0.92 ) . as mentioned in table 5 multivariate analyses with 95% confidence interval t=0.004 ) where t=2.87 , where girls participants wpb were ( coef.=4.600939 units ) less the boys . the pwb was found significant relationship with age of the participants ( p > t=0.000 ) the pwb was decreasing ( coef.=3.366921 unit ) with the increases in age . the association was strongly significant for pwb of school adolescents to their classes * ( p>=0.000 , p > t=0.000 , p>=0.001 ) as class were increasing wpb was decreasing . last grade ( p>=0.015 ) , father education ( p > t = 0.028 ) with t=2.21 and number of family members ( p > t = 0.000 ) were found strong important association with pwb . multivariable analysis for psychological wellbeing ( adjusted r2=0.15 ) with some socio - demographic covariate gender , age , class , last grade , father education and family member some other results to take note of were , first the mean of pa score in boys were 0.05 more than girls , although this difference was not statistically different while the mean of pa score in students with different class was significantly different from each other , the association by kruskal - wallis was statistically significant ( p=0.0034 ) . this study also found fathers education ( p=0.03 ) and mothers education ( p=0.025 ) important for ap . in conclusion our study demonstrated that , school adolescents pwb by with participants general levels of pa was nt correlated but at the same time the result shows pwb was significantly associated with some important socio - demographic covariate , especially a strong association was observed with gender , age , class , parents education , parents with types of jobs and family members . a total of 345 adolescents were participated , the mean age of adolescents was 14.64 ( sd=1.275 ) years ; 55.4% were female and without gender differences that the majority ( 43.4% ) of adolescents were shown moderate level of pwb , ( 33.3% ) high while ( 23.2% ) participants were revealed low level of pwb and were likely to have depression ( table 1 ) . descriptive statistics for participants pa score and standardized scores of ( who-5 ) pwb this study estimated the mean score of pwb ( m=69.96 ) for adolescents , was observed moderate with standard deviation ( sd=14.914 ) but the maximum score was 100 complete high and minimum pwb was 28.00 found in some of the adolescents , it indicates depression . while the mean general level of pa was found ( m=2.087 ) with standard deviation ( sd=1.041 ) socio - demographics variables examined for group differences on the pwb measures as well as for the general levels of pa ( paq - a ) among voluntarily participated school adolescents . the mean of high pwb ( m=85.1765 ) , among class 6th was higher than other classes with standard deviation ( sd=11.38110 ) and ( ci=95% ) confidence interval , although the mean pwb ( m=66.7755 ) of class 6th was moderate with ( sd= 15.36265 ) . almost for all the demographic variables the mean pwb was remained moderate but shows distribution of sample according to pa score in relation to the gender , is showing that the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m=2.0666 ) with ( sd=1.05998 ) among females in the present investigation . the below table 2 in present study found that pwb score in categories ( 1-low , 2-moderate and 3-high ) a pwb was low in ( n=80 ) participants and the mean pa ( m=2.108 ) with standard deviation ( sd= 1.00758 ) was moderate among subjects with low pwb its shows depression or low mood , while participants ( n=115 ) were have high categorical pwb found with moderate level of mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) . this study finds that pa did nt have any big difference in participants with high and low pwb . by spearman s , pwb ( score a ) of adolescents and general levels of pa ( paq _ score ) the correlation coefficient = 0.36 was attainable value ( b / w 01 ) but the association was not significant ( p = 0.511 ) figures are mentioned in the table 3 . but according to literature many studies reported the strong relation between individuals pwb and pa however for this study it did nt showed . secondly as presented in table 4 it s also demonstrated the associations between measured covariates and standardized scores of pwb along with their corresponding p - value in the bivariate a total score was significantly correlated with gender and class as stated school adolescent boys aged 12 to 18 ( 14.1% ) were have high mean pwb ( m=71.74 ) than the mean of pwb ( m=68.5 ) among girls ( 15.4% ) . the spearman s rho correlation between paq - score and score - a the bivariate analysis of the association between some measured covariates and standardized scores of ( who - five ) pwb in school adolescents there was positive correlation between two factors pwb and gender it was statistically significant at ( p = 0.046 ) . in the kruskal - wallis test , was found strong indirect important association between pwb and classes of the participant with ( p = 0.0001 ) . but it was nt found any major association among last grade , language with pwb ( p = 0.92 ) . as mentioned in table 5 multivariate analyses with 95% confidence interval the relationship between participants pwb and gender was also significant ( p > t=0.004 ) where t=2.87 , where girls participants wpb were ( coef.=4.600939 units ) less the boys . t=0.000 ) the pwb was decreasing ( coef.=3.366921 unit ) with the increases in age . the association was strongly significant for pwb of school adolescents to their classes * ( p>=0.000 , p > t=0.000 , p>=0.001 ) as class were increasing wpb was decreasing . last grade ( p>=0.015 ) , father education ( p > t = 0.028 ) with t=2.21 and number of family members ( p > t = 0.000 ) were found strong important association with pwb . multivariable analysis for psychological wellbeing ( adjusted r2=0.15 ) with some socio - demographic covariate gender , age , class , last grade , father education and family member some other results to take note of were , first the mean of pa score in boys were 0.05 more than girls , although this difference was not statistically different while the mean of pa score in students with different class was significantly different from each other , the association by kruskal - wallis was statistically significant ( p=0.0034 ) . this study also found fathers education ( p=0.03 ) and mothers education ( p=0.025 ) important for ap . in conclusion our study demonstrated that , school adolescents pwb by with participants general levels of pa was nt correlated but at the same time the result shows pwb was significantly associated with some important socio - demographic covariate , especially a strong association was observed with gender , age , class , parents education , parents with types of jobs and family members . the purpose of the current study was to provide a quantitative assessment for the relationship among some measures of school adolescent pwb for both pa and socio - demographic factors . another purpose was to understand the association was to comprehend the general levels of pa between boys and girls and to recognize , if there is an association of pa to a specific gender for this age group . moreover this research project aim was to extend the research on this current topic to understand the association in a pakistani context . to investigator s knowledge , this is the first study which applied ( who - five ) pwb and relating to the relationship of these variables in north of pakistan . according to the national health survey of pakistan ( 19901994 ) consideration to mental health , very high prevalence of depression and anxiety has been revealed especially in reference to the student population ( 31 ) . while two other studies indicated 43.7% prevalence of anxiety and 19.5 % of depression among the medical students in pakistan ( 32 ) . this research found 23.2% school students with low pwb which indicates depression or low mood while 43.5% participants with moderate pwb and 33.3% with high . the moderate mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) this study find that pa did nt have any big difference in participant with high and low pwb , it does nt showed any statistical significance between pa and pwb of school students . interestingly , one fourth of the students ( 25% ) in the present study revealed to be psychologically distressed . the similar results were presented in prior studies among students in pakistan ( 3335 ) . while this research analysis composite the relationship between pwb and pa was attainable values 01 but correlation between these two variables was not significant ( p > .05 ) , thus indicating that pwb has a moderate negative association according to the study . whereas pwb and pa had a strong significant association with some of demographic variable including gender , age , class , father s educational status , number of family members and with last month illness . the negative association between pwb and pa suggests that as pwb scores raise but pa scores diereses even though the two variables were not significantly correlated which may be due to other factors but it was nt sure . in other study they find consideration to the intensity of pa displayed a significant positive association with mental health , physical health and quality of life ; although a negative relation was revealed with the psychological distress ( 36 ) . this study therefore could not make hypothesis that ; pwb would significantly correlate with pa , and approves of the null hypothesis . much of the recent research has consistently found that pa has a positive correlation with pwb ( 37 , 38 ) . in another study they found more than 60% of adolescents with mood disorders and these numbers correspond with other findings of low levels of pa in adults and adolescents with depression and anxiety ( 39 , 40 ) . this research project has backed up and is consistent , to a certain extent , with previous literature on the topic . nevertheless some research conducted on pa and pwb has led to contrasted conclusions relating to research has consistently found that pa has a positive correlation with pwb and mood ( 41 ) . one study reported 9 of their 11 studies reporting a negative association of pa with depression . even though the association in this study was negative but it was still a considerable one ( 42 ) , this result is similar to other findings related to pwb , for instance some researcher found a small effect size with pa and anxiety , a symptom of negative pwb and also found a small effect of exercise on wb , a relation of pwb , in a nonclinical population ( 43 , 44 ) . high percentage of female medical students reported feelings of anxiety and depression 43.7% and 19.5% respectively ( 45 ) , the present research demonstrates 15.4 % females population have moderate level of mean pwb while 14.1% male school adolescent were have high mean pwb . the third hypothesis of the present research indicated that males would have a higher participation in pa than females would . the result was one of significance , leading to association between the two variables and the reason important gender difference was also observed in relation to physical activity in the in the literature . the overall pa index was significantly higher for male students as compared to the female . the gender , was showing among adolescents that , the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m= 2.0666 ) with ( sd= 1.05998 ) among females in the present investigation . in another study also school student from class sixth and ninth boys generally reported higher levels of pa than girls ( 46 ) . this was also consistent with the previous findings as studies from us , uk , norway and australia also reported south asian females to be less physically active as compared to the white counterparts ( 47 , 48 ) . socioeconomic and housing status for pwb was nt found important but one other study says , male children , adolescents and those who were in less wealthy families were significantly associated with mental health problems ( 49 ) . even the association was found significant for pwb of adolescents to their classes and parents education , students with educated father were 4.8 units more than the students with non - educated fathers . the reason for the non - significant result found in this study maybe due to none inclusion of home based physical activity and some local sports , so in order to preserve a pwb responses may be influenced by the other social factor which not included in the study ( they may not well tell about their true attitudes , but answer in a way that they feel socially acceptable ) , this is known as the social desirability theory . in conclusion , this study is first cross sectional ( who - five ) pwb assessment framework for the evaluation of unmet levels of pwb and pa of school adolescent both boys and girls in gilgit city , pakistan . its findings may facilitate innovative efforts by all stakeholders , to improve self - management and psychosocial support to this age group of people , thus improving acceptance of change , reducing the psychological problems like feeling of being overwhelmed , through increasing the opportunities to sports in adolescent population as well as health education and promotion programs in community . according to ( who - five ) wb index applied in general , a majority of school adolescents aged 12 to 18 in gilgit , pakistan were perceived moderate level of pwb , while pa level was decreasing with the increasing of age , class and family members . some school students found with low pwb which indicates low mood or depression and 23.6 elevated prevalence of low pwb including low mood , depression . male adolescents are more physically inactive and having a high wb in comparison to the female students . the results of present study are invaluable in addressing low , moderate and high levels of pwb , inadequate pa with some of demographic factors as a crucial health issue , especially among female adolescents in pakistani society . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : adolescence is a critical developmental stage marked by complex transitions . the purpose of study was to assess school adolescents pwb , examine the relationship of pa and socio - demographic factors with pwb.methods:a cross sectional study conducted in five randomly selected schools with 345 adolescents ( aged 1218 ) from grade 6th10th . a self - administered well - being index was adapted to measure pwb and questionnaire for adolescents pa ( paq - a ) . socio - demographic variables determined : age , gender , household income and parental education . bivariate and multivariate regression analyses performed to examine the association between pwb , pa and covariates.results:findings indicated the mean age 14.64 ( sd=1.275 ) , 55.4% were female . without gender difference the majority ( 43.4% ) of adolescents showed moderate , while ( 23.2% ) revealed low level of pwb . participants with low level likely to have depression but scores were significantly not different between low , moderate and high pwb with pa . socio - demographic trends of adolescents were observed significant ( p < 0.005 ) for pwb . in multivariable analysis the mean wellbeing in females adjusted for other covariates was significant ( p = 0.004 ) than males . pwb importantly ( p < 0.001 ) decreased by 3.36 units as its covariates increased and pa score in boys found 0.05 unit more than girls.conclusion:the study results are invaluable in addressing low , moderate and high levels of pwb . inadequate pa and pwb decreasing with some socio - demographic covariates is crucial health issue among female adolescents in pakistan . further studies need to find barrier , social indicators of pwb and implication of health among adolescents .

Introduction Methods Research Design Site and Population Sampling Research Tools and data collection Statistical Analysis Descriptive Analytical Results Descriptive Analytical Discussion Conclusion Ethical considerations

, gender , age , ethnicity , parental education , economic status and physical activity etc . ) from a broad perspective , the measurement and promotion of adolescent well - being is a desirable social and political objective ( 14 , 15 ) . psychological wellbeing of adolescents means being content with life and understanding an abundance of positive emotions , when joined with the absence of psychopathology , is linked with greatest academic function , social skills and support and physical health , being a stage that lays strong foundation for future personality , and a critical period during human development in which life goals , values , direction and purpose in life are created ( 16 , 17 ) , guaranteeing psychological wellbeing of adolescents is a socio psychological necessity . however , no particular study in pakistan has been noticed to assess the association between general levels of physical activity and ( who - five ) psychological wellbeing in adolescents . the purpose of this research is to provide a broad picture of the psychological well - being of school adolescents and its relationship with general levels of physical activity and socio - demographic factors . the study analyses have evaluated the degree of co - existence of demographic factors , and to determine any gender , age , grade , physical activity specific differences in psychological wellbeing among school adolescents in gilgit , pakistan . a population - based cross - sectional quantitative correlational ( descriptive and analytic ) research design employed in this study to examine to understand if there is significant relationship between the psychological well - being , physical activity and socio - demographic factors carried out using a self - administered questionnaire . the subjects of sample size of three hundred and forty five [ n=345 ] was drawn for school adolescent ( aged 1218 ) , among them , one hundred and ninety one ( n = 191 ) were girls and one hundred and fifty four ( n = 154 ) boys , mean age was 14.64 year . data used in the thesis is based on self - reported data from the questionnaire and consisted of three sections ( a ) socio - demographic part along with ( b ) well - being ( who five index ) and ( c ) physical activity questionnaire for adolescents ( paq - a ) . in the present study 28 was for likely depression , 50 was for low mood or low level of emotional well - being in categorical is , it comes between 50 and 75 percentile was show moderate / medium and > 75th was show high level of well - being . the paq - a was used to measure general levels of physical activity in adolescents . the paq - a was self - administer it provides last seven days summary of physical activity score derived from eight items , each scored on a 5-point scale ( 30 ) . a population - based cross - sectional quantitative correlational ( descriptive and analytic ) research design employed in this study to examine to understand if there is significant relationship between the psychological well - being , physical activity and socio - demographic factors carried out using a self - administered questionnaire . the subjects of sample size of three hundred and forty five [ n=345 ] was drawn for school adolescent ( aged 1218 ) , among them , one hundred and ninety one ( n = 191 ) were girls and one hundred and fifty four ( n = 154 ) boys , mean age was 14.64 year . data used in the thesis is based on self - reported data from the questionnaire and consisted of three sections ( a ) socio - demographic part along with ( b ) well - being ( who five index ) and ( c ) physical activity questionnaire for adolescents ( paq - a ) . the ( who - five index ) captures emotional , psychological well - being and was developed from the world health organization - ten well - being index ( 28 ) . a score < 50 suggests poor emotional well - being and is a sign for further testing . in the present study 28 was for likely depression , 50 was for low mood or low level of emotional well - being in categorical is , it comes between 50 and 75 percentile was show moderate / medium and > 75th was show high level of well - being . the paq - a was used to measure general levels of physical activity in adolescents . the paq - a was self - administer it provides last seven days summary of physical activity score derived from eight items , each scored on a 5-point scale ( 30 ) . associations between well - being and socio - demographic variables were investigated in regression models and linear regression , with well - being as the dependent variable and socio - demographic variables as independents . the bivariate analysis mann - whitney is nonparametric test for two groups , kruskal - wallis test was used to compare three or more samples and spearman s rank correlation coefficients ( rho ) were calculated for correlations of pwb ( score - a ) and pa ( paq - score ) . multivariate analysis were used after adjusted ( r2= 0.15 ) to control the effect of the other confounders and multiple comparisons were performed to compare participants pwb with different classes using bonferroni method , while this opportunity is not possible in bivariate analysis . associations between well - being and socio - demographic variables were investigated in regression models and linear regression , with well - being as the dependent variable and socio - demographic variables as independents . the bivariate analysis mann - whitney is nonparametric test for two groups , kruskal - wallis test was used to compare three or more samples and spearman s rank correlation coefficients ( rho ) were calculated for correlations of pwb ( score - a ) and pa ( paq - score ) . multivariate analysis were used after adjusted ( r2= 0.15 ) to control the effect of the other confounders and multiple comparisons were performed to compare participants pwb with different classes using bonferroni method , while this opportunity is not possible in bivariate analysis . a total of 345 adolescents were participated , the mean age of adolescents was 14.64 ( sd=1.275 ) years ; 55.4% were female and without gender differences that the majority ( 43.4% ) of adolescents were shown moderate level of pwb , ( 33.3% ) high while ( 23.2% ) participants were revealed low level of pwb and were likely to have depression ( table 1 ) . descriptive statistics for participants pa score and standardized scores of ( who-5 ) pwb this study estimated the mean score of pwb ( m=69.96 ) for adolescents , was observed moderate with standard deviation ( sd=14.914 ) but the maximum score was 100 complete high and minimum pwb was 28.00 found in some of the adolescents , it indicates depression . while the mean general level of pa was found ( m=2.087 ) with standard deviation ( sd=1.041 ) socio - demographics variables examined for group differences on the pwb measures as well as for the general levels of pa ( paq - a ) among voluntarily participated school adolescents . the mean of high pwb ( m=85.1765 ) , among class 6th was higher than other classes with standard deviation ( sd=11.38110 ) and ( ci=95% ) confidence interval , although the mean pwb ( m=66.7755 ) of class 6th was moderate with ( sd= 15.36265 ) . almost for all the demographic variables the mean pwb was remained moderate but shows distribution of sample according to pa score in relation to the gender , is showing that the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m=2.0666 ) with ( sd=1.05998 ) among females in the present investigation . the below table 2 in present study found that pwb score in categories ( 1-low , 2-moderate and 3-high ) a pwb was low in ( n=80 ) participants and the mean pa ( m=2.108 ) with standard deviation ( sd= 1.00758 ) was moderate among subjects with low pwb its shows depression or low mood , while participants ( n=115 ) were have high categorical pwb found with moderate level of mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) . descriptive for pwb in categorical and general level of pa as a continuous variable by spearman s , pwb ( score a ) of adolescents and general levels of pa ( paq _ score ) the correlation coefficient = 0.36 was attainable value ( b / w 01 ) but the association was not significant ( p = 0.511 ) figures are mentioned in the table 3 . secondly as presented in table 4 it s also demonstrated the associations between measured covariates and standardized scores of pwb along with their corresponding p - value in the bivariate a total score was significantly correlated with gender and class as stated school adolescent boys aged 12 to 18 ( 14.1% ) were have high mean pwb ( m=71.74 ) than the mean of pwb ( m=68.5 ) among girls ( 15.4% ) . the spearman s rho correlation between paq - score and score - a the bivariate analysis of the association between some measured covariates and standardized scores of ( who - five ) pwb in school adolescents there was positive correlation between two factors pwb and gender it was statistically significant at ( p = 0.046 ) . in the kruskal - wallis test , was found strong indirect important association between pwb and classes of the participant with ( p = 0.0001 ) . but it was nt found any major association among last grade , language with pwb ( p = 0.92 ) . the association was strongly significant for pwb of school adolescents to their classes * ( p>=0.000 , p > t=0.000 , p>=0.001 ) as class were increasing wpb was decreasing . last grade ( p>=0.015 ) , father education ( p > t = 0.028 ) with t=2.21 and number of family members ( p > t = 0.000 ) were found strong important association with pwb . multivariable analysis for psychological wellbeing ( adjusted r2=0.15 ) with some socio - demographic covariate gender , age , class , last grade , father education and family member some other results to take note of were , first the mean of pa score in boys were 0.05 more than girls , although this difference was not statistically different while the mean of pa score in students with different class was significantly different from each other , the association by kruskal - wallis was statistically significant ( p=0.0034 ) . in conclusion our study demonstrated that , school adolescents pwb by with participants general levels of pa was nt correlated but at the same time the result shows pwb was significantly associated with some important socio - demographic covariate , especially a strong association was observed with gender , age , class , parents education , parents with types of jobs and family members . a total of 345 adolescents were participated , the mean age of adolescents was 14.64 ( sd=1.275 ) years ; 55.4% were female and without gender differences that the majority ( 43.4% ) of adolescents were shown moderate level of pwb , ( 33.3% ) high while ( 23.2% ) participants were revealed low level of pwb and were likely to have depression ( table 1 ) . descriptive statistics for participants pa score and standardized scores of ( who-5 ) pwb this study estimated the mean score of pwb ( m=69.96 ) for adolescents , was observed moderate with standard deviation ( sd=14.914 ) but the maximum score was 100 complete high and minimum pwb was 28.00 found in some of the adolescents , it indicates depression . while the mean general level of pa was found ( m=2.087 ) with standard deviation ( sd=1.041 ) socio - demographics variables examined for group differences on the pwb measures as well as for the general levels of pa ( paq - a ) among voluntarily participated school adolescents . the mean of high pwb ( m=85.1765 ) , among class 6th was higher than other classes with standard deviation ( sd=11.38110 ) and ( ci=95% ) confidence interval , although the mean pwb ( m=66.7755 ) of class 6th was moderate with ( sd= 15.36265 ) . almost for all the demographic variables the mean pwb was remained moderate but shows distribution of sample according to pa score in relation to the gender , is showing that the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m=2.0666 ) with ( sd=1.05998 ) among females in the present investigation . the below table 2 in present study found that pwb score in categories ( 1-low , 2-moderate and 3-high ) a pwb was low in ( n=80 ) participants and the mean pa ( m=2.108 ) with standard deviation ( sd= 1.00758 ) was moderate among subjects with low pwb its shows depression or low mood , while participants ( n=115 ) were have high categorical pwb found with moderate level of mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) . by spearman s , pwb ( score a ) of adolescents and general levels of pa ( paq _ score ) the correlation coefficient = 0.36 was attainable value ( b / w 01 ) but the association was not significant ( p = 0.511 ) figures are mentioned in the table 3 . secondly as presented in table 4 it s also demonstrated the associations between measured covariates and standardized scores of pwb along with their corresponding p - value in the bivariate a total score was significantly correlated with gender and class as stated school adolescent boys aged 12 to 18 ( 14.1% ) were have high mean pwb ( m=71.74 ) than the mean of pwb ( m=68.5 ) among girls ( 15.4% ) . the spearman s rho correlation between paq - score and score - a the bivariate analysis of the association between some measured covariates and standardized scores of ( who - five ) pwb in school adolescents there was positive correlation between two factors pwb and gender it was statistically significant at ( p = 0.046 ) . in the kruskal - wallis test , was found strong indirect important association between pwb and classes of the participant with ( p = 0.0001 ) . but it was nt found any major association among last grade , language with pwb ( p = 0.92 ) . as mentioned in table 5 multivariate analyses with 95% confidence interval the relationship between participants pwb and gender was also significant ( p > t=0.004 ) where t=2.87 , where girls participants wpb were ( coef.=4.600939 units ) less the boys . the association was strongly significant for pwb of school adolescents to their classes * ( p>=0.000 , p > t=0.000 , p>=0.001 ) as class were increasing wpb was decreasing . last grade ( p>=0.015 ) , father education ( p > t = 0.028 ) with t=2.21 and number of family members ( p > t = 0.000 ) were found strong important association with pwb . multivariable analysis for psychological wellbeing ( adjusted r2=0.15 ) with some socio - demographic covariate gender , age , class , last grade , father education and family member some other results to take note of were , first the mean of pa score in boys were 0.05 more than girls , although this difference was not statistically different while the mean of pa score in students with different class was significantly different from each other , the association by kruskal - wallis was statistically significant ( p=0.0034 ) . in conclusion our study demonstrated that , school adolescents pwb by with participants general levels of pa was nt correlated but at the same time the result shows pwb was significantly associated with some important socio - demographic covariate , especially a strong association was observed with gender , age , class , parents education , parents with types of jobs and family members . the purpose of the current study was to provide a quantitative assessment for the relationship among some measures of school adolescent pwb for both pa and socio - demographic factors . another purpose was to understand the association was to comprehend the general levels of pa between boys and girls and to recognize , if there is an association of pa to a specific gender for this age group . this research found 23.2% school students with low pwb which indicates depression or low mood while 43.5% participants with moderate pwb and 33.3% with high . the moderate mean pa ( m=2.0515 ) with standard deviation ( sd= .97959 ) this study find that pa did nt have any big difference in participant with high and low pwb , it does nt showed any statistical significance between pa and pwb of school students . while this research analysis composite the relationship between pwb and pa was attainable values 01 but correlation between these two variables was not significant ( p > .05 ) , thus indicating that pwb has a moderate negative association according to the study . whereas pwb and pa had a strong significant association with some of demographic variable including gender , age , class , father s educational status , number of family members and with last month illness . the negative association between pwb and pa suggests that as pwb scores raise but pa scores diereses even though the two variables were not significantly correlated which may be due to other factors but it was nt sure . in another study they found more than 60% of adolescents with mood disorders and these numbers correspond with other findings of low levels of pa in adults and adolescents with depression and anxiety ( 39 , 40 ) . nevertheless some research conducted on pa and pwb has led to contrasted conclusions relating to research has consistently found that pa has a positive correlation with pwb and mood ( 41 ) . even though the association in this study was negative but it was still a considerable one ( 42 ) , this result is similar to other findings related to pwb , for instance some researcher found a small effect size with pa and anxiety , a symptom of negative pwb and also found a small effect of exercise on wb , a relation of pwb , in a nonclinical population ( 43 , 44 ) . the gender , was showing among adolescents that , the mean pa ( m=2.1117 ) with ( sd=1.020275 ) was higher in male school adolescents than the mean level of pa ( m= 2.0666 ) with ( sd= 1.05998 ) among females in the present investigation . even the association was found significant for pwb of adolescents to their classes and parents education , students with educated father were 4.8 units more than the students with non - educated fathers . in conclusion , this study is first cross sectional ( who - five ) pwb assessment framework for the evaluation of unmet levels of pwb and pa of school adolescent both boys and girls in gilgit city , pakistan . according to ( who - five ) wb index applied in general , a majority of school adolescents aged 12 to 18 in gilgit , pakistan were perceived moderate level of pwb , while pa level was decreasing with the increasing of age , class and family members . the results of present study are invaluable in addressing low , moderate and high levels of pwb , inadequate pa with some of demographic factors as a crucial health issue , especially among female adolescents in pakistani society .

symptoms of crohn s disease in patients range on a spectrum from mild to very severe . symptoms include diarrhea , abdominal pain , intermittent fever , rectal bleeding , loss of appetite , significant weight loss , arthralgias , fatigue , malaise , and headaches . involvement of other organ systems beyond the intestinal tract , such as eyes , skin , and liver , may be present.1 as there is currently no known cure , treatment is focused on symptom control . complications secondary to medications prescribed for symptom control may occur . when the disease fails to respond to the milder medications , medication complications can be severe , including infections , serum sickness , drug - induced lupus , diabetes , cancers , and even death.2 this paper documents a case study of a patient with severe crohn s disease . the patient had suffered with crohn s disease of progressing severity for 22 years , during which time no sustained remission of symptoms was noted . he experienced no sustained response from mesalamine , low - dose naltrexone , or dietary modification . the patient s clinical course was complicated by steroid - induced insulin - dependent diabetes . he also suffered from severe weight loss , depression , fatigue , malaise , headaches , purulent - mucinous diarrhea , rectal bleeding , bilious vomiting , and diffuse arthralgias . complaints of back pain resulted in back surgery with negative operative findings and no relief of symptoms . the pathologist s report of tissue submitted from the gallbladder surgery was negative for any pathology . in february 2004 , the patient had progressed to the most severe state of his disease , losing 25% of his body weight . he experienced constant symptoms of crohn s disease despite attempts at medication alteration . at all times from his first confirmed attack of crohn s disease in 1990 at age 19 years , he was on one or more prescription drugs to try to control the disease symptoms . the patient achieved full remission of symptoms in a matter of days once the proper orally administered serotonin and dopamine amino acid precursor dosing values were established with the guidance of urinary organic cation transporter ( oct ) functional status determination ( herein referred to as oct assay interpretation ) . the patient was treated with a novel treatment protocol developed by neuroresearch clinics ( duluth , minnesota , mn , usa ) . peer - reviewed publications from 20093,4 and 201057 outlined a novel three - phase model of oct response to simultaneous administration of serotonin and dopamine amino acid precursors in significant amounts , which is the basis for oct assay interpretation . outlined in this paper is a proposed novel oct model that potentially describes the etiology of the three - phase response of serotonin and dopamine during simultaneous administration of their amino acid precursors in varied daily dosing values.5 serotonin and dopamine exist in two states . the competitive inhibition state is found when significant amounts of amino acid precursors of both serotonin and dopamine are administered simultaneously . this novel approach places serotonin and dopamine in the competitive inhibition state and then optimizes their transport in proper balance through the octs with oct analysis interpretation . peer - reviewed research covering methodology , applications , and the scientific foundation of this novel approach was published in 20093,4 and 2010.57 optimization of the serotonin dopamine system has applications in any condition where an imbalance between serotonin and dopamine in transport , synthesis , or metabolism is present . the potential scope of applications is far - reaching . the protocol utilized for treatment of crohn s disease consisted of the amino acid dosing values listed in table 1 . this protocol has been covered in previous peer - reviewed research.3,7 the initial step of the protocol is the simultaneous administration of serotonin and dopamine amino acid precursors with no oct functional status determination in order to place the system into a competitive inhibition state . at the first visit , the patient was started on level 1 amino acid dosing . the patient was then followed weekly for evaluation of response to the start or change in amino acid dosing levels . as described in the results section of this paper , the patients took the amino acid dosing values of each level at the times indicated in table 1 . if the patient failed to achieve full relief of symptoms on level 3 dosing , a urine sample was collected and submitted for urinary serotonin and dopamine laboratory assay . based on oct assay interpretation , the amino acid precursors of serotonin and dopamine were adjusted in an effort to achieve full relief of symptoms or a balance of urinary serotonin and dopamine in the phase 3 therapeutic range , whichever came first.3,7 the serotonin and dopamine filtered at the glomerulous are metabolized by the kidneys , and significant amounts do not make it to the final urine . serotonin and dopamine found in the urine are monoamines synthesized in the proximal convoluted renal tubule cells and have never been found in the central nervous system or peripheral system . serotonin and dopamine that are newly synthesized by the kidneys meet one of two fates . urinary serotonin and dopamine levels are primarily dependent on the interaction of the basolateral monoamine transporters ( oct2s ) and the apical monoamine transporters ( octn2s ) of the proximal convoluted renal tubule cells of the kidneys.5,8 the octn2s8 of the proximal convoluted renal tubule cells transport serotonin and dopamine that is not transported by the oct2.5 while in the competitive inhibition state , serotonin and dopamine not transported by the oct2s are found in the final urine as waste.6 although there are numerous other forces that interact with the newly synthesized renal monoamines , they are small compared with the effects of these transporters.5 proper interpretation of urinary serotonin and dopamine levels in the competitive inhibition state determines the functional status of the oct2s of the proximal convoluted renal tubule cells of the kidneys , known as oct assay interpretation . the oct2s exist in three different phases dependent on the status of the entrance gate and lumen saturation.37 table 2 outlines the correlation between entrance gate status and lumen saturation . the basis for oct assay interpretation requires that the system be placed into the competitive inhibition state and then two or more urinary serotonin and dopamine assays performed while taking serotonin and dopamine amino acid precursors at significantly varied dosing values . the results are then compared in order to determine the change in urinary serotonin and dopamine levels in response to the change in amino acid precursor dosing values.37 urinary serotonin and dopamine values found on assay were reported in micrograms of monoamine per gram of creatinine in order to compensate for fluctuations in urinary - specific gravity . a urinary serotonin or dopamine value less than 80 or 475 g of monoamine per 1 g of creatinine , respectively , is defined as a phase 2 response . a urinary serotonin or dopamine value greater than 80 or 475 g of monoamine per 1 g of creatinine , if a direct relationship is found between amino acid dosing and urinary assay response , it is referred to as a phase 3 response . the phase 3 therapeutic range for urinary serotonin is defined as 80240 g of serotonin per 1 g of creatinine . the phase 3 therapeutic range for urinary dopamine is defined as 4751100 g of dopamine per 1 g of creatinine.3,57 processing , management , and assay of the urine samples collected for this study were as follows . urine samples were collected 6 hours prior to bedtime with 4:00 pm being the most frequent collection time point . the samples were stabilized in 6 n hydrochloric acid to preserve the dopamine and serotonin . the urine samples were collected after a minimum of 1 week , during which the patient was taking a specific daily dosing of amino acid precursors of serotonin and dopamine . urinary dopamine and serotonin were assayed utilizing commercially available radioimmunoassay kits ( 3 cat ria ib88501 and ib89527 , both from immuno biological laboratories , inc . , the dbs laboratory is accredited by clinical laboratory improvement amendments as a high - complexity laboratory . results were reported in micrograms of monoamine per gram of creatinine to compensate for specific gravity variances in the urine . the competitive inhibition state is found when significant amounts of amino acid precursors of both serotonin and dopamine are administered simultaneously . this novel approach places serotonin and dopamine in the competitive inhibition state and then optimizes their transport in proper balance through the octs with oct analysis interpretation . peer - reviewed research covering methodology , applications , and the scientific foundation of this novel approach was published in 20093,4 and 2010.57 optimization of the serotonin dopamine system has applications in any condition where an imbalance between serotonin and dopamine in transport , synthesis , or metabolism is present . the potential scope of applications is far - reaching . the protocol utilized for treatment of crohn s disease consisted of the amino acid dosing values listed in table 1 . this protocol has been covered in previous peer - reviewed research.3,7 the initial step of the protocol is the simultaneous administration of serotonin and dopamine amino acid precursors with no oct functional status determination in order to place the system into a competitive inhibition state . at the first visit , the patient was started on level 1 amino acid dosing . the patient was then followed weekly for evaluation of response to the start or change in amino acid dosing levels . as described in the results section of this paper , the patients took the amino acid dosing values of each level at the times indicated in table 1 . if the patient failed to achieve full relief of symptoms on level 3 dosing , a urine sample was collected and submitted for urinary serotonin and dopamine laboratory assay . based on oct assay interpretation , the amino acid precursors of serotonin and dopamine were adjusted in an effort to achieve full relief of symptoms or a balance of urinary serotonin and dopamine in the phase 3 therapeutic range , whichever came first.3,7 the serotonin and dopamine filtered at the glomerulous are metabolized by the kidneys , and significant amounts do not make it to the final urine . serotonin and dopamine found in the urine are monoamines synthesized in the proximal convoluted renal tubule cells and have never been found in the central nervous system or peripheral system . serotonin and dopamine that are newly synthesized by the kidneys meet one of two fates . urinary serotonin and dopamine levels are primarily dependent on the interaction of the basolateral monoamine transporters ( oct2s ) and the apical monoamine transporters ( octn2s ) of the proximal convoluted renal tubule cells of the kidneys.5,8 the octn2s8 of the proximal convoluted renal tubule cells transport serotonin and dopamine that is not transported by the oct2.5 while in the competitive inhibition state , serotonin and dopamine not transported by the oct2s are found in the final urine as waste.6 although there are numerous other forces that interact with the newly synthesized renal monoamines , they are small compared with the effects of these transporters.5 proper interpretation of urinary serotonin and dopamine levels in the competitive inhibition state determines the functional status of the oct2s of the proximal convoluted renal tubule cells of the kidneys , known as oct assay interpretation . the oct2s exist in three different phases dependent on the status of the entrance gate and lumen saturation.37 table 2 outlines the correlation between entrance gate status and lumen saturation . the basis for oct assay interpretation requires that the system be placed into the competitive inhibition state and then two or more urinary serotonin and dopamine assays performed while taking serotonin and dopamine amino acid precursors at significantly varied dosing values . the results are then compared in order to determine the change in urinary serotonin and dopamine levels in response to the change in amino acid precursor dosing values.37 urinary serotonin and dopamine values found on assay were reported in micrograms of monoamine per gram of creatinine in order to compensate for fluctuations in urinary - specific gravity . a urinary serotonin or dopamine value less than 80 or 475 g of monoamine per 1 g of creatinine , respectively , is defined as a phase 2 response . a urinary serotonin or dopamine value greater than 80 or 475 g of monoamine per 1 g of creatinine , if a direct relationship is found between amino acid dosing and urinary assay response , it is referred to as a phase 3 response . the phase 3 therapeutic range for urinary serotonin is defined as 80240 g of serotonin per 1 g of creatinine . the phase 3 therapeutic range for urinary dopamine is defined as 4751100 g of dopamine per 1 g of creatinine.3,57 processing , management , and assay of the urine samples collected for this study were as follows . urine samples were collected 6 hours prior to bedtime with 4:00 pm being the most frequent collection time point . the samples were stabilized in 6 n hydrochloric acid to preserve the dopamine and serotonin . the urine samples were collected after a minimum of 1 week , during which the patient was taking a specific daily dosing of amino acid precursors of serotonin and dopamine . urinary dopamine and serotonin were assayed utilizing commercially available radioimmunoassay kits ( 3 cat ria ib88501 and ib89527 , both from immuno biological laboratories , inc . , the dbs laboratory is accredited by clinical laboratory improvement amendments as a high - complexity laboratory . results were reported in micrograms of monoamine per gram of creatinine to compensate for specific gravity variances in the urine . an endoscopy examination , prior to treatment with amino acids while the disease was active , was performed in september 2005 . , the patient was still taking mesalamine , low - dose naltrexone , and escitalopram . the escitalopram was discontinued at the start of amino acid treatment , and the mesalamine and low - dose naltrexone were continued . at the first visit , the patient was started on level 1 amino acid dosing as per table 1 . one week later there was no change in symptoms , and the patient s amino acid dosing values were increased to level 2 ( see table 1 ) . the patient achieved lessening of the symptoms when he was on level 2 amino acid dosing . at that point , the patient revealed that he felt that this approach was the best treatment he had experienced during the course of his 22-year illness . the amino acids were increased to level 3 dosing ( see table 1 ) , with no further change in symptoms . after 1 week of level 3 dosing , a urine sample was obtained and analyzed . when the first urine sample was collected for oct assay interpretation , the patient was taking level 3 dosing : 900 mg 5-hydroxytryptophan ( 5-htp ) , 5000 mg l - tyrosine , and 4500 mg l - cysteine with cofactors . the first urinary assay revealed serotonin to be in phase 3 ( table 2 ) with a reported value of 5150.7 g of serotonin per 1 g of creatinine , and a dopamine in phase 2 ( table 2 ) with a reported value of 206.4 g of dopamine per 1 g of creatinine . after the first oct assay interpretation , the patient s daily amino acid dosing was increased by 1000 mg of l - tyrosine and 240 mg of l - dopa . at that point , the patient was taking the following in divided daily doses : 900 mg 5-htp , 6000 mg l - tyrosine , 240 mg l - dopa , and 4500 mg l - cysteine with cofactors . after 1 week taking these new amino acid dosing values , there was no change in the patient s symptoms . this revealed that the patient s urinary serotonin was in phase 3 ( table 2 ) at 12,611.1 g of serotonin per 1 g of creatinine , and his dopamine was in phase 3 ( table 2 ) at 741.3 g of dopamine per 1 g of creatinine . the recommendation was to decrease the daily 5-htp dosing by 300 mg per day , increase l - tyrosine by 1000 mg per day , and continue other amino acids as before . the patient was then taking the following in divided daily doses : 600 mg 5-htp , 7000 mg l - tyrosine , 240 mg l - dopa , and 4500 mg l - cysteine with cofactors.5 within 1 week of this dosing value change , the patient became asymptomatic , indicating that adequate oct balance of the serotonin the patient s response and remission with amino acid treatment was very impressive and relatively abrupt compared with the 22-year course of his disease . this profound resolution of symptoms was achieved within 6 weeks of the first clinic visit . the patient noted the return of solid stools , no further vomiting , restored energy , increased motivation , and resolution of depression symptoms . all prescription medications that the patient had been taking since the start of amino acid treatment were discontinued after 6 weeks of amino acid treatment , including mesalamine and naltrexone , with no return of symptoms . the amino acid dosing values that had induced relief of symptoms were continued . following remission of symptoms , his weight stabilized at approximately 20 pounds above the lowest weight attained while disease symptoms were present . the patient found that if he missed a dose of the amino acids , some of the crohn s disease symptoms would return . a third oct assay interpretation was obtained 5 months later with amino acid dosing values that induced relief of symptoms . urinary serotonin was reported as 9019.5 g of serotonin per 1 g of creatinine and urinary dopamine was 604.3 g of dopamine per 1 g of creatinine ; both were in phase 3 ( table 2 ) . at this point , the recommendation was to decrease the daily 5-htp dosage to 300 mg , decrease l - tyrosine dosing by 1000 mg per day , and continue other amino acids as before . after this dosing value change , the patient was then taking the following in divided daily doses : 300 mg 5-htp , 6000 mg l - tyrosine , 240 mg l - dopa , and 4500 mg l - cysteine with cofactors . following this change in amino acid dosing values , the patient continued to be asymptomatic , a state that exists to this day as long as he is compliant with the prescribed amino acid dosing values . this was 26 months after starting the amino acid protocol guided by oct assay interpretation and 24 months after achieving relief of symptoms . this endoscopy was performed by the same gastroenterologist that performed endoscopy prior to remission of symptoms . at this endoscopy he was taking no insulin or oral hypoglycemic agents , and his hba1c had returned to normal . he had returned to full - time gainful employment , after a period of over 4 years during which he was fully disabled . the gastroenterologist reported that for the first time in 10 years of caring for the patient , the crohn s disease was in complete remission . this finding was verified by the pathologist after review of tissue samples submitted . as of the time of writing this paper , the patient continues to do well with no infections or adverse reactions . the authors have documented a number of patients with crohn s disease who experienced similar remission of symptoms with this approach . this case was selected for this paper due to the severity of disease in the patient . serotonin and dopamine levels inside and outside of the cell structures containing them are primarily a function of transporter status.5 the question raised is how oct assay interpretation of renal transporters relates to the octs of the gastrointestinal ( gi ) tract . the hypothesis is that performing oct assay interpretation on one set of octs will give insight into transport of serotonin , dopamine , and their precursors at other octs throughout the body . within 35 days of starting or changing amino acid precursor dosing values , serotonin , dopamine , and their precursors reach equilibrium throughout the body.3,57 at equilibrium , amino acid precursors , serotonin , and dopamine exert similar effects at cation transporters throughout the body . in the competitive inhibition state , the serotonin and dopamine systems function as one system in transport , synthesis , and metabolism . serotonin , dopamine , and their amino acid precursors compete for transport at the octs . significant increases in one monoamine will decrease monoamine and precursor transport of the other system through competitive inhibition . transport of precursors into the cells is required in order to place them in an environment where synthesis takes place . the same enzyme , the l - aromatic amino acid decarboxylase enzyme ( aaad ) , is responsible for synthesis of serotonin and dopamine . creating an environment where precursors of one system are significantly increased without significantly increasing the precursors of the other system leads to decreased access to the aaad by precursors of the other system , with associated decreased synthesis or depletion due to competitive inhibition . both serotonin and dopamine are metabolized by the monoamine oxidase ( mao ) enzyme system . a significant increase in levels of one system will increase mao activity , leading to increased metabolism and depletion of the other system.2,5,6 in the intestinal tract of crohn s patients there is excessive synthesis with associated increased tissue levels of serotonin.8,9 in crohn s disease , high levels of serotonin dominate synthesis , metabolism , and transport , leading to dopamine and catecholamine levels that are low relative to the balance needed to function properly with the serotonin levels present.3,5 as noted in previous peer - reviewed research by the authors , oct phase determination defines the status of the serotonin and dopamine gates at the entrance to the basolateral monoamine oct ( open or partially closed ) of the proximal convoluted renal tubule cells of the kidneys and the status of serotonin and dopamine saturation in these transporters ( see table 2).5 proper interpretation of the findings requires the following explanation . serotonin and dopamine both need to be in the competitive inhibition state when oct assay interpretation is performed . this means that significant dosing values of both serotonin and dopamine need to be administered simultaneously . when in the competitive inhibition state , serotonin and dopamine are in full competition for transport , synthesis , and metabolism.3,5 testing of the urine is only done after amino acid precursors of the monoamines are started in accordance with the protocol , placing the serotonin baseline testing in the endogenous state prior to administration of amino acid precursors is of no value , as these assay levels correlate with nothing . as noted in previous peer - reviewed literature , baseline testing of urinary serotonin and dopamine does not correlate with baseline assays performed on subsequent days in the same individual.6 simply giving the patient one or more amino acid precursors is not the key to optimal outcomes . the oct needs to be challenged with serotonin and dopamine precursors in significant amounts to place transport in the competitive inhibition state so that proper oct assay interpretation can be realized.5 there is a known genetic defect of octn1 and octn2 in the colon of patients suffering from crohn s diease.9 all oct and octn transporters are capable of transporting organic cations , including serotonin , dopamine , and their precursors.8 in crohn s disease , the serotonin content of the mucosa and submucosa of the proximal and distal colon is increased.10 increased synthesis of serotonin is known to be associated with crohn s disease.11 no reasonable explanation of the etiology of serotonin elevation in the colon tissue of crohn s disease patients has been put forth previously . it is postulated that the known octn1 and octn2 genetic deficit may be tied to the increased synthesis and tissue levels of serotonin seen with crohn s disease . based on oct assay interpretation , it appears that a severe imbalance between serotonin and dopamine transport , synthesis , and metabolism is at the heart of crohn s disease . dopamine transport system has been linked to numerous diseases.3,57 it is proposed that much of the clinical constellation found with crohn s disease may be induced by a serotonin toxicity of the colon exacerbated by relatively low levels of dopamine resulting from defective octn transport . in the gi tract , serotonin is contained primarily in the enteroendocrine cells ( ecs ) . the serotonin autocrine and/or endocrine mediators that modulate gi function.12 it is asserted that proper treatment needs to include correct management of the serotonin and dopamine imbalance in transport , synthesis , and metabolism . the only definitive way to address these problems optimally is with oct analysis interpretation in the competitive inhibition state that is established with proper amino acid precursor administration . it is postulated that the patient s crohn s disease was impacted in a positive manner as follows . it is known that there is increased synthesis of serotonin with increased serotonin levels in the proximal and distal colon.11 levels of the serotonin dopamine system are impacted primarily by synthesis , uptake , and metabolism . for serotonin and dopamine to be synthesized , their amino acid precursors need to be transported into the structures where this occurs . serotonin precursors are transported preferentially at the exclusion of dopamine precursors , leading to high levels of synthesis , high levels of serotonin in portions of the colon , and compromise of catecholamine synthesis . properly balancing the serotonin and dopamine precursor transport leads to a decrease in serotonin synthesis , less serotonin in the tissue of the proximal and distal colon , and an increase in synthesis of dopamine , norepinephrine , and epinephrine . increased serotonin levels of crohn s disease lead to increased mao activity , which without reciprocal increases of the catecholamines leads to increased metabolism of the catecholamines , further exacerbating the imbalance . with a case study such as this this patient had a 22-year history of progressively worsening crohn s with no remissions and has been free of crohn s disease symptoms clinically and on biopsy for 2.5 years since the appropriate dosing values of serotonin and dopamine amino acids were established . we leave it to the reader to speculate as to the odds of this being a spontaneous coincidental remission versus a response to properly balanced amino acids . previously published peer - reviewed literature by the authors indicates that this same approach with oct assay interpretation for treatment of depression is effective.3,7 in this case study , the patient s depression resolved when the serotonin and dopamine were balanced to the degree needed for relief of crohn s disease symptoms . it is asserted that it was no coincidence that the patient s depression resolved simultaneously with the resolution of the symptoms of crohn s disease . the authors have documented a number of patients with crohn s disease who experienced similar remission of symptoms with this approach . this case was selected for this paper due to the severity of disease in the patient . serotonin and dopamine levels inside and outside of the cell structures containing them are primarily a function of transporter status.5 the question raised is how oct assay interpretation of renal transporters relates to the octs of the gastrointestinal ( gi ) tract . the hypothesis is that performing oct assay interpretation on one set of octs will give insight into transport of serotonin , dopamine , and their precursors at other octs throughout the body . within 35 days of starting or changing amino acid precursor dosing values , serotonin , dopamine , and their precursors reach equilibrium throughout the body.3,57 at equilibrium , amino acid precursors , serotonin , and dopamine exert similar effects at cation transporters throughout the body . in the competitive inhibition state , the serotonin and dopamine systems function as one system in transport , synthesis , and metabolism . serotonin , dopamine , and their amino acid precursors compete for transport at the octs . significant increases in one monoamine will decrease monoamine and precursor transport of the other system through competitive inhibition . transport of precursors into the cells is required in order to place them in an environment where synthesis takes place . the same enzyme , the l - aromatic amino acid decarboxylase enzyme ( aaad ) , is responsible for synthesis of serotonin and dopamine . creating an environment where precursors of one system are significantly increased without significantly increasing the precursors of the other system leads to decreased access to the aaad by precursors of the other system , with associated decreased synthesis or depletion due to competitive inhibition . both serotonin and dopamine are metabolized by the monoamine oxidase ( mao ) enzyme system . a significant increase in levels of one system will increase mao activity , leading to increased metabolism and depletion of the other system.2,5,6 in the intestinal tract of crohn s patients there is excessive synthesis with associated increased tissue levels of serotonin.8,9 in crohn s disease , high levels of serotonin dominate synthesis , metabolism , and transport , leading to dopamine and catecholamine levels that are low relative to the balance needed to function properly with the serotonin levels present.3,5 as noted in previous peer - reviewed research by the authors , oct phase determination defines the status of the serotonin and dopamine gates at the entrance to the basolateral monoamine oct ( open or partially closed ) of the proximal convoluted renal tubule cells of the kidneys and the status of serotonin and dopamine saturation in these transporters ( see table 2).5 proper interpretation of the findings requires the following explanation . serotonin and dopamine both need to be in the competitive inhibition state when oct assay interpretation is performed . this means that significant dosing values of both serotonin and dopamine need to be administered simultaneously . when in the competitive inhibition state , serotonin and dopamine are in full competition for transport , synthesis , and metabolism.3,5 testing of the urine is only done after amino acid precursors of the monoamines are started in accordance with the protocol , placing the serotonin baseline testing in the endogenous state prior to administration of amino acid precursors is of no value , as these assay levels correlate with nothing . as noted in previous peer - reviewed literature , baseline testing of urinary serotonin and dopamine does not correlate with baseline assays performed on subsequent days in the same individual.6 simply giving the patient one or more amino acid precursors is not the key to optimal outcomes . the oct needs to be challenged with serotonin and dopamine precursors in significant amounts to place transport in the competitive inhibition state so that proper oct assay interpretation can be realized.5 there is a known genetic defect of octn1 and octn2 in the colon of patients suffering from crohn s diease.9 all oct and octn transporters are capable of transporting organic cations , including serotonin , dopamine , and their precursors.8 in crohn s disease , the serotonin content of the mucosa and submucosa of the proximal and distal colon is increased.10 increased synthesis of serotonin is known to be associated with crohn s disease.11 no reasonable explanation of the etiology of serotonin elevation in the colon tissue of crohn s disease patients has been put forth previously . it is postulated that the known octn1 and octn2 genetic deficit may be tied to the increased synthesis and tissue levels of serotonin seen with crohn s disease . based on oct assay interpretation , it appears that a severe imbalance between serotonin and dopamine transport , synthesis , and metabolism is at the heart of crohn s disease . dopamine transport system has been linked to numerous diseases.3,57 it is proposed that much of the clinical constellation found with crohn s disease may be induced by a serotonin toxicity of the colon exacerbated by relatively low levels of dopamine resulting from defective octn transport . in the gi tract , serotonin is contained primarily in the enteroendocrine cells ( ecs ) . the serotonin autocrine and/or endocrine mediators that modulate gi function.12 it is asserted that proper treatment needs to include correct management of the serotonin and dopamine imbalance in transport , synthesis , and metabolism . the only definitive way to address these problems optimally is with oct analysis interpretation in the competitive inhibition state that is established with proper amino acid precursor administration . it is postulated that the patient s crohn s disease was impacted in a positive manner as follows . it is known that there is increased synthesis of serotonin with increased serotonin levels in the proximal and distal colon.11 levels of the serotonin dopamine system are impacted primarily by synthesis , uptake , and metabolism . for serotonin and dopamine to be synthesized , their amino acid precursors need to be transported into the structures where this occurs . serotonin precursors are transported preferentially at the exclusion of dopamine precursors , leading to high levels of synthesis , high levels of serotonin in portions of the colon , and compromise of catecholamine synthesis . properly balancing the serotonin and dopamine precursor transport leads to a decrease in serotonin synthesis , less serotonin in the tissue of the proximal and distal colon , and an increase in synthesis of dopamine , norepinephrine , and epinephrine . increased serotonin levels of crohn s disease lead to increased mao activity , which without reciprocal increases of the catecholamines leads to increased metabolism of the catecholamines , further exacerbating the imbalance . with a case study such as this there is always the possibility that remission was coincidental to treatment . this patient had a 22-year history of progressively worsening crohn s with no remissions and has been free of crohn s disease symptoms clinically and on biopsy for 2.5 years since the appropriate dosing values of serotonin and dopamine amino acids were established . we leave it to the reader to speculate as to the odds of this being a spontaneous coincidental remission versus a response to properly balanced amino acids . previously published peer - reviewed literature by the authors indicates that this same approach with oct assay interpretation for treatment of depression is effective.3,7 in this case study , the patient s depression resolved when the serotonin and dopamine were balanced to the degree needed for relief of crohn s disease symptoms . it is asserted that it was no coincidence that the patient s depression resolved simultaneously with the resolution of the symptoms of crohn s disease . in recent years , a genetic defect of the octn1 and octn2 of the colon has been identified in patients with crohn s disease . the octn1 and octn2 are responsible for transport of cations , including the monoamines of the serotonin it is known with crohn s disease patients that there is a marked increased in serotonin levels of the proximal and distal colon associated with a defect in serotonin synthesis . dopamine system induced by the octn1 and octn2 genetic defect found in crohn s disease . for clearly , further studies relating to oct analysis interpretation and the octn transporters of the colon as they relate to other abnormal findings associated with crohn s disease are indicated . this paper potentially opens the door to a new area of treatment and study in crohn s disease patients . the goal of the paper is to stimulate further interest in these findings in order to duplicate , confirm , and invite scrutiny of these results .

purpose : this paper reviews the clinical course of a case of severe crohn s disease and discusses the scientific ramifications of a novel treatment approach.patients and methods : a case study of a 37-year - old male with a 22-year history of crohn s disease whose clinical course had experienced no sustained remissions . the patient was treated with a protocol that utilized serotonin and dopamine amino acid precursors administered under the guidance of organic cation transporter assay interpretation.results:within 5 days of achieving the necessary balance of serotonin and dopamine , the patient experienced remission of symptoms . this remission has been sustained without the use of any crohn s disease medications.conclusion:in crohn s disease , it is known that there is an increase of both synthesis and tissue levels of serotonin in specific locations . it is asserted that this is prima facie evidence of a significant imbalance in the serotonin dopamine system , leading to serotonin toxicity . the hypothesis formulated is that improperly balanced serotonin and dopamine transport , synthesis , and metabolism is a primary defect contributing to the pathogenesis of crohn s disease .

Introduction Material and methods The protocol OCT assay interpretation Results Discussion Scientific basis OCT assay interpretation The OCTN Other implications Conclusion

when the disease fails to respond to the milder medications , medication complications can be severe , including infections , serum sickness , drug - induced lupus , diabetes , cancers , and even death.2 this paper documents a case study of a patient with severe crohn s disease . the patient had suffered with crohn s disease of progressing severity for 22 years , during which time no sustained remission of symptoms was noted . the patient achieved full remission of symptoms in a matter of days once the proper orally administered serotonin and dopamine amino acid precursor dosing values were established with the guidance of urinary organic cation transporter ( oct ) functional status determination ( herein referred to as oct assay interpretation ) . the patient was treated with a novel treatment protocol developed by neuroresearch clinics ( duluth , minnesota , mn , usa ) . peer - reviewed publications from 20093,4 and 201057 outlined a novel three - phase model of oct response to simultaneous administration of serotonin and dopamine amino acid precursors in significant amounts , which is the basis for oct assay interpretation . outlined in this paper is a proposed novel oct model that potentially describes the etiology of the three - phase response of serotonin and dopamine during simultaneous administration of their amino acid precursors in varied daily dosing values.5 serotonin and dopamine exist in two states . the competitive inhibition state is found when significant amounts of amino acid precursors of both serotonin and dopamine are administered simultaneously . peer - reviewed research covering methodology , applications , and the scientific foundation of this novel approach was published in 20093,4 and 2010.57 optimization of the serotonin dopamine system has applications in any condition where an imbalance between serotonin and dopamine in transport , synthesis , or metabolism is present . the protocol utilized for treatment of crohn s disease consisted of the amino acid dosing values listed in table 1 . this protocol has been covered in previous peer - reviewed research.3,7 the initial step of the protocol is the simultaneous administration of serotonin and dopamine amino acid precursors with no oct functional status determination in order to place the system into a competitive inhibition state . at the first visit , the patient was started on level 1 amino acid dosing . as described in the results section of this paper , the patients took the amino acid dosing values of each level at the times indicated in table 1 . based on oct assay interpretation , the amino acid precursors of serotonin and dopamine were adjusted in an effort to achieve full relief of symptoms or a balance of urinary serotonin and dopamine in the phase 3 therapeutic range , whichever came first.3,7 the serotonin and dopamine filtered at the glomerulous are metabolized by the kidneys , and significant amounts do not make it to the final urine . urinary serotonin and dopamine levels are primarily dependent on the interaction of the basolateral monoamine transporters ( oct2s ) and the apical monoamine transporters ( octn2s ) of the proximal convoluted renal tubule cells of the kidneys.5,8 the octn2s8 of the proximal convoluted renal tubule cells transport serotonin and dopamine that is not transported by the oct2.5 while in the competitive inhibition state , serotonin and dopamine not transported by the oct2s are found in the final urine as waste.6 although there are numerous other forces that interact with the newly synthesized renal monoamines , they are small compared with the effects of these transporters.5 proper interpretation of urinary serotonin and dopamine levels in the competitive inhibition state determines the functional status of the oct2s of the proximal convoluted renal tubule cells of the kidneys , known as oct assay interpretation . the basis for oct assay interpretation requires that the system be placed into the competitive inhibition state and then two or more urinary serotonin and dopamine assays performed while taking serotonin and dopamine amino acid precursors at significantly varied dosing values . the urine samples were collected after a minimum of 1 week , during which the patient was taking a specific daily dosing of amino acid precursors of serotonin and dopamine . the competitive inhibition state is found when significant amounts of amino acid precursors of both serotonin and dopamine are administered simultaneously . peer - reviewed research covering methodology , applications , and the scientific foundation of this novel approach was published in 20093,4 and 2010.57 optimization of the serotonin dopamine system has applications in any condition where an imbalance between serotonin and dopamine in transport , synthesis , or metabolism is present . this protocol has been covered in previous peer - reviewed research.3,7 the initial step of the protocol is the simultaneous administration of serotonin and dopamine amino acid precursors with no oct functional status determination in order to place the system into a competitive inhibition state . at the first visit , the patient was started on level 1 amino acid dosing . as described in the results section of this paper , the patients took the amino acid dosing values of each level at the times indicated in table 1 . if the patient failed to achieve full relief of symptoms on level 3 dosing , a urine sample was collected and submitted for urinary serotonin and dopamine laboratory assay . based on oct assay interpretation , the amino acid precursors of serotonin and dopamine were adjusted in an effort to achieve full relief of symptoms or a balance of urinary serotonin and dopamine in the phase 3 therapeutic range , whichever came first.3,7 the serotonin and dopamine filtered at the glomerulous are metabolized by the kidneys , and significant amounts do not make it to the final urine . the basis for oct assay interpretation requires that the system be placed into the competitive inhibition state and then two or more urinary serotonin and dopamine assays performed while taking serotonin and dopamine amino acid precursors at significantly varied dosing values . the results are then compared in order to determine the change in urinary serotonin and dopamine levels in response to the change in amino acid precursor dosing values.37 urinary serotonin and dopamine values found on assay were reported in micrograms of monoamine per gram of creatinine in order to compensate for fluctuations in urinary - specific gravity . the urine samples were collected after a minimum of 1 week , during which the patient was taking a specific daily dosing of amino acid precursors of serotonin and dopamine . , the patient was still taking mesalamine , low - dose naltrexone , and escitalopram . at the first visit , the patient was started on level 1 amino acid dosing as per table 1 . at that point , the patient revealed that he felt that this approach was the best treatment he had experienced during the course of his 22-year illness . when the first urine sample was collected for oct assay interpretation , the patient was taking level 3 dosing : 900 mg 5-hydroxytryptophan ( 5-htp ) , 5000 mg l - tyrosine , and 4500 mg l - cysteine with cofactors . after the first oct assay interpretation , the patient s daily amino acid dosing was increased by 1000 mg of l - tyrosine and 240 mg of l - dopa . at that point , the patient was taking the following in divided daily doses : 900 mg 5-htp , 6000 mg l - tyrosine , 240 mg l - dopa , and 4500 mg l - cysteine with cofactors . the patient was then taking the following in divided daily doses : 600 mg 5-htp , 7000 mg l - tyrosine , 240 mg l - dopa , and 4500 mg l - cysteine with cofactors.5 within 1 week of this dosing value change , the patient became asymptomatic , indicating that adequate oct balance of the serotonin the patient s response and remission with amino acid treatment was very impressive and relatively abrupt compared with the 22-year course of his disease . the gastroenterologist reported that for the first time in 10 years of caring for the patient , the crohn s disease was in complete remission . the hypothesis is that performing oct assay interpretation on one set of octs will give insight into transport of serotonin , dopamine , and their precursors at other octs throughout the body . within 35 days of starting or changing amino acid precursor dosing values , serotonin , dopamine , and their precursors reach equilibrium throughout the body.3,57 at equilibrium , amino acid precursors , serotonin , and dopamine exert similar effects at cation transporters throughout the body . in the competitive inhibition state , the serotonin and dopamine systems function as one system in transport , synthesis , and metabolism . the same enzyme , the l - aromatic amino acid decarboxylase enzyme ( aaad ) , is responsible for synthesis of serotonin and dopamine . a significant increase in levels of one system will increase mao activity , leading to increased metabolism and depletion of the other system.2,5,6 in the intestinal tract of crohn s patients there is excessive synthesis with associated increased tissue levels of serotonin.8,9 in crohn s disease , high levels of serotonin dominate synthesis , metabolism , and transport , leading to dopamine and catecholamine levels that are low relative to the balance needed to function properly with the serotonin levels present.3,5 as noted in previous peer - reviewed research by the authors , oct phase determination defines the status of the serotonin and dopamine gates at the entrance to the basolateral monoamine oct ( open or partially closed ) of the proximal convoluted renal tubule cells of the kidneys and the status of serotonin and dopamine saturation in these transporters ( see table 2).5 proper interpretation of the findings requires the following explanation . serotonin and dopamine both need to be in the competitive inhibition state when oct assay interpretation is performed . when in the competitive inhibition state , serotonin and dopamine are in full competition for transport , synthesis , and metabolism.3,5 testing of the urine is only done after amino acid precursors of the monoamines are started in accordance with the protocol , placing the serotonin baseline testing in the endogenous state prior to administration of amino acid precursors is of no value , as these assay levels correlate with nothing . as noted in previous peer - reviewed literature , baseline testing of urinary serotonin and dopamine does not correlate with baseline assays performed on subsequent days in the same individual.6 simply giving the patient one or more amino acid precursors is not the key to optimal outcomes . the oct needs to be challenged with serotonin and dopamine precursors in significant amounts to place transport in the competitive inhibition state so that proper oct assay interpretation can be realized.5 there is a known genetic defect of octn1 and octn2 in the colon of patients suffering from crohn s diease.9 all oct and octn transporters are capable of transporting organic cations , including serotonin , dopamine , and their precursors.8 in crohn s disease , the serotonin content of the mucosa and submucosa of the proximal and distal colon is increased.10 increased synthesis of serotonin is known to be associated with crohn s disease.11 no reasonable explanation of the etiology of serotonin elevation in the colon tissue of crohn s disease patients has been put forth previously . it is postulated that the known octn1 and octn2 genetic deficit may be tied to the increased synthesis and tissue levels of serotonin seen with crohn s disease . based on oct assay interpretation , it appears that a severe imbalance between serotonin and dopamine transport , synthesis , and metabolism is at the heart of crohn s disease . dopamine transport system has been linked to numerous diseases.3,57 it is proposed that much of the clinical constellation found with crohn s disease may be induced by a serotonin toxicity of the colon exacerbated by relatively low levels of dopamine resulting from defective octn transport . the serotonin autocrine and/or endocrine mediators that modulate gi function.12 it is asserted that proper treatment needs to include correct management of the serotonin and dopamine imbalance in transport , synthesis , and metabolism . it is postulated that the patient s crohn s disease was impacted in a positive manner as follows . it is known that there is increased synthesis of serotonin with increased serotonin levels in the proximal and distal colon.11 levels of the serotonin dopamine system are impacted primarily by synthesis , uptake , and metabolism . for serotonin and dopamine to be synthesized , their amino acid precursors need to be transported into the structures where this occurs . serotonin precursors are transported preferentially at the exclusion of dopamine precursors , leading to high levels of synthesis , high levels of serotonin in portions of the colon , and compromise of catecholamine synthesis . properly balancing the serotonin and dopamine precursor transport leads to a decrease in serotonin synthesis , less serotonin in the tissue of the proximal and distal colon , and an increase in synthesis of dopamine , norepinephrine , and epinephrine . with a case study such as this this patient had a 22-year history of progressively worsening crohn s with no remissions and has been free of crohn s disease symptoms clinically and on biopsy for 2.5 years since the appropriate dosing values of serotonin and dopamine amino acids were established . previously published peer - reviewed literature by the authors indicates that this same approach with oct assay interpretation for treatment of depression is effective.3,7 in this case study , the patient s depression resolved when the serotonin and dopamine were balanced to the degree needed for relief of crohn s disease symptoms . it is asserted that it was no coincidence that the patient s depression resolved simultaneously with the resolution of the symptoms of crohn s disease . the authors have documented a number of patients with crohn s disease who experienced similar remission of symptoms with this approach . this case was selected for this paper due to the severity of disease in the patient . serotonin and dopamine levels inside and outside of the cell structures containing them are primarily a function of transporter status.5 the question raised is how oct assay interpretation of renal transporters relates to the octs of the gastrointestinal ( gi ) tract . the hypothesis is that performing oct assay interpretation on one set of octs will give insight into transport of serotonin , dopamine , and their precursors at other octs throughout the body . within 35 days of starting or changing amino acid precursor dosing values , serotonin , dopamine , and their precursors reach equilibrium throughout the body.3,57 at equilibrium , amino acid precursors , serotonin , and dopamine exert similar effects at cation transporters throughout the body . in the competitive inhibition state , the serotonin and dopamine systems function as one system in transport , synthesis , and metabolism . the same enzyme , the l - aromatic amino acid decarboxylase enzyme ( aaad ) , is responsible for synthesis of serotonin and dopamine . a significant increase in levels of one system will increase mao activity , leading to increased metabolism and depletion of the other system.2,5,6 in the intestinal tract of crohn s patients there is excessive synthesis with associated increased tissue levels of serotonin.8,9 in crohn s disease , high levels of serotonin dominate synthesis , metabolism , and transport , leading to dopamine and catecholamine levels that are low relative to the balance needed to function properly with the serotonin levels present.3,5 as noted in previous peer - reviewed research by the authors , oct phase determination defines the status of the serotonin and dopamine gates at the entrance to the basolateral monoamine oct ( open or partially closed ) of the proximal convoluted renal tubule cells of the kidneys and the status of serotonin and dopamine saturation in these transporters ( see table 2).5 proper interpretation of the findings requires the following explanation . serotonin and dopamine both need to be in the competitive inhibition state when oct assay interpretation is performed . when in the competitive inhibition state , serotonin and dopamine are in full competition for transport , synthesis , and metabolism.3,5 testing of the urine is only done after amino acid precursors of the monoamines are started in accordance with the protocol , placing the serotonin baseline testing in the endogenous state prior to administration of amino acid precursors is of no value , as these assay levels correlate with nothing . as noted in previous peer - reviewed literature , baseline testing of urinary serotonin and dopamine does not correlate with baseline assays performed on subsequent days in the same individual.6 simply giving the patient one or more amino acid precursors is not the key to optimal outcomes . the oct needs to be challenged with serotonin and dopamine precursors in significant amounts to place transport in the competitive inhibition state so that proper oct assay interpretation can be realized.5 there is a known genetic defect of octn1 and octn2 in the colon of patients suffering from crohn s diease.9 all oct and octn transporters are capable of transporting organic cations , including serotonin , dopamine , and their precursors.8 in crohn s disease , the serotonin content of the mucosa and submucosa of the proximal and distal colon is increased.10 increased synthesis of serotonin is known to be associated with crohn s disease.11 no reasonable explanation of the etiology of serotonin elevation in the colon tissue of crohn s disease patients has been put forth previously . it is postulated that the known octn1 and octn2 genetic deficit may be tied to the increased synthesis and tissue levels of serotonin seen with crohn s disease . based on oct assay interpretation , it appears that a severe imbalance between serotonin and dopamine transport , synthesis , and metabolism is at the heart of crohn s disease . dopamine transport system has been linked to numerous diseases.3,57 it is proposed that much of the clinical constellation found with crohn s disease may be induced by a serotonin toxicity of the colon exacerbated by relatively low levels of dopamine resulting from defective octn transport . the serotonin autocrine and/or endocrine mediators that modulate gi function.12 it is asserted that proper treatment needs to include correct management of the serotonin and dopamine imbalance in transport , synthesis , and metabolism . it is known that there is increased synthesis of serotonin with increased serotonin levels in the proximal and distal colon.11 levels of the serotonin dopamine system are impacted primarily by synthesis , uptake , and metabolism . for serotonin and dopamine to be synthesized , their amino acid precursors need to be transported into the structures where this occurs . serotonin precursors are transported preferentially at the exclusion of dopamine precursors , leading to high levels of synthesis , high levels of serotonin in portions of the colon , and compromise of catecholamine synthesis . properly balancing the serotonin and dopamine precursor transport leads to a decrease in serotonin synthesis , less serotonin in the tissue of the proximal and distal colon , and an increase in synthesis of dopamine , norepinephrine , and epinephrine . increased serotonin levels of crohn s disease lead to increased mao activity , which without reciprocal increases of the catecholamines leads to increased metabolism of the catecholamines , further exacerbating the imbalance . with a case study such as this there is always the possibility that remission was coincidental to treatment . this patient had a 22-year history of progressively worsening crohn s with no remissions and has been free of crohn s disease symptoms clinically and on biopsy for 2.5 years since the appropriate dosing values of serotonin and dopamine amino acids were established . previously published peer - reviewed literature by the authors indicates that this same approach with oct assay interpretation for treatment of depression is effective.3,7 in this case study , the patient s depression resolved when the serotonin and dopamine were balanced to the degree needed for relief of crohn s disease symptoms . it is asserted that it was no coincidence that the patient s depression resolved simultaneously with the resolution of the symptoms of crohn s disease . the octn1 and octn2 are responsible for transport of cations , including the monoamines of the serotonin it is known with crohn s disease patients that there is a marked increased in serotonin levels of the proximal and distal colon associated with a defect in serotonin synthesis .

the human gene mutation database ( hgmd ) represents an attempt to collate all known gene lesions responsible for causing human inherited disease together with disease - associated / functional polymorphisms that have been published in the peer - reviewed literature . these data comprise single base - pair substitutions in coding , regulatory and splicing - relevant ( both intronic and exonic ) regions of human nuclear genes , as well as micro - deletions and micro - insertions , combined micro - insertions / micro - deletions ( indels ) of 20 bp or less , repeat variations , gross lesions ( deletions , insertions and duplications of greater than 20 bp , up to and including a single characterized gene or group of contiguous genes that are directly involved in the aetiology of the disease / phenotype ) and complex rearrangements ( including inversions , translocations and complex indels ) . mutation data are summarized in table 1.table 1numbers of different mutations by mutation type present in hgmd professional 2013.2 and the publicly available version of the database ( june 28th 2013)mutation typetotal numbers of mutationshgmd professionalwith chromosomal coordinatespublicly availablemissense substitutions62,36861,84544,933nonsense substitutions15,78115,57411,306splicing substitutions13,03012,5389,467regulatory substitutions2,7512,7131,753micro - deletions 20 bp21,68121,13415,796micro - insertions 20 bp2,0832,0041,459gross deletions > 20 bp10,26706,156gross insertions / duplications > 20 bp2,37601,253complex rearrangements1,4090946repeat variations4210305totals141,161124,52999,868 numbers of different mutations by mutation type present in hgmd professional 2013.2 and the publicly available version of the database ( june 28th 2013 ) hgmd does not include either somatic or mitochondrial mutations , which are well covered by cosmic ( forbes et al . 2011 ) and mitomap ( ruiz - pesini et al . hgmd also does not attempt to provide comprehensive coverage of pharmacological variants ( except for those variants where evidence supporting a functional impairment has been provided ) ; such variants are covered by pharmgkb ( thorn et al . 2010 ) . finally , hgmd is not a general genetic variation database ; users interested in this type of variant should visit dbsnp ( sherry et al . hgmd was originally established for the scientific study of mutational mechanisms in human genes causing inherited disease ( cooper et al . 2010 ) , but has since acquired a much broader utility as a central unified repository for germ - line disease - related functional variation . it is now routinely accessed and utilized by next generation sequencing ( ngs ) project researchers , human molecular geneticists , molecular biologists , clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals , bioinformatics and personalized genomics . the public version of hgmd ( http://www.hgmd.org ) is freely available to registered users from academic institutions / non - profit organizations . this version is , however , maintained in a basic form that is only updated twice per annum , is permanently 3 years out of date , and does not contain any of the additional annotations or extra features present in hgmd professional ( such as grch37/hg19 genomic chromosomal coordinates , hgvs nomenclature and additional literature references , see table 2 ) . the professional version is available to both commercial and academic / non - profit users via subscription from biobase gmbh ( http://www.biobase-international.com).table 2differences between hgmd professional and hgmd publichgmd professionalhgmd publicup - to - date mutation datacurator commentsquarterly updates gene - oriented searchmutation - oriented searchreference - oriented searchbatch search modechromosomal coordinateshgvs nomenclatureadditional literature referencestracked variant historydbsnp identifiersenhanced search optionsadvanced search featuresdisease ontology terms*data in vcf*downloadable version hgmd public is updated on a 6-monthly basis * download customers only differences between hgmd professional and hgmd public hgmd public is updated on a 6-monthly basis * download customers only identification of relevant literature reports is carried out via a combination of manual journal screening and automated procedures . the database currently contains mutation entries obtained from over 41,000 primary and 15,000 additional ( supplementary ) literature reports published in more than 1,950 different journals . of > 10,000 identified articles screened for mutation data during 2012 , 35 % contained novel mutation data , 29 % contained additional useful information ( e.g. in vitro functional data or further clinical or phenotypic information ) and were therefore cited as additional references , whilst the remaining 36 % of articles contained no novel mutation data or supporting information to warrant their inclusion as either primary or supplementary references in hgmd . the number of articles screened by hgmd is increasing on a yearly basis ; however , we impose no prior limit upon the number of articles we include as supplementary references for a given mutation . for ~4 % of all the missense / nonsense mutations reported in the literature during 2012 , it was necessary for the hgmd curators to contact the original authors to obtain correction and/or clarification of the nature or precise location of the mutations in question . however , only half of the mutations that required author contact were satisfactorily resolved by these means , leading to their inclusion in hgmd ; the ~2 % of unresolved missense / nonsense mutations will not be entered into hgmd unless or until the nature or precise location of the mutation(s ) in question is determined to the satisfaction of the hgmd curators . such data ( currently 366 entries ) are , however , retained indefinitely by hgmd as part of a bad bank of inadequately described mutations . there are six different classes of variant present in hgmd ( figs . 1 , 2 ) . disease - causing mutations ( dm ) are entered into hgmd where the authors of the corresponding report(s ) have demonstrated that the reported mutation(s ) are involved in conferring the associated clinical phenotype upon the individuals concerned . the dm classification may , however , also appear with a question mark ( dm ? ) , denoting a probable / possible pathological mutation , reported to be pathogenic in the corresponding report , but where ( 1 ) the author has indicated that there may be some degree of uncertainty ; ( 2 ) the hgmd curators believe greater interpretational caution is warranted ; or ( 3 ) subsequent evidence has appeared in the literature which has called the putatively deleterious nature of the variant into question.fig . 15,734 genes are listed in hgmd professional 2013.2 , subdivided here by variant classfig . 2013 figures to june 28th 5,734 genes are listed in hgmd professional 2013.2 , subdivided here by variant class hgmd annual mutation totals subdivided by variant class . * 2013 figures to june 28th disease - associated polymorphisms ( dp ) are entered into hgmd where there is evidence for a significant association with a disease / clinical phenotype along with additional evidence that the polymorphism is itself likely to be of functional relevance ( e.g. as a consequence of genic / genomic location , evolutionary conservation , transcription factor binding potential , etc . ) , although there may be no direct evidence ( e.g. from an expression study ) of a functional effect . functional polymorphisms ( fp ) are included in hgmd where the reporting authors have shown that the polymorphism in question exerts a direct functional effect ( e.g. by means of an in vitro reporter gene assay or alternatively by protein structure , function or expression studies ) , but with no disease association reported as yet . disease - associated polymorphisms with supporting functional evidence ( dfp ) must meet both of the above criteria in that the polymorphism should not only be reported to be significantly associated with disease but should also display evidence of being of direct functional relevance . copy number variations ( cnvs ) represent an important subset of potentially functional disease - associated variation . while hgmd does not wish to replicate the excellent curatorial work of other resources ( e.g. the database of genomic variants http://dgv.tcag.ca/dgv/app/home , decipher http://decipher.sanger.ac.uk/ and copy number variation in disease http://202.97.205.78/cnvd/ ) , we are nevertheless interested in including such variants ( as gross deletions or duplications ) if they meet certain criteria . therefore , hgmd will include such variations if they have been shown to be both of functional significance and associated with disease , and involve a single characterized gene that has itself been directly implicated in the disease association . such variants would be entered under one of the above - mentioned polymorphism categories , depending upon the supporting evidence provided by the authors of the original reporting article . in the opinion of the hgmd curators , the polymorphism data present in hgmd should be viewed with a considerable degree of caution owing to ( 1 ) the possibility that the observed disease association may be simply due to a linkage disequilibrium effect rather than a bona fide underlying functional mechanism and ( 2 ) the fact that in vitro studies are not invariably accurate indicators of in vivo functionality ( cirulli and goldstein 2007 ; dimas et al . finally , frameshift or truncating variants ( ftv ) are polymorphic or rare variants reported in the literature that are predicted to truncate or otherwise alter the length of the gene product ( i.e. a stop - gain , stop - loss or frameshift variant ) but with no disease association reported as yet . most known ftvs have been identified during the course of large - scale genome / exome screening studies ( involving either patient panels or apparently healthy individuals from the general population ) . they may be considered to represent either latent protein deficiencies or , potentially , heterozygous carrier states for recessive disorders . coverage of ftvs is far from being comprehensive at this juncture , and it remains unclear what proportion will turn out to be clinically significant . the hgmd curators have adopted a policy of continual reassessment of the curated content within the database . if and when additional and important new information pertaining to a specific mutation entry becomes available ( e.g. questionable pathogenicity , confirmed pathogenicity , additional clinical or laboratory phenotypes , population frequency data , supporting functional studies , etc . ) , then the mutation entry may be revised or even re - categorized . alternatively , a comment or additional reference may be added in order to communicate this new information to users . where new information becomes available which suggests that a given disease - causing mutation ( dm ) is likely to be of questionable pathological relevance or possibly a neutral polymorphism ( on the basis of additional case reports , genome / population screening studies , presence in dbsnp with reliable population frequency data , etc . ) , it may be flagged with a question mark ( dm ? ) or even removed from the database entirely if it turns out to have been erroneously included ab initio . in a recent re - curation exercise , a total of 539 mutations were re - examined due to their presence in the 1000 genomes project dataset at a frequency of > 1 % . of the total re - examined , 33 mutations were removed from hgmd , 109 were re - categorized and 220 had additional comments or references added to further justify their inclusion in hgmd ( xue et al . one reason why some hgmd - listed mutations are often to be found among 1000 genomes project data is that many pathogenic lesions are found quite frequently in the population at large ( nishiguchi and rivolta 2012 ; andreasen et al . in addition to internal curation , users of hgmd professional may utilize a feedback function in order to inform the hgmd curators of relevant new or missing information , to request corrections or to ask for the reclassification or removal of a listed variant . most of the clinical phenotypes attributed to dms in hgmd represent individually rare conditions that are generally regarded as monogenic diseases . however , it is important to note that hgmd also considers a few silent protein deficiencies or biochemical phenotypes ( e.g. butrylcholinesterase deficiency , reduced oxygen affinity haemoglobin , etc . ) to be worthy of inclusion since they are potentially disease - relevant ( even if they are relatively common in the general population ) ; such variants may well be assigned to the dm category . for individual mutations in hgmd , the provision of zygosity information ( heterozygous , homozygous or compound heterozygous ) has not been attempted . reasons for this include ( 1 ) the fact that this information is not always unambiguously provided in the corresponding article ; ( 2 ) the possibility that a given mutation may be pathogenic irrespective of the zygosity in which it is found ; ( 3 ) the clinical consequences of zygosity may often be modified by other genetic variants either in cis or in trans and ( 4 ) the general phenomenon of variable or reduced penetrance which ensures that the genotype is not invariably predictive of the phenotype ( cooper et al . thus , information pertaining to zygosity would not always be helpful or informative with regard to ascertaining or predicting the clinical phenotype , and indeed might even prove inaccurate or misleading . hgmd users should not assume that just because a mutation is labelled dm , that it automatically follows that the mutation is known or believed to be pathogenic in all individuals harbouring it ( i.e. that the mutation exhibits 100 % penetrance ) . this is not invariably going to be the case and many disease - causing mutations will display reduced or variable penetrance for a variety of different reasons ( reviewed by cooper et al . next generation sequencing programmes ( such as the 1000 genomes project ) are now identifying considerable numbers of dm mutations in apparently healthy individuals ( macarthur et al . such lesions should not automatically be regarded as being clinically irrelevant because it is quite possible that they represent low - penetrance , mild or late onset , or more complex disease susceptibility alleles , as opposed to neutral variants ( cooper et al . it has always been hgmd policy to enter a variant into the database even if its pathological relevance may be questionable ( while indicating this fact wherever feasible to our users ) , rather than run the risk of inadvertently excluding a variant that may be directly ( or indirectly ) relevant to disease . variant class , a dbsnp 1000 genomes frequency flag ( to highlight hgmd variants that are also present in dbsnp , with allele frequency information included ; see below ) and the provision of additional literature citations where the pathogenicity of the variant may have been subsequently either questioned or confirmed . this latter point is particularly pertinent in the clinical setting , where a greater burden of proof may be required for use in diagnostic and predictive medicine , and when considering the return of incidental findings to patients after testing ( green et al . hgmd professional has been developed to serve as the subscription version of hgmd , and is available to both commercial and academic customers under license from biobase gmbh . hgmd professional allows access to up - to - date mutation data with a quarterly release cycle ; this version is therefore essential for checking the novelty of newly found mutations . hgmd professional contains many features not available in the free public version ( table 2 ) . more powerful search tools in the form of an expanded search engine with full text boolean searching are provided . a batch search mode has recently been developed to allow users to search hgmd using gene ( e.g. omim ids ) and variant ( e.g. dbsnp ids ) oriented lists . users can employ these tools to perform additional searches for gene - specific ( e.g. chromosomal locations , gene names / aliases and gene ontology ) , mutation - specific ( e.g. chromosomal coordinates , hgvs nomenclature , dbsnp i d ) or citation - specific ( e.g. first author , publication year , pubmed i d ) information . the provision of chromosomal coordinates ( hg19 ) for the vast majority of our nucleotide substitutions ( 98.7 % coverage ) and other micro - lesions ( 97.3 % coverage ) has made hgmd an invaluable tool for the large - scale analysis of ngs datasets such as the 1000 genomes project ( 1000 genomes project consortium 2010 , 2012 ) . additional information is also provided on a mutation - specific basis including curatorial comments pertaining to particular mutations ( for example , if the mutation data presented required in - house correction in relation to the data presented in the original publication [ 510 % of entries ] , or if the clinical phenotype is associated with a more complex , i.e. a digenic or snp in - cis inheritance pattern ) , additional reports comprising functional characterisation , further phenotypic information , comparative biochemical parameters , evolutionary conservation and sift ( sim et al . recently , hgmd clinical phenotypes have been annotated against the unified medical language system ( umls ) using a combination of manual curation and natural language processing . the umls is a comprehensive collection of biomedical concepts and the relationships between them ( http://www.nlm.nih.gov/research/umls/ ) . thus , searches using alternative disease names will return the same result - set , e.g. a search for breast cancer would yield identical results to a search for malignant breast tumour . in addition , utilizing the umls allows for powerful semantic searching ( e.g. searches for all mutations linked to blood disorders or all immune disorders ) . another new feature involves the highlighting of hgmd entries where the pathogenicity of the variant may have been cast into doubt by virtue of its allele frequency . hgmd professional now displays a frequency flag when a listed variant is also found in dbsnp , and population frequency data from the 1000 genomes project are also provided . hgmd data have also recently been made available in variant call format or vcf ( danecek et al . in addition to searching and viewing mutation data in a variety of ways , users of hgmd professional may utilize a new feedback facility to submit corrections to the database curators or to request additional features . hgmd professional also contains an advanced search suite which has been designed to enhance mutation searching , viewing and retrieval . two of the main types of mutation in hgmd ( single - nucleotide substitutions and micro - lesions ) can be interrogated with this toolset . datasets for more than one mutation type may be combined ( for example , micro - deletions , micro - insertions and indels ) to enable more powerful searching across comparable types of mutation . when using the advanced search , users can tailor their queries with more specific criteria , including functional profile ( e.g. in vitro and in silico characterized transcription factor binding sites , post - translational modifications , microrna binding sites , upstream orfs , and catalytic residues , see fig . 3 ) ; amino - acid change ; nucleotide substitution ; size and/or sequence composition of micro - deletions , micro - insertions or indels ; pre- or user - defined sequence motifs ( both those created and those abolished by the mutation ) ; dbsnp number ; keywords found in the article title or abstract . results returned by the advanced search can be downloaded as tab - delimited text or a genome browser track , ready to be used in different applications . the advanced search also includes a batch mode called mutation mart to query hgmd via multiple identifiers including dbsnp , entrez gene ( http://www.ncbi.nlm.nih.gov/gene ) and pubmed . hgmd professional is available to subscribers either as an online only package or in downloadable form enabling users to incorporate hgmd data into their local variant analysis pipelines ( http://www.biobase-international.com).fig . 3advanced nucleotide substitutions search in hgmd professional advanced nucleotide substitutions search in hgmd professional several other databases are available that attempt to record disease - causing or disease - associated ( i.e. pathogenic ) variation . these include the online mendelian inheritance in man , omim ( http://www.omim.org/ ; amberger et al . 2009 ) , clinvar ( http://www.ncbi.nlm.nih.gov/clinvar/ ) , dbsnp ( http://www.ncbi.nlm.nih.gov/snp/ ; sherry et al . 2001 ) and an assorted collection of locus - specific mutation databases ( lsdbs ) ( http://www.hgvs.org/dblist/glsdb.html ) . omim does not provide statistics for allelic variants on its website ; however , 22,901 germline omim variants appear to have been added to clinvar , which itself contains a total of 25,375 pathogenic and probable pathogenic germline variants , while dbsnp contains 23,973 pathogenic or probable pathogenic germline variants ( all databases were accessed july 10th 2013 ) . owing to the highly dispersed nature of the lsdbs and the potential for duplication between databases , accurate statistics with regard to like - for - like bona fide germline disease - causing ( not merely neutral ) variation is difficult to obtain . since omim only records a limited number of variants per gene , and clinvar is still in its infancy , hgmd is the only database of human pathological mutations that approaches comprehensive coverage of the peer - reviewed literature ( peterson et al . contain unpublished ( non - peer reviewed ) mutation data , the question has arisen as to whether hgmd should also include these data ( patrinos et al . however , several obstacles have been encountered by the lsdbs , including serious problems pertaining to data quality as well as issues of data provenance and consent . however , mutation data may be made available at the discretion of the curators for non - commercial research purposes . potential collaborators who wish to access hgmd data in full are required to sign a confidentiality agreement . hgmd data have been used to perform an extensive series of meta - analyses on different types of gene mutation causing human inherited disease . these studies have helped to improve our understanding of mutational spectra and the molecular mechanisms underlying human inherited disease ( cooper et al . they have served to demonstrate not only that human gene mutation is an inherently non - random process but also that the nature , location and frequency of different types of mutation are shaped in large part by the local dna sequence environment ( cooper et al . hgmd data have been used extensively in several international collaborative research projects including the 1000 genomes project ( 1000 genomes project 2010 , 2012 ) , where a surprising number of hgmd variants were found in apparently healthy individuals . they have also been used in the comparative analysis of several orthologous genomes including gorilla ( scally et al . 2012 ) , cynomolgus and chinese macaque ( yan et al . 2011 ) , rhesus macaque ( rhesus macaque genome sequencing and analysis consortium 2007 ) and rat ( rat genome sequencing project consortium 2004 ) , in which many apparently disease - causing mutations in human were found as wild type ( compensated mutations ) . in a clinical setting , hgmd is widely utilized by many groups in ongoing ngs diagnostic ( johnston et al . 2012 , bell et al . 2011 ) and human genome sequencing ( tong et al . 2010 ; kim et al . 2009 ) programmes . hgmd has also been used by a number of different groups to aid the development of post - ngs variant interpretation algorithms including mutpred ( li et al . 2009 ) , provean ( choi et al . 2012 ) , carol ( lopes et al . 2012 ) , cravat ( douville et al . 2013 ) , nest ( carter et al . 2013 ) and fathmm ( shihab et al . 2013 ) . finally , hgmd has been used as a resource for structural biologists in the reconstruction of protein interaction networks ( wang et al . 2013 ) . a more complete list of articles which have utilized hgmd data or expertise in their production can be found on the hgmd website ( http://www.hgmd.cf.ac.uk/docs/articles.html ) . a limited hgmd data set , containing both chromosomal coordinates and hgmd identifiers , has been made available via academic data exchange programmes to the gen2phen project ( webb et al . 2011 ) , the european bioinformatics institute ( ebi)/ensembl ( flicek et al . 2013 ) and the university of california , santa cruz ( ucsc ) ( meyer et al . 2013 ) and may be viewed in these projects respective genome browsers . data from hgmd professional have additionally been made available to hgmd subscribers via genome trax ( biobase gmbh ) and alamut ( interactive biosoftware ) , but are also accessible as part of the hgmd professional stand - alone package ( biobase gmbh ) . allowing free access to the bulk of the mutation data present in hgmd , while generating sufficient income from its commercial distribution to support its maintenance and expansion , represents a business model that should maximize the availability of hgmd at the same time as ensuring its long - term sustainability . although we are necessarily obliged to be prudent with regard to data sharing with public data repositories , we have always taken the view that making as much data as possible publicly available is generally beneficial to both hgmd and its users worldwide . the provision of chromosomal coordinates for the vast majority of coding region micro - lesions in hgmd is now complete . expanding this provision to include micro - lesions in non - coding regions and the gross and complex lesion dataset ( where feasible ) is a high priority , as is expanding the provision of genomic coordinates to include popularly utilized ngs formats such as general feature format ( gff ) ( http://www.sanger.ac.uk/resources/software/gff/ ) and bed format , to complement the recently added hgmd variant call format ( vcf ) ( danecek et al . mutations will also be mapped to the new genome build ( grch38 ) in due course . provision of genomic reference sequences based on the ncbi refseqgene project ( pruitt et al . 2009 ) , links to available protein structures and homology models , and mapping hgmd phenotypes to the human phenotype ontology ( hpo ) are also regarded as priorities . in its current state of development , hgmd provides the user with a unique resource that can be utilized not only to obtain evidence to support the pathological authenticity and/or novelty of detected gene lesions and to acquire an overview of the mutational spectra for specific genes , but also as a knowledgebase for use in the bioinformatics and whole genome screening projects that underpin personalized genomics .

the human gene mutation database ( hgmd ) is a comprehensive collection of germline mutations in nuclear genes that underlie , or are associated with , human inherited disease . by june 2013 , the database contained over 141,000 different lesions detected in over 5,700 different genes , with new mutation entries currently accumulating at a rate exceeding 10,000 per annum . hgmd was originally established in 1996 for the scientific study of mutational mechanisms in human genes . however , it has since acquired a much broader utility as a central unified disease - oriented mutation repository utilized by human molecular geneticists , genome scientists , molecular biologists , clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals , bioinformatics and personalized genomics . the public version of hgmd ( http://www.hgmd.org ) is freely available to registered users from academic institutions / non - profit organizations whilst the subscription version ( hgmd professional ) is available to academic , clinical and commercial users under license via biobase gmbh .

Introduction Acquisition of mutation data Classes of variant listed in HGMD HGMD Professional Other variant databases How HGMD is utilized Data sharing Future plans

the human gene mutation database ( hgmd ) represents an attempt to collate all known gene lesions responsible for causing human inherited disease together with disease - associated / functional polymorphisms that have been published in the peer - reviewed literature . these data comprise single base - pair substitutions in coding , regulatory and splicing - relevant ( both intronic and exonic ) regions of human nuclear genes , as well as micro - deletions and micro - insertions , combined micro - insertions / micro - deletions ( indels ) of 20 bp or less , repeat variations , gross lesions ( deletions , insertions and duplications of greater than 20 bp , up to and including a single characterized gene or group of contiguous genes that are directly involved in the aetiology of the disease / phenotype ) and complex rearrangements ( including inversions , translocations and complex indels ) . mutation data are summarized in table 1.table 1numbers of different mutations by mutation type present in hgmd professional 2013.2 and the publicly available version of the database ( june 28th 2013)mutation typetotal numbers of mutationshgmd professionalwith chromosomal coordinatespublicly availablemissense substitutions62,36861,84544,933nonsense substitutions15,78115,57411,306splicing substitutions13,03012,5389,467regulatory substitutions2,7512,7131,753micro - deletions 20 bp21,68121,13415,796micro - insertions 20 bp2,0832,0041,459gross deletions > 20 bp10,26706,156gross insertions / duplications > 20 bp2,37601,253complex rearrangements1,4090946repeat variations4210305totals141,161124,52999,868 numbers of different mutations by mutation type present in hgmd professional 2013.2 and the publicly available version of the database ( june 28th 2013 ) hgmd does not include either somatic or mitochondrial mutations , which are well covered by cosmic ( forbes et al . 2011 ) and mitomap ( ruiz - pesini et al . hgmd was originally established for the scientific study of mutational mechanisms in human genes causing inherited disease ( cooper et al . 2010 ) , but has since acquired a much broader utility as a central unified repository for germ - line disease - related functional variation . it is now routinely accessed and utilized by next generation sequencing ( ngs ) project researchers , human molecular geneticists , molecular biologists , clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals , bioinformatics and personalized genomics . the public version of hgmd ( http://www.hgmd.org ) is freely available to registered users from academic institutions / non - profit organizations . this version is , however , maintained in a basic form that is only updated twice per annum , is permanently 3 years out of date , and does not contain any of the additional annotations or extra features present in hgmd professional ( such as grch37/hg19 genomic chromosomal coordinates , hgvs nomenclature and additional literature references , see table 2 ) . the professional version is available to both commercial and academic / non - profit users via subscription from biobase gmbh ( http://www.biobase-international.com).table 2differences between hgmd professional and hgmd publichgmd professionalhgmd publicup - to - date mutation datacurator commentsquarterly updates gene - oriented searchmutation - oriented searchreference - oriented searchbatch search modechromosomal coordinateshgvs nomenclatureadditional literature referencestracked variant historydbsnp identifiersenhanced search optionsadvanced search featuresdisease ontology terms*data in vcf*downloadable version hgmd public is updated on a 6-monthly basis * download customers only differences between hgmd professional and hgmd public hgmd public is updated on a 6-monthly basis * download customers only identification of relevant literature reports is carried out via a combination of manual journal screening and automated procedures . the database currently contains mutation entries obtained from over 41,000 primary and 15,000 additional ( supplementary ) literature reports published in more than 1,950 different journals . in vitro functional data or further clinical or phenotypic information ) and were therefore cited as additional references , whilst the remaining 36 % of articles contained no novel mutation data or supporting information to warrant their inclusion as either primary or supplementary references in hgmd . the number of articles screened by hgmd is increasing on a yearly basis ; however , we impose no prior limit upon the number of articles we include as supplementary references for a given mutation . for ~4 % of all the missense / nonsense mutations reported in the literature during 2012 , it was necessary for the hgmd curators to contact the original authors to obtain correction and/or clarification of the nature or precise location of the mutations in question . however , only half of the mutations that required author contact were satisfactorily resolved by these means , leading to their inclusion in hgmd ; the ~2 % of unresolved missense / nonsense mutations will not be entered into hgmd unless or until the nature or precise location of the mutation(s ) in question is determined to the satisfaction of the hgmd curators . such data ( currently 366 entries ) are , however , retained indefinitely by hgmd as part of a bad bank of inadequately described mutations . there are six different classes of variant present in hgmd ( figs . 1 , 2 ) . disease - causing mutations ( dm ) are entered into hgmd where the authors of the corresponding report(s ) have demonstrated that the reported mutation(s ) are involved in conferring the associated clinical phenotype upon the individuals concerned . the dm classification may , however , also appear with a question mark ( dm ? ) 15,734 genes are listed in hgmd professional 2013.2 , subdivided here by variant classfig . 2013 figures to june 28th 5,734 genes are listed in hgmd professional 2013.2 , subdivided here by variant class hgmd annual mutation totals subdivided by variant class . * 2013 figures to june 28th disease - associated polymorphisms ( dp ) are entered into hgmd where there is evidence for a significant association with a disease / clinical phenotype along with additional evidence that the polymorphism is itself likely to be of functional relevance ( e.g. as a consequence of genic / genomic location , evolutionary conservation , transcription factor binding potential , etc . ) disease - associated polymorphisms with supporting functional evidence ( dfp ) must meet both of the above criteria in that the polymorphism should not only be reported to be significantly associated with disease but should also display evidence of being of direct functional relevance . copy number variations ( cnvs ) represent an important subset of potentially functional disease - associated variation . while hgmd does not wish to replicate the excellent curatorial work of other resources ( e.g. the database of genomic variants http://dgv.tcag.ca/dgv/app/home , decipher http://decipher.sanger.ac.uk/ and copy number variation in disease http://202.97.205.78/cnvd/ ) , we are nevertheless interested in including such variants ( as gross deletions or duplications ) if they meet certain criteria . therefore , hgmd will include such variations if they have been shown to be both of functional significance and associated with disease , and involve a single characterized gene that has itself been directly implicated in the disease association . in the opinion of the hgmd curators , the polymorphism data present in hgmd should be viewed with a considerable degree of caution owing to ( 1 ) the possibility that the observed disease association may be simply due to a linkage disequilibrium effect rather than a bona fide underlying functional mechanism and ( 2 ) the fact that in vitro studies are not invariably accurate indicators of in vivo functionality ( cirulli and goldstein 2007 ; dimas et al . the hgmd curators have adopted a policy of continual reassessment of the curated content within the database . where new information becomes available which suggests that a given disease - causing mutation ( dm ) is likely to be of questionable pathological relevance or possibly a neutral polymorphism ( on the basis of additional case reports , genome / population screening studies , presence in dbsnp with reliable population frequency data , etc . ) , it may be flagged with a question mark ( dm ? ) or even removed from the database entirely if it turns out to have been erroneously included ab initio . in a recent re - curation exercise , a total of 539 mutations were re - examined due to their presence in the 1000 genomes project dataset at a frequency of > 1 % . of the total re - examined , 33 mutations were removed from hgmd , 109 were re - categorized and 220 had additional comments or references added to further justify their inclusion in hgmd ( xue et al . in addition to internal curation , users of hgmd professional may utilize a feedback function in order to inform the hgmd curators of relevant new or missing information , to request corrections or to ask for the reclassification or removal of a listed variant . however , it is important to note that hgmd also considers a few silent protein deficiencies or biochemical phenotypes ( e.g. to be worthy of inclusion since they are potentially disease - relevant ( even if they are relatively common in the general population ) ; such variants may well be assigned to the dm category . for individual mutations in hgmd , the provision of zygosity information ( heterozygous , homozygous or compound heterozygous ) has not been attempted . this is not invariably going to be the case and many disease - causing mutations will display reduced or variable penetrance for a variety of different reasons ( reviewed by cooper et al . next generation sequencing programmes ( such as the 1000 genomes project ) are now identifying considerable numbers of dm mutations in apparently healthy individuals ( macarthur et al . such lesions should not automatically be regarded as being clinically irrelevant because it is quite possible that they represent low - penetrance , mild or late onset , or more complex disease susceptibility alleles , as opposed to neutral variants ( cooper et al . it has always been hgmd policy to enter a variant into the database even if its pathological relevance may be questionable ( while indicating this fact wherever feasible to our users ) , rather than run the risk of inadvertently excluding a variant that may be directly ( or indirectly ) relevant to disease . variant class , a dbsnp 1000 genomes frequency flag ( to highlight hgmd variants that are also present in dbsnp , with allele frequency information included ; see below ) and the provision of additional literature citations where the pathogenicity of the variant may have been subsequently either questioned or confirmed . hgmd professional has been developed to serve as the subscription version of hgmd , and is available to both commercial and academic customers under license from biobase gmbh . hgmd professional allows access to up - to - date mutation data with a quarterly release cycle ; this version is therefore essential for checking the novelty of newly found mutations . hgmd professional contains many features not available in the free public version ( table 2 ) . omim ids ) and variant ( e.g. the provision of chromosomal coordinates ( hg19 ) for the vast majority of our nucleotide substitutions ( 98.7 % coverage ) and other micro - lesions ( 97.3 % coverage ) has made hgmd an invaluable tool for the large - scale analysis of ngs datasets such as the 1000 genomes project ( 1000 genomes project consortium 2010 , 2012 ) . additional information is also provided on a mutation - specific basis including curatorial comments pertaining to particular mutations ( for example , if the mutation data presented required in - house correction in relation to the data presented in the original publication [ 510 % of entries ] , or if the clinical phenotype is associated with a more complex , i.e. the umls is a comprehensive collection of biomedical concepts and the relationships between them ( http://www.nlm.nih.gov/research/umls/ ) . a search for breast cancer would yield identical results to a search for malignant breast tumour . another new feature involves the highlighting of hgmd entries where the pathogenicity of the variant may have been cast into doubt by virtue of its allele frequency . hgmd professional now displays a frequency flag when a listed variant is also found in dbsnp , and population frequency data from the 1000 genomes project are also provided . in addition to searching and viewing mutation data in a variety of ways , users of hgmd professional may utilize a new feedback facility to submit corrections to the database curators or to request additional features . hgmd professional also contains an advanced search suite which has been designed to enhance mutation searching , viewing and retrieval . two of the main types of mutation in hgmd ( single - nucleotide substitutions and micro - lesions ) can be interrogated with this toolset . the advanced search also includes a batch mode called mutation mart to query hgmd via multiple identifiers including dbsnp , entrez gene ( http://www.ncbi.nlm.nih.gov/gene ) and pubmed . hgmd professional is available to subscribers either as an online only package or in downloadable form enabling users to incorporate hgmd data into their local variant analysis pipelines ( http://www.biobase-international.com).fig . 3advanced nucleotide substitutions search in hgmd professional advanced nucleotide substitutions search in hgmd professional several other databases are available that attempt to record disease - causing or disease - associated ( i.e. these include the online mendelian inheritance in man , omim ( http://www.omim.org/ ; amberger et al . 2009 ) , clinvar ( http://www.ncbi.nlm.nih.gov/clinvar/ ) , dbsnp ( http://www.ncbi.nlm.nih.gov/snp/ ; sherry et al . 2001 ) and an assorted collection of locus - specific mutation databases ( lsdbs ) ( http://www.hgvs.org/dblist/glsdb.html ) . omim does not provide statistics for allelic variants on its website ; however , 22,901 germline omim variants appear to have been added to clinvar , which itself contains a total of 25,375 pathogenic and probable pathogenic germline variants , while dbsnp contains 23,973 pathogenic or probable pathogenic germline variants ( all databases were accessed july 10th 2013 ) . owing to the highly dispersed nature of the lsdbs and the potential for duplication between databases , accurate statistics with regard to like - for - like bona fide germline disease - causing ( not merely neutral ) variation is difficult to obtain . contain unpublished ( non - peer reviewed ) mutation data , the question has arisen as to whether hgmd should also include these data ( patrinos et al . however , several obstacles have been encountered by the lsdbs , including serious problems pertaining to data quality as well as issues of data provenance and consent . however , mutation data may be made available at the discretion of the curators for non - commercial research purposes . potential collaborators who wish to access hgmd data in full are required to sign a confidentiality agreement . hgmd data have been used to perform an extensive series of meta - analyses on different types of gene mutation causing human inherited disease . these studies have helped to improve our understanding of mutational spectra and the molecular mechanisms underlying human inherited disease ( cooper et al . they have served to demonstrate not only that human gene mutation is an inherently non - random process but also that the nature , location and frequency of different types of mutation are shaped in large part by the local dna sequence environment ( cooper et al . hgmd data have been used extensively in several international collaborative research projects including the 1000 genomes project ( 1000 genomes project 2010 , 2012 ) , where a surprising number of hgmd variants were found in apparently healthy individuals . they have also been used in the comparative analysis of several orthologous genomes including gorilla ( scally et al . 2011 ) , rhesus macaque ( rhesus macaque genome sequencing and analysis consortium 2007 ) and rat ( rat genome sequencing project consortium 2004 ) , in which many apparently disease - causing mutations in human were found as wild type ( compensated mutations ) . in a clinical setting , hgmd is widely utilized by many groups in ongoing ngs diagnostic ( johnston et al . 2013 ) , nest ( carter et al . finally , hgmd has been used as a resource for structural biologists in the reconstruction of protein interaction networks ( wang et al . a more complete list of articles which have utilized hgmd data or expertise in their production can be found on the hgmd website ( http://www.hgmd.cf.ac.uk/docs/articles.html ) . a limited hgmd data set , containing both chromosomal coordinates and hgmd identifiers , has been made available via academic data exchange programmes to the gen2phen project ( webb et al . 2011 ) , the european bioinformatics institute ( ebi)/ensembl ( flicek et al . data from hgmd professional have additionally been made available to hgmd subscribers via genome trax ( biobase gmbh ) and alamut ( interactive biosoftware ) , but are also accessible as part of the hgmd professional stand - alone package ( biobase gmbh ) . allowing free access to the bulk of the mutation data present in hgmd , while generating sufficient income from its commercial distribution to support its maintenance and expansion , represents a business model that should maximize the availability of hgmd at the same time as ensuring its long - term sustainability . the provision of chromosomal coordinates for the vast majority of coding region micro - lesions in hgmd is now complete . expanding this provision to include micro - lesions in non - coding regions and the gross and complex lesion dataset ( where feasible ) is a high priority , as is expanding the provision of genomic coordinates to include popularly utilized ngs formats such as general feature format ( gff ) ( http://www.sanger.ac.uk/resources/software/gff/ ) and bed format , to complement the recently added hgmd variant call format ( vcf ) ( danecek et al . mutations will also be mapped to the new genome build ( grch38 ) in due course . provision of genomic reference sequences based on the ncbi refseqgene project ( pruitt et al . 2009 ) , links to available protein structures and homology models , and mapping hgmd phenotypes to the human phenotype ontology ( hpo ) are also regarded as priorities . in its current state of development , hgmd provides the user with a unique resource that can be utilized not only to obtain evidence to support the pathological authenticity and/or novelty of detected gene lesions and to acquire an overview of the mutational spectra for specific genes , but also as a knowledgebase for use in the bioinformatics and whole genome screening projects that underpin personalized genomics .

this study was approved by the duke university medical center institutional review board ( durham , nc , usa ) , was conducted in compliance with the health insurance portability and accountability act ( hipaa ) and adhered to the tenets of the declaration of helsinki . subjects with ga secondary to dry amd who received faf imaging as part of their routine care between 1/1/2005 and 12/31/10 were identified . subjects with a history of neovascular amd , myopia greater than 6 diopters ( d ) , or other macular pathology were excluded . subjects were required to have total ga area exceeding 1 mm at baseline and to have undergone faf imaging at least 6 months apart . based on these criteria , 43 subjects were identified and their faf images were obtained for analysis . image quality was graded based on the definition of vascular structures and classified as good , fair , or poor . images that were of poor quality or in which the ga lesion extended beyond the edge of the image were excluded from analysis . ultimately , 49 eyes from 30 subjects were included in the study . all images for this study were obtained using the heidelberg retina angiograph 2 ( hra2 ; heidelberg , germany ) device and the standard 30 field of view ( 768 768 pixels ) as previously described . briefly , the standard image acquisition protocol used excitation at 488 nm via an optically pumped solid state laser and a barrier filter set for detection above 500 nm . all images were obtained using the automatic real time ( art ) mode . for each eye , two faf images obtained at least 6 months apart were analyzed . for each image , the vessels and optic nerve head ( onh ) were first segmented out . vessels were automatically detected using dijkstra forest based automatic vessel segmentation and then removed using in - paint technology using a traditional in - painting method by solving laplace 's equation . next , the onh was automatically identified by taking advantage of the fact that it is a round hypoautofluorescent region located at the border of the image . specifically , the algorithm first obtained the average intensity of the image by filtering the image with a disk averaging filter . the radius of the disk filter is 80 pixels , which corresponds to the approximate size of the onh . after disk filtering , the areas comprising the lowest 5% average intensity were taken as candidate onh regions . finally , the largest of these low - intensity regions located at the border of the image was selected as the onh region . representative baseline images and output from the semiautomated software analysis algorithm from subjects of each rafh tertile . geographic atrophy is outlined in red and the outer border of the rim area is outlined in green . subject one and two had high and medium rafh , respectively , and significant progression in lesion size . simple thresholding can not be used for segmentation as faf images often have uneven illumination . this artifact is due to an optical aberration called vignetting or due to the optics system used to capture the images and can hinder direct interpretation of the pixel intensity information . we detected ga by using both an absolute pixel intensity threshold and by considering local variation in pixel intensity . in detail , after obtaining the mean intensity of the onh , monh , we first generated a binary image ibw1 by using monh as reference threshold . a second binary image , ibw2 , was generated using a local pixel intensity comparison as below : here x(r , c ) represents the faf intensity value ( ranging from 0255 ) in position ( r , c ) . m(r , c ) represents the mean intensity value within a 400 400 pixel window centered at ( r , c ) . many factors including settings during image acquisition , media opacity , eye movements , and subject positioning cause variation in faf intensity between images . to compensate for these variations between images , our software provides a slider allowing manual adjustment of the threshold parameter by the grader . when both binary images ( ibw1 and ibw2 ) had been generated , we calculated their union as a pilot candidate ga region estimate . we next employed a series of modified morphologic close operations to fill the holes in the region , combine neighboring regions , and smooth the region border . to achieve this , we first eroded the image using a disk structuring element with a radius of 10 . then , we dilated the image using a disk structuring element with a radius of 12 , followed by eroding the image again using a disk structuring element with a radius of 12 . finally , we removed small regions spanning less than 3000 pixels . segmentation of ga in some cases required manual adjustment especially when ga is contiguous with peripapillary atrophy and cases of normal foveal hypoautofluorescence . accordingly , the software contains tools that allow the grader to manually segment portions of the image , a slider to adjust the threshold of ga detection at the automatically determined lesion borders and a custom region selector that permits local pixel intensity analysis of a user selected region . after the ga border had been defined , the software automatically generated a second line 40 pixels ( approximately 440 m ) beyond the ga border . we defined the space between this line and the ga border as the rim area . a rim area of 440 m was selected based on two factors : the prior observation by bearelly et al . that increased rafh within approximately 500 m of the ga lesion border was associated with increased rate of lesion growth , and based on the observation in faf images not part of the study that nearly all rafh was contained within 450 m of the lesion borders . a second local intensity comparison using a threshold of + 50 pixels is performed to identify the hyperautofluorescent pixels within the rim area . rim area focal hyperautofluorescence was automatically calculated as the ratio of hyperautofluorescent rim area to total rim area . images were segmented by three graders ; two retina clinicians ( ma and el ) and one nonclinician high school student ( di ) . all graders were familiarized with the use of the software and allowed to practice on faf images , which were not part of this study prior to grading . in addition , the nonclinician grader was given a 2-hour lecture regarding ga , the technical aspects of faf , normal , and pathologic findings on faf , particularly those present in patients with amd and ga . graders were instructed to adjust the automatic ga lesion segmentation if needed by first adjusting the sensitivity threshold for detecting ga using a slider in the software . next small areas of ga , which were left unsegmented could be incorporated using a selection tool allowing automatic local pixel analysis of a user selected region . finally , in cases where foveal hypoautofluorescence was segmented as ga or when ga was contiguous with the optic nerve , manual segmentation was allowed . following this training , all graders segmented each image independently using the same computer under identical lighting conditions . correlation between rafh at study entry and ga progression measured in millimeters squared ( mm ) per month was analyzed using spearman correlation . median lesion size or ga growth rate among rafh tertiles were compared using kruskal - wallis testing after accounting for correlation between eyes from the same individual using generalized estimating equations . statistical analysis was performed using sas software ( sas institute , cary , nc , usa ) . this study was approved by the duke university medical center institutional review board ( durham , nc , usa ) , was conducted in compliance with the health insurance portability and accountability act ( hipaa ) and adhered to the tenets of the declaration of helsinki . subjects with ga secondary to dry amd who received faf imaging as part of their routine care between 1/1/2005 and 12/31/10 were identified . subjects with a history of neovascular amd , myopia greater than 6 diopters ( d ) , or other macular pathology were excluded . subjects were required to have total ga area exceeding 1 mm at baseline and to have undergone faf imaging at least 6 months apart . based on these criteria , 43 subjects were identified and their faf images were obtained for analysis . image quality was graded based on the definition of vascular structures and classified as good , fair , or poor . images that were of poor quality or in which the ga lesion extended beyond the edge of the image were excluded from analysis . all images for this study were obtained using the heidelberg retina angiograph 2 ( hra2 ; heidelberg , germany ) device and the standard 30 field of view ( 768 768 pixels ) as previously described . briefly , the standard image acquisition protocol used excitation at 488 nm via an optically pumped solid state laser and a barrier filter set for detection above 500 nm . for each eye , two faf images obtained at least 6 months apart were analyzed . representative samples of images and image analysis are shown in figure 1 . for each image , vessels were automatically detected using dijkstra forest based automatic vessel segmentation and then removed using in - paint technology using a traditional in - painting method by solving laplace 's equation . next , the onh was automatically identified by taking advantage of the fact that it is a round hypoautofluorescent region located at the border of the image . specifically , the algorithm first obtained the average intensity of the image by filtering the image with a disk averaging filter . the radius of the disk filter is 80 pixels , which corresponds to the approximate size of the onh . after disk filtering , the areas comprising the lowest 5% average intensity were taken as candidate onh regions . finally , the largest of these low - intensity regions located at the border of the image was selected as the onh region . representative baseline images and output from the semiautomated software analysis algorithm from subjects of each rafh tertile . geographic atrophy is outlined in red and the outer border of the rim area is outlined in green . subject one and two had high and medium rafh , respectively , and significant progression in lesion size . simple thresholding can not be used for segmentation as faf images often have uneven illumination . this artifact is due to an optical aberration called vignetting or due to the optics system used to capture the images and can hinder direct interpretation of the pixel intensity information . we detected ga by using both an absolute pixel intensity threshold and by considering local variation in pixel intensity . in detail , after obtaining the mean intensity of the onh , monh , we first generated a binary image ibw1 by using monh as reference threshold . a second binary image , ibw2 , was generated using a local pixel intensity comparison as below : here x(r , c ) represents the faf intensity value ( ranging from 0255 ) in position ( r , c ) . m(r , c ) represents the mean intensity value within a 400 400 pixel window centered at ( r , c ) . many factors including settings during image acquisition , media opacity , eye movements , and subject positioning cause variation in faf intensity between images . to compensate for these variations between images , our software provides a slider allowing manual adjustment of the threshold parameter by the grader . when both binary images ( ibw1 and ibw2 ) had been generated , we calculated their union as a pilot candidate ga region estimate . we next employed a series of modified morphologic close operations to fill the holes in the region , combine neighboring regions , and smooth the region border . to achieve this , we first eroded the image using a disk structuring element with a radius of 10 . then , we dilated the image using a disk structuring element with a radius of 12 , followed by eroding the image again using a disk structuring element with a radius of 12 . finally , we removed small regions spanning less than 3000 pixels . segmentation of ga in some cases required manual adjustment especially when ga is contiguous with peripapillary atrophy and cases of normal foveal hypoautofluorescence . accordingly , the software contains tools that allow the grader to manually segment portions of the image , a slider to adjust the threshold of ga detection at the automatically determined lesion borders and a custom region selector that permits local pixel intensity analysis of a user selected region . after the ga border had been defined , the software automatically generated a second line 40 pixels ( approximately 440 m ) beyond the ga border . we defined the space between this line and the ga border as the rim area . a rim area of 440 m was selected based on two factors : the prior observation by bearelly et al . that increased rafh within approximately 500 m of the ga lesion border was associated with increased rate of lesion growth , and based on the observation in faf images not part of the study that nearly all rafh was contained within 450 m of the lesion borders . a second local intensity comparison using a threshold of + 50 pixels is performed to identify the hyperautofluorescent pixels within the rim area . rim area focal hyperautofluorescence was automatically calculated as the ratio of hyperautofluorescent rim area to total rim area . images were segmented by three graders ; two retina clinicians ( ma and el ) and one nonclinician high school student ( di ) . all graders were familiarized with the use of the software and allowed to practice on faf images , which were not part of this study prior to grading . in addition , the nonclinician grader was given a 2-hour lecture regarding ga , the technical aspects of faf , normal , and pathologic findings on faf , particularly those present in patients with amd and ga . graders were instructed to adjust the automatic ga lesion segmentation if needed by first adjusting the sensitivity threshold for detecting ga using a slider in the software . next small areas of ga , which were left unsegmented could be incorporated using a selection tool allowing automatic local pixel analysis of a user selected region . finally , in cases where foveal hypoautofluorescence was segmented as ga or when ga was contiguous with the optic nerve , manual segmentation was allowed . following this training , all graders segmented each image independently using the same computer under identical lighting conditions . correlation between rafh at study entry and ga progression measured in millimeters squared ( mm ) per month was analyzed using spearman correlation . median lesion size or ga growth rate among rafh tertiles were compared using kruskal - wallis testing after accounting for correlation between eyes from the same individual using generalized estimating equations . statistical analysis was performed using sas software ( sas institute , cary , nc , usa ) . all subjects except one were self - identified as caucasian ; the non - caucasian patient did not have their race recorded in the medical record . the mean time between acquisition of the first and second faf images was 15.6 months ( median , 13 months ; interquartile range [ iqr ] , 821 ; range , 632 months ) . mean lesion size at the first time - point was 7.55 mm ( median , 6.25 mm ) . thirty eyes had unifocal lesions and 19 had multifocal lesions . in eyes with multifocal lesions , the mean number of lesions was four and maximum was eight . the mean growth rate was 0.16 mm / mo and 1.92 mm / y ( median , 1.68 mm / y ) . have previously demonstrated that ga lesions subjectively graded as having high rafh by expert graders have a higher rate of ga progression . to confirm this finding , we examined the correlation between rafh and the rate of ga lesion growth and found a positive correlation between rafh and rate of ga lesion growth ( spearman correlation coefficient 0.49 , p < 0.001 ) . a scatterplot of ga lesion growth rate versus rafh at the first time - point is shown in figure 2 . eyes were next separated into tertiles by rafh and the median lesion size and growth rate were compared ( table 1 ) . ga growth rate by tertile of rim area focal hyperautofluorescence the difference between the lowest rafh tertile and the middle and upper rafh tertiles were statistically significant ( p = 0.005 for medium versus low and p = 0.001 for high versus low ) . global comparison of tertiles was also statistically significant ( p = 0.001 ) . in order to determine reproducibility of our algorithm and whether our software could be used by nonexpert graders , two expert retina clinician graders , and one nonclinician nonexpert grader used the software to segment ga and rafh in all study eyes . all graders reported being able to use the software without difficulty and took under 1 minute to segment each image on average . all three graders made some adjustment in the automatic ga segmentation of most eyes ( range , 8088 of 98 total images ) . most adjustments were minor alterations to the ga lesion border and only 13 images required use of the hand drawing tool due to foveal hypoautofluorescence or ga contiguous with the optic nerve . to compare the results of segmentation performed by expert and nonexpert graders , we used bland - altman analysis ( table 2 ) . mean difference in rafh was 0.012 between expert graders and 0.005 to 0.017 between expert and nonexpert graders . mean difference in lesion size was 0.11 mm between expert graders and 0.29 to 0.41 mm between expert and nonexpert graders . mean difference in lesion growth rate was 0.0098 mm / mo between expert graders and 0.027 to 0.037 mm / mo between expert and nonexpert graders . mean differences and 95% confidence intervals for interobserver agreements for initial lesion size , lesion growth rate , and rafh mean agreements for interobserver variability for lesion size are similar to previously reported values using commercial software for ga segmentation using trained graders in a reading center . agreement between graders for lesion progression was somewhat less than those of schmitz - valckenberg et al . when comparing expert and nonexpert graders . importantly , mean agreement between all graders for rafh was extremely high , which resulted in risk stratification based on rafh tertile being 96% identical across all three graders . specifically , there was complete agreement between the nonexpert grader and one expert and there was discrepancy in only 2 of 49 eyes when comparing the nonexpert grader and the other expert . this demonstrates that , following minimal training , a nonexpert can easily learn to operate the software and can generate predictions comparable with an expert . finally , we compared the predictions of our software with those based on patterns of faf described by holz and colleagues . two expert retina clinicians ( pm and sc ) were masked to the output of our software and were asked to classify by consensus the faf pattern present in the first image of each eye using the stock images and algorithm published by holz and colleagues . classification of study subjects by holz qualitative analysis of fundus autofluorescence patterns in our cohort , eight eyes had low risk patterns of faf ( none or focal ) . of these , seven were in the lowest rafh tertile and five fell into the lowest decile of rafh . this suggests that our software with the holz algorithm in identifying eyes at low risk of progression . holz and colleagues have delineated six patterns of faf which are associated with increased ga progression ; banded , and five subtypes of diffuse pattern faf . in our cohort , pattern of faf . of these , 71.9% were in the middle or upper rafh tertiles . these results suggest that our software correctly identifies eyes likely to experience rapid ga progression and that eyes with very low rafh are at low risk of progression . the purpose of this study was to develop a rapid , objective means of quantifying ga and rafh that is usable by nonexpert graders and to demonstrate that predictions based on quantification of rafh are comparable to those of the current gold standard qualitative classification system . the current study is unique in its inclusion of a nonexpert grader . when compared with our two expert graders , the nonexpert grader was able to use the software to generate nearly identical rafh - based predictions of ga progression . importantly , the predictions of our software correlate well with those based on the holz faf pattern system . while rafh does correlate with ga progression , this technology does not completely replace the qualitative categorization algorithms , which have been previously validated . there are certain diffuse faf patterns such as branching and fine granular , which are less well segmented by our software . our findings are complementary to those of holz and support the concept that hyperautofluorescence at ga lesion borders is correlated with ga progression and that faf may be a marker of rpe dysfunction in ga . our software could serve as a ga risk prediction algorithm that is useful when it is impractical to have images evaluated by expert graders in a reading center . analysis of images from our study cohort demonstrates a statistically significant , positive correlation between rafh and rate of ga lesion growth . study subjects in the top or middle tertile of rafh had a significantly greater rate of ga growth when compared with those in the bottom tertile . this is in agreement with previous studies which have demonstrated a positive correlation between hyperautofluorescence at the borders of ga lesions and growth of ga . in contrast to our findings , hwang et al . reported that total area of hyperautofluorescence in the rim area surrounding ga fails to predict progression . first , the study cohort used by hwang et al . included only eight eyes of six subjects raising the possibility that our larger cohort was capable of identifying correlations not detectable in smaller groups . second , the algorithm used by hwang et al . uses a single threshold across the entire image to define rafh , whereas ours uses a combination of a global threshold and a local area intensity measurement to compensate for variations in autofluorescence intensity across individual images . thus , rafh as defined by our algorithm differs substantially from previously published work and may therefore have superior predictive value . for instance , ga has been shown to grow more rapidly along its peripheral borders compared with those adjacent to the fovea and lesions , which are extrafoveal grow more rapidly than those already involving the fovea . additionally , multifocal ga has been found to progress more rapidly than unifocal ga . in our cohort , the growth rate in multifocal and unifocal lesions was 0.14 ( iqr , 0.080.28 ) and 0.14 ( iqr , 0.080.25 ) , respectively ( p = 0.89 ) . the median growth rate in foveal and extrafoveal lesions was 0.11 ( iqr , 0.070.24 ) and 0.15 ( iqr , 0.080.28 ) , respectively ( p = 0.74 ) . the reason for the discrepancy between our study and previously published work is not immediately apparent . however , it may be that due to its relatively small size our study was not powered to detect these differences . while faf has long been used as a means to quantify ga and predict progression , numerous recent studies have used morphologic findings from sd - oct to predict the rate or location of ga lesion growth . several markers have been variably associated with ga progression including outer retinal tubulations ( ort ) , ellipsoid zone disruption at ga borders , alterations in reflectivity of the outer nuclear layer , and various alterations in the rpe / bruch 's membrane complex . outer retinal tubulations have been associated with numerous retinal pathologies including ga . in a study of 43 eyes with ga , moussa et al . identified ort within the atrophic zone , but not at the ga border as a risk factor for ga progression . however , hariri et al . found that in a study of 108 eyes presence of ort was associated with slower progression of ga . ellipsoid zone ( ez ) loss at the ga border region was initially described by bearelly et al . who performed analysis of sd - oct b - scans to characterize the morphology of ga lesion borders . more recently , en face analysis of sd - oct has been used to quantify ez loss as a potential particularly those without reticular pseudodrusen , it was not a robust predictor in a real world cohort . interestingly however , nunes et al . found that in select cases , ez loss very accurately predicts the specific location of future ga within 12 months . while this methodology holds promise , its use is limited because it is unknown whether areas with ez loss are irreversibly destined to become atrophic and if so over what time period atrophy will develop . intriguingly , stetson and colleagues found that increased reflectivity of the outer nuclear layer or henle fiber layer ( onl / hfl ) , which manifests as higher minimum pixel intensity along individual a - scans between the inner limiting membrane and rpe is correlated with rate of ga progression . they speculate that this change in the onh / hfl may be due to early degenerative changes in the photoreceptors that precede development of ga . finally , several different abnormalities of the rpe and subrpe space have been associated with risk of ga progression . irregular elevation of rpe / bruch 's membrane complex within the atrophic zone of ga as well as splitting of rpe / bruch 's membrane complex at two lesion borders has been associated with increased progression . recently , folgar et al . used semiautomated segmentation of the rpe / drusen complex in subjects from the areds2 ancillary sd - oct study and found that abnormal thinning of the rpe / drusen complex was associated with new onset ga as well as development of central ga but did not specifically examine the impact on ga growth rate . of note , brar et al . found that alterations in the rpe / bruch 's membrane complex at ga borders correspond to increased faf , which suggests that there may be some redundancy in the predictive information provided by faf and sd - oct . the use of sd - oct in identifying morphologic signatures of specific areas , which will progress to ga is a significant potential advantage when compared with faf , which has not been shown to predict the location of ga lesion growth . fundus autofluorescence is advantageous in that it can be used to predict ga progression and is amenable to segmentation and quantification , even by nonexpert graders . thus , additional studies will be needed to determine , which combination of structural and functional imaging findings best predict the rate and location of ga lesion growth . one was reliance on a semiautomated software rather than a fully automated one to quantify ga . we note that a recent large prospective natural history study used semiautomated software and that no such fully automated software has been tested on a large noncherry - picked clinical dataset , which often suffer from a variety of distortions common in real - world clinical imaging . thus , while our quantitative results are generated using semiautomated software , it is less subjective and time consuming than the previous studies based on manual image analysis . additionally , the quantification of rafh was fully automated , which removed subjectivity from the primary predictive component of the algorithm . our study was also limited by relatively small size . however , ga progression rates in our study are similar to published values reported in other studies using faf or color fundus photos to quantify ga suggesting that ours is a representative cohort . in our cohort , the median lesion size and rate of ga progression were 6.25 mm and 1.68 mm / y , respectively . previously , holz et al . reported a median lesion size of 7.04 mm and median growth rate of 1.52 mm / y in 195 eyes and bearelly et al . reported a median growth rate of 1.49 mm / y in 45 eyes based on faf imaging . reported a median growth rate of 2.1 mm / y in 212 eyes based on analysis of color fundus photos . another limitation is that our study is retrospective and therefore demonstrates an association between rafh and ga progression , but can not prove that biology measured by rafh is causative in ga progression . in the future , we plan a larger prospective multimodal imaging study to validate our algorithm and its findings and to compare predictions based on sd - oct and faf . in conclusion , we have developed robust segmentation software that can be used to accurately predict risk of ga progression , and which makes predictions similar to validated but more cumbersome prediction algorithms . we also confirm and extend the previous finding that rafh is correlated with rate of ga progression . compared with other software available for ga quantification , ours has the advantage that it can be effectively used by nonexperts and requires minimal grader training . the use of semiautomated segmentation software allows rapid , accurate measurement of ga area and rafh , and could aid both clinicians and researchers in predicting which patients will experience rapid progression in their ga . future work will be focused on validating our software and the predictive value of rafh in a larger , prospective observational study .

purposeto develop image analysis software usable by nonexpert graders to segment geographic atrophy ( ga ) from dry amd and to quantify rim area focal hyperautofluorescence ( rafh ) surrounding ga on fundus autofluorescence ( faf ) images . to compare the ga progression predictions based on rafh with those of a validated qualitative classification system.methodsretrospective analysis of serial faf images from 49 eyes of 30 subjects with ga was performed using matlab - based software ( mathworks , natick , ma , usa ) . correlation between rafh and progression of ga was analyzed using spearman correlation . comparisons of lesion growth rate between rafh tertiles used generalized estimating equations and kruskal - wallis testing . interobserver variability in lesion size , growth rate and rafh were compared between two expert and one nonexpert grader using bland - altman statistics.resultsrim area focal hyperautofluorescence was positively correlated with ga progression rate ( = 0.49 , p < 0.001 ) . subjects in the middle or highest rafh tertile were at greater risk of progression ( p = 0.005 and p = 0.001 , respectively ) . mean difference in rafh was 0.012 between expert and 0.005 to 0.017 between expert and nonexperts . mean difference in lesion size ( mm2 ) was 0.11 between expert and 0.29 to 0.41 between expert and nonexperts . mean difference in lesion growth rate ( mm2/mo ) was 0.0098 between expert and 0.027 to 0.037 between expert and nonexperts . risk stratification based on rafh tertile was 96% identical across all graders.conclusionsour semiautomated image analysis software facilitates stratification of progression risk based on rafh and enabled a nonexpert grader with minimal training to obtain results comparable to expert graders . predictions based on rafh were similar to those of a validated qualitative classification system .

Methods Subjects Image Acquisition Image Analysis Image Segmentation by Human Graders Statistical Analysis Results Discussion

this study was approved by the duke university medical center institutional review board ( durham , nc , usa ) , was conducted in compliance with the health insurance portability and accountability act ( hipaa ) and adhered to the tenets of the declaration of helsinki . subjects with ga secondary to dry amd who received faf imaging as part of their routine care between 1/1/2005 and 12/31/10 were identified . subjects with a history of neovascular amd , myopia greater than 6 diopters ( d ) , or other macular pathology were excluded . subjects were required to have total ga area exceeding 1 mm at baseline and to have undergone faf imaging at least 6 months apart . based on these criteria , 43 subjects were identified and their faf images were obtained for analysis . images that were of poor quality or in which the ga lesion extended beyond the edge of the image were excluded from analysis . ultimately , 49 eyes from 30 subjects were included in the study . for each eye , two faf images obtained at least 6 months apart were analyzed . representative baseline images and output from the semiautomated software analysis algorithm from subjects of each rafh tertile . geographic atrophy is outlined in red and the outer border of the rim area is outlined in green . subject one and two had high and medium rafh , respectively , and significant progression in lesion size . simple thresholding can not be used for segmentation as faf images often have uneven illumination . we next employed a series of modified morphologic close operations to fill the holes in the region , combine neighboring regions , and smooth the region border . segmentation of ga in some cases required manual adjustment especially when ga is contiguous with peripapillary atrophy and cases of normal foveal hypoautofluorescence . accordingly , the software contains tools that allow the grader to manually segment portions of the image , a slider to adjust the threshold of ga detection at the automatically determined lesion borders and a custom region selector that permits local pixel intensity analysis of a user selected region . we defined the space between this line and the ga border as the rim area . a rim area of 440 m was selected based on two factors : the prior observation by bearelly et al . that increased rafh within approximately 500 m of the ga lesion border was associated with increased rate of lesion growth , and based on the observation in faf images not part of the study that nearly all rafh was contained within 450 m of the lesion borders . a second local intensity comparison using a threshold of + 50 pixels is performed to identify the hyperautofluorescent pixels within the rim area . rim area focal hyperautofluorescence was automatically calculated as the ratio of hyperautofluorescent rim area to total rim area . all graders were familiarized with the use of the software and allowed to practice on faf images , which were not part of this study prior to grading . in addition , the nonclinician grader was given a 2-hour lecture regarding ga , the technical aspects of faf , normal , and pathologic findings on faf , particularly those present in patients with amd and ga . graders were instructed to adjust the automatic ga lesion segmentation if needed by first adjusting the sensitivity threshold for detecting ga using a slider in the software . next small areas of ga , which were left unsegmented could be incorporated using a selection tool allowing automatic local pixel analysis of a user selected region . finally , in cases where foveal hypoautofluorescence was segmented as ga or when ga was contiguous with the optic nerve , manual segmentation was allowed . following this training , all graders segmented each image independently using the same computer under identical lighting conditions . correlation between rafh at study entry and ga progression measured in millimeters squared ( mm ) per month was analyzed using spearman correlation . median lesion size or ga growth rate among rafh tertiles were compared using kruskal - wallis testing after accounting for correlation between eyes from the same individual using generalized estimating equations . statistical analysis was performed using sas software ( sas institute , cary , nc , usa ) . this study was approved by the duke university medical center institutional review board ( durham , nc , usa ) , was conducted in compliance with the health insurance portability and accountability act ( hipaa ) and adhered to the tenets of the declaration of helsinki . subjects with ga secondary to dry amd who received faf imaging as part of their routine care between 1/1/2005 and 12/31/10 were identified . subjects with a history of neovascular amd , myopia greater than 6 diopters ( d ) , or other macular pathology were excluded . based on these criteria , 43 subjects were identified and their faf images were obtained for analysis . images that were of poor quality or in which the ga lesion extended beyond the edge of the image were excluded from analysis . for each eye , two faf images obtained at least 6 months apart were analyzed . representative samples of images and image analysis are shown in figure 1 . geographic atrophy is outlined in red and the outer border of the rim area is outlined in green . subject one and two had high and medium rafh , respectively , and significant progression in lesion size . simple thresholding can not be used for segmentation as faf images often have uneven illumination . we next employed a series of modified morphologic close operations to fill the holes in the region , combine neighboring regions , and smooth the region border . segmentation of ga in some cases required manual adjustment especially when ga is contiguous with peripapillary atrophy and cases of normal foveal hypoautofluorescence . accordingly , the software contains tools that allow the grader to manually segment portions of the image , a slider to adjust the threshold of ga detection at the automatically determined lesion borders and a custom region selector that permits local pixel intensity analysis of a user selected region . after the ga border had been defined , the software automatically generated a second line 40 pixels ( approximately 440 m ) beyond the ga border . we defined the space between this line and the ga border as the rim area . a rim area of 440 m was selected based on two factors : the prior observation by bearelly et al . that increased rafh within approximately 500 m of the ga lesion border was associated with increased rate of lesion growth , and based on the observation in faf images not part of the study that nearly all rafh was contained within 450 m of the lesion borders . rim area focal hyperautofluorescence was automatically calculated as the ratio of hyperautofluorescent rim area to total rim area . images were segmented by three graders ; two retina clinicians ( ma and el ) and one nonclinician high school student ( di ) . all graders were familiarized with the use of the software and allowed to practice on faf images , which were not part of this study prior to grading . in addition , the nonclinician grader was given a 2-hour lecture regarding ga , the technical aspects of faf , normal , and pathologic findings on faf , particularly those present in patients with amd and ga . next small areas of ga , which were left unsegmented could be incorporated using a selection tool allowing automatic local pixel analysis of a user selected region . following this training , all graders segmented each image independently using the same computer under identical lighting conditions . correlation between rafh at study entry and ga progression measured in millimeters squared ( mm ) per month was analyzed using spearman correlation . median lesion size or ga growth rate among rafh tertiles were compared using kruskal - wallis testing after accounting for correlation between eyes from the same individual using generalized estimating equations . statistical analysis was performed using sas software ( sas institute , cary , nc , usa ) . all subjects except one were self - identified as caucasian ; the non - caucasian patient did not have their race recorded in the medical record . the mean time between acquisition of the first and second faf images was 15.6 months ( median , 13 months ; interquartile range [ iqr ] , 821 ; range , 632 months ) . mean lesion size at the first time - point was 7.55 mm ( median , 6.25 mm ) . the mean growth rate was 0.16 mm / mo and 1.92 mm / y ( median , 1.68 mm / y ) . have previously demonstrated that ga lesions subjectively graded as having high rafh by expert graders have a higher rate of ga progression . to confirm this finding , we examined the correlation between rafh and the rate of ga lesion growth and found a positive correlation between rafh and rate of ga lesion growth ( spearman correlation coefficient 0.49 , p < 0.001 ) . a scatterplot of ga lesion growth rate versus rafh at the first time - point is shown in figure 2 . eyes were next separated into tertiles by rafh and the median lesion size and growth rate were compared ( table 1 ) . ga growth rate by tertile of rim area focal hyperautofluorescence the difference between the lowest rafh tertile and the middle and upper rafh tertiles were statistically significant ( p = 0.005 for medium versus low and p = 0.001 for high versus low ) . global comparison of tertiles was also statistically significant ( p = 0.001 ) . in order to determine reproducibility of our algorithm and whether our software could be used by nonexpert graders , two expert retina clinician graders , and one nonclinician nonexpert grader used the software to segment ga and rafh in all study eyes . all graders reported being able to use the software without difficulty and took under 1 minute to segment each image on average . to compare the results of segmentation performed by expert and nonexpert graders , we used bland - altman analysis ( table 2 ) . mean difference in rafh was 0.012 between expert graders and 0.005 to 0.017 between expert and nonexpert graders . mean difference in lesion size was 0.11 mm between expert graders and 0.29 to 0.41 mm between expert and nonexpert graders . mean difference in lesion growth rate was 0.0098 mm / mo between expert graders and 0.027 to 0.037 mm / mo between expert and nonexpert graders . mean differences and 95% confidence intervals for interobserver agreements for initial lesion size , lesion growth rate , and rafh mean agreements for interobserver variability for lesion size are similar to previously reported values using commercial software for ga segmentation using trained graders in a reading center . agreement between graders for lesion progression was somewhat less than those of schmitz - valckenberg et al . when comparing expert and nonexpert graders . importantly , mean agreement between all graders for rafh was extremely high , which resulted in risk stratification based on rafh tertile being 96% identical across all three graders . specifically , there was complete agreement between the nonexpert grader and one expert and there was discrepancy in only 2 of 49 eyes when comparing the nonexpert grader and the other expert . this demonstrates that , following minimal training , a nonexpert can easily learn to operate the software and can generate predictions comparable with an expert . finally , we compared the predictions of our software with those based on patterns of faf described by holz and colleagues . two expert retina clinicians ( pm and sc ) were masked to the output of our software and were asked to classify by consensus the faf pattern present in the first image of each eye using the stock images and algorithm published by holz and colleagues . classification of study subjects by holz qualitative analysis of fundus autofluorescence patterns in our cohort , eight eyes had low risk patterns of faf ( none or focal ) . of these , seven were in the lowest rafh tertile and five fell into the lowest decile of rafh . this suggests that our software with the holz algorithm in identifying eyes at low risk of progression . holz and colleagues have delineated six patterns of faf which are associated with increased ga progression ; banded , and five subtypes of diffuse pattern faf . of these , 71.9% were in the middle or upper rafh tertiles . these results suggest that our software correctly identifies eyes likely to experience rapid ga progression and that eyes with very low rafh are at low risk of progression . the purpose of this study was to develop a rapid , objective means of quantifying ga and rafh that is usable by nonexpert graders and to demonstrate that predictions based on quantification of rafh are comparable to those of the current gold standard qualitative classification system . the current study is unique in its inclusion of a nonexpert grader . when compared with our two expert graders , the nonexpert grader was able to use the software to generate nearly identical rafh - based predictions of ga progression . importantly , the predictions of our software correlate well with those based on the holz faf pattern system . while rafh does correlate with ga progression , this technology does not completely replace the qualitative categorization algorithms , which have been previously validated . our findings are complementary to those of holz and support the concept that hyperautofluorescence at ga lesion borders is correlated with ga progression and that faf may be a marker of rpe dysfunction in ga . our software could serve as a ga risk prediction algorithm that is useful when it is impractical to have images evaluated by expert graders in a reading center . analysis of images from our study cohort demonstrates a statistically significant , positive correlation between rafh and rate of ga lesion growth . study subjects in the top or middle tertile of rafh had a significantly greater rate of ga growth when compared with those in the bottom tertile . this is in agreement with previous studies which have demonstrated a positive correlation between hyperautofluorescence at the borders of ga lesions and growth of ga . reported that total area of hyperautofluorescence in the rim area surrounding ga fails to predict progression . included only eight eyes of six subjects raising the possibility that our larger cohort was capable of identifying correlations not detectable in smaller groups . uses a single threshold across the entire image to define rafh , whereas ours uses a combination of a global threshold and a local area intensity measurement to compensate for variations in autofluorescence intensity across individual images . for instance , ga has been shown to grow more rapidly along its peripheral borders compared with those adjacent to the fovea and lesions , which are extrafoveal grow more rapidly than those already involving the fovea . in our cohort , the growth rate in multifocal and unifocal lesions was 0.14 ( iqr , 0.080.28 ) and 0.14 ( iqr , 0.080.25 ) , respectively ( p = 0.89 ) . the median growth rate in foveal and extrafoveal lesions was 0.11 ( iqr , 0.070.24 ) and 0.15 ( iqr , 0.080.28 ) , respectively ( p = 0.74 ) . while faf has long been used as a means to quantify ga and predict progression , numerous recent studies have used morphologic findings from sd - oct to predict the rate or location of ga lesion growth . several markers have been variably associated with ga progression including outer retinal tubulations ( ort ) , ellipsoid zone disruption at ga borders , alterations in reflectivity of the outer nuclear layer , and various alterations in the rpe / bruch 's membrane complex . identified ort within the atrophic zone , but not at the ga border as a risk factor for ga progression . found that in a study of 108 eyes presence of ort was associated with slower progression of ga . who performed analysis of sd - oct b - scans to characterize the morphology of ga lesion borders . more recently , en face analysis of sd - oct has been used to quantify ez loss as a potential particularly those without reticular pseudodrusen , it was not a robust predictor in a real world cohort . intriguingly , stetson and colleagues found that increased reflectivity of the outer nuclear layer or henle fiber layer ( onl / hfl ) , which manifests as higher minimum pixel intensity along individual a - scans between the inner limiting membrane and rpe is correlated with rate of ga progression . they speculate that this change in the onh / hfl may be due to early degenerative changes in the photoreceptors that precede development of ga . finally , several different abnormalities of the rpe and subrpe space have been associated with risk of ga progression . used semiautomated segmentation of the rpe / drusen complex in subjects from the areds2 ancillary sd - oct study and found that abnormal thinning of the rpe / drusen complex was associated with new onset ga as well as development of central ga but did not specifically examine the impact on ga growth rate . found that alterations in the rpe / bruch 's membrane complex at ga borders correspond to increased faf , which suggests that there may be some redundancy in the predictive information provided by faf and sd - oct . the use of sd - oct in identifying morphologic signatures of specific areas , which will progress to ga is a significant potential advantage when compared with faf , which has not been shown to predict the location of ga lesion growth . fundus autofluorescence is advantageous in that it can be used to predict ga progression and is amenable to segmentation and quantification , even by nonexpert graders . thus , additional studies will be needed to determine , which combination of structural and functional imaging findings best predict the rate and location of ga lesion growth . one was reliance on a semiautomated software rather than a fully automated one to quantify ga . thus , while our quantitative results are generated using semiautomated software , it is less subjective and time consuming than the previous studies based on manual image analysis . however , ga progression rates in our study are similar to published values reported in other studies using faf or color fundus photos to quantify ga suggesting that ours is a representative cohort . in our cohort , the median lesion size and rate of ga progression were 6.25 mm and 1.68 mm / y , respectively . reported a median lesion size of 7.04 mm and median growth rate of 1.52 mm / y in 195 eyes and bearelly et al . reported a median growth rate of 1.49 mm / y in 45 eyes based on faf imaging . reported a median growth rate of 2.1 mm / y in 212 eyes based on analysis of color fundus photos . another limitation is that our study is retrospective and therefore demonstrates an association between rafh and ga progression , but can not prove that biology measured by rafh is causative in ga progression . in the future , we plan a larger prospective multimodal imaging study to validate our algorithm and its findings and to compare predictions based on sd - oct and faf . in conclusion , we have developed robust segmentation software that can be used to accurately predict risk of ga progression , and which makes predictions similar to validated but more cumbersome prediction algorithms . we also confirm and extend the previous finding that rafh is correlated with rate of ga progression . the use of semiautomated segmentation software allows rapid , accurate measurement of ga area and rafh , and could aid both clinicians and researchers in predicting which patients will experience rapid progression in their ga .

chronic obstructive pulmonary disease ( copd ) is a leading cause of morbidity and mortality worldwide and results in an economic and social burden that is both substantial and increasing.13 copd is one of the world s most common noncommunicable health problems.2 the world health organization reported that copd is the third leading cause of death in the world and is presently the fifth leading cause of death among high - income countries , with a rate of 31 deaths per 100,000 people.4 furthermore , the burden of copd is expected to continue increasing.4 copd is considered to be a disease of later years ; typical onset is in middle adulthood ( > 55 years ) . estimates suggest that 50% of those with copd are younger than 65 years of age,5,6 and many of them are likely to be under paid employment.7 copd is a debilitating disease affecting the daily lives of patients . physical activity levels are low even in patients in the early stages of copd.8 increasing severity of copd is associated with decreasing physical activity.8,9 although data exist on the physical activity levels of those with copd , data on its impact on presenteeism and absenteeism among the working population are limited . few studies have examined the association between copd and work ability as well as use of sick leave.7,1014 employed adults with copd reported significantly lower quality of life and work productivity and increased health care resource utilization than employed adults without copd in the usa.12 workers diagnosed with copd had significantly higher levels of presenteeism and overall work impairment.12 furthermore , there are no data regarding the association between airflow limitation ( al ) severity and productivity at work or use of sick leave . we hypothesized that al could be an important factor associated with loss of productivity at work and use of sick leave . this study aimed to reveal the associations between al severity and productivity at work as well as use of sick leave among japanese workers . figure 1 demonstrates the flow chart for selection of the participants with either al or normal lung function . participants were employees who underwent medical health checkups at the japanese red cross kumamoto health care center and the medical health checkups included interview questionnaires , a physical examination , blood sampling , and spirometry , as previously described.1518 the interview questionnaires were conducted by a trained public health nurse to obtain data regarding medical history , smoking status , sleep duration , work hours per day , workplace smoking environment , productivity loss at work , and sickness absence . pack - years were calculated by multiplying the number of years of smoking by the average number of cigarettes smoked per day and dividing it by 20 . according to the interview questionnaires , sleep duration was categorized into < 6 hours , 68 hours , and > 8 hours . work hours per day were categorized into 8 hours , 810 hours , and > 10 hours . workplace smoking environment was categorized into smoke - free policies , restrictive policies ( prohibitive policies using designated smoking rooms ) , and nonrestrictive ( nonprohibitive ) policies . those without jobs ( n=288 ) were excluded , as were those without lung function data ( n=210 ) and those for whom there was insufficient information regarding loss of work productivity ( n=67 ) . complete data on lung function tests , productivity loss at work , and sickness absence were obtained from 1,866 workers . additionally , participants with forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) > 70% and fev1 predicted < 80% ( n=126 ) were excluded from this study . we excluded data obtained from participants younger than 30 years ( n=8 ) and older than 75 years of age ( n=2 ) . we further excluded participants with a history or clinical evidence of mental disorder , allergic disease , respiratory disease except for copd and asthma , cancer , cerebrovascular disease , brain tumor , cardiac disease , and collagen disease . those with mnire s disease , vertigo , chronic headache , and chronic headache with vertigo were also excluded . data from a total of 1,378 participants ( 921 males and 457 females ) aged 3074 years were included in the final analysis . the main occupation of the participants consisted of managers ( n=105 ) , professionals and technicians ( n=375 ) , clerks ( n=545 ) , sales ( n=110 ) , service ( n=57 ) , agriculture and fishery ( n=79 ) , manufacturing ( n=57 ) , construction ( n=13 ) , and others ( n=37 ) . subjects were divided according to lung function ( 1,280 had normal lung function , 48 had mild al , and 50 had moderate - to - severe al ) ( table 1 ) . all study participants gave their written informed consent regarding all aspects of the study and to undergo this examination . our research protocol was approved by the human ethics committee of kumamoto university ( numbers 436 , 816 , and 870 ) and the japanese red cross kumamoto health care center . tokyo , japan ) , as previously described,1518 using equipment and quality criteria that complied with international recommendations.19 reversibility tests were not performed for this study , and the classifications were based on prebronchodilator levels . according to guidelines from the global initiative for chronic obstructive pulmonary disease ( gold ) , we defined al as an fev1/fvc ratio < 70%.1 the predicted values were determined from the prediction equations published by the japanese respiratory society:20 males , 0.036 height ( cm ) 0.028 age 1.178 ; females , 0.022 height ( cm ) 0.022 age 0.005 . the criteria used for the al staging were also developed according to the gold guidelines and were as follows : stage i ( mild al ) : fev1/fvc < 70% and fev1 80% of predicted value ; stage ii ( moderate al ) : fev1/fvc < 70% and 50%fev1<80% of predicted value ; stage iii ( severe al ) : fev1/fvc < 70% and 30%fev1<50% of predicted value ; and stage iv ( very severe al ) : fev1/fvc < 70% and fev1 < 30% of predicted value . the subjects were divided into three groups : a control group ( normal lung function ) , gold stage i ( mild al ) , and gold stages ii the participants with normal lung function were defined as having a fev1/fvc > 70% and fev1 > productivity loss at work was measured according to the quality and quantity scale of the quantity and quality ( qq ) method,21 as described by robroek et al.22,23 participants were requested to indicate how much work they actually performed during regular hours on their most recent regular workday compared to that on a normal workday . the amount of the quality and quantity of productivity was measured on a scale from 0 ( nothing ) to 10 ( regular amount ) . the outcome productivity loss at work was classified into two categories : no productivity loss ( score = 10 ) and productivity loss at work ( score of 9 or lower ) . participants were asked how many days in the past 12 months they were not able to work due to health problems.22,23 those reporting sick leave were categorized into two categories : no sick leave ( 0 day ) and use of sick leave ( 1 day or more ) . systolic and diastolic blood pressure were measured by trained nurses using an automated digital sphygmomanometer ( hem-904 ; omron , kyoto , japan ) placed on the upper arm at heart height while the participant was seated , following 5 minutes of rest . hypertension was defined as antihypertensive medication use , systolic blood pressure of 130 mmhg or more , or diastolic blood pressure of 85 mmhg or more . dyslipidemia was defined as medication use , triglyceride level of 150 mg / dl or more , low - density lipoprotein cholesterol level of 140 mg / dl or more , or high - density lipoprotein cholesterol level of < 40 mg / dl . hyperglycemia was defined as blood glucose - lowering medication use or fasting glucose level of 110 mg / dl or more , as previously described.15 the results are given as mean ( standard deviation ) . differences among normal lung function , mild al , and moderate - to - severe al were compared using an analysis of variance , the kruskal wallis test for continuous variables , or the chi - square test for categorical variables . the post hoc scheffe s test was used to assess the difference in characteristics according to lung function status . a multivariate logistic regression model adjusted for sex , age , body mass index ( bmi ) , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment was used to assess the relationship between severity of al and productivity loss at work as well as use of sick leave , with normal lung function as the reference . the odds ratios ( ors ) were estimated as measures of association with corresponding 95% confidence intervals ( 95% cis ) . adjustments were made according to sex , age , and bmi ( model 1 ) . in order to study the influence of lifestyle - related factors ( such as models 2 [ model 1 + smoking status ] , 3 [ model 2 + hypertension , hyperglycemia , and dyslipidemia ] , and 4 [ model 3 + sleep duration ] ) and work - related factors ( such as models 5 [ model 4 + work hours per day ] and 6 [ model 5 + workplace smoking environment ] ) , these factors were added separately to the basic statistical model ( model 1 ) describing the association between al severity and productivity loss at work or the presence of sick leave . all statistical analyses were performed using ibm spss statistics 22.0 software ( ibm corporation , armonk , ny , usa ) . whether the data showed normal distribution was assessed by shapiro figure 1 demonstrates the flow chart for selection of the participants with either al or normal lung function . participants were employees who underwent medical health checkups at the japanese red cross kumamoto health care center and the medical health checkups included interview questionnaires , a physical examination , blood sampling , and spirometry , as previously described.1518 the interview questionnaires were conducted by a trained public health nurse to obtain data regarding medical history , smoking status , sleep duration , work hours per day , workplace smoking environment , productivity loss at work , and sickness absence . pack - years were calculated by multiplying the number of years of smoking by the average number of cigarettes smoked per day and dividing it by 20 . according to the interview questionnaires , sleep duration was categorized into < 6 hours , 68 hours , and > 8 hours . work hours per day were categorized into 8 hours , 810 hours , and > 10 hours . workplace smoking environment was categorized into smoke - free policies , restrictive policies ( prohibitive policies using designated smoking rooms ) , and nonrestrictive ( nonprohibitive ) policies . those without jobs ( n=288 ) were excluded , as were those without lung function data ( n=210 ) and those for whom there was insufficient information regarding loss of work productivity ( n=67 ) . complete data on lung function tests , productivity loss at work , and sickness absence were obtained from 1,866 workers . additionally , participants with forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) > 70% and fev1 predicted < 80% ( n=126 ) were excluded from this study . we excluded data obtained from participants younger than 30 years ( n=8 ) and older than 75 years of age ( n=2 ) . we further excluded participants with a history or clinical evidence of mental disorder , allergic disease , respiratory disease except for copd and asthma , cancer , cerebrovascular disease , brain tumor , cardiac disease , and collagen disease . those with mnire s disease , vertigo , chronic headache , and chronic headache with vertigo were also excluded . data from a total of 1,378 participants ( 921 males and 457 females ) aged 3074 years were included in the final analysis . the main occupation of the participants consisted of managers ( n=105 ) , professionals and technicians ( n=375 ) , clerks ( n=545 ) , sales ( n=110 ) , service ( n=57 ) , agriculture and fishery ( n=79 ) , manufacturing ( n=57 ) , construction ( n=13 ) , and others ( n=37 ) . subjects were divided according to lung function ( 1,280 had normal lung function , 48 had mild al , and 50 had moderate - to - severe al ) ( table 1 ) . all study participants gave their written informed consent regarding all aspects of the study and to undergo this examination . our research protocol was approved by the human ethics committee of kumamoto university ( numbers 436 , 816 , and 870 ) and the japanese red cross kumamoto health care center . tokyo , japan ) , as previously described,1518 using equipment and quality criteria that complied with international recommendations.19 reversibility tests were not performed for this study , and the classifications were based on prebronchodilator levels . according to guidelines from the global initiative for chronic obstructive pulmonary disease ( gold ) , we defined al as an fev1/fvc ratio < 70%.1 the predicted values were determined from the prediction equations published by the japanese respiratory society:20 males , 0.036 height ( cm ) 0.028 age 1.178 ; females , 0.022 height ( cm ) 0.022 age 0.005 . the criteria used for the al staging were also developed according to the gold guidelines and were as follows : stage i ( mild al ) : fev1/fvc < 70% and fev1 80% of predicted value ; stage ii ( moderate al ) : fev1/fvc < 70% and 50%fev1<80% of predicted value ; stage iii ( severe al ) : fev1/fvc < 70% and 30%fev1<50% of predicted value ; and stage iv ( very severe al ) : fev1/fvc the subjects were divided into three groups : a control group ( normal lung function ) , gold stage i ( mild al ) , and gold stages ii the participants with normal lung function were defined as having a fev1/fvc > 70% and fev1 > 80% of the predicted values . productivity loss at work was measured according to the quality and quantity scale of the quantity and quality ( qq ) method,21 as described by robroek et al.22,23 participants were requested to indicate how much work they actually performed during regular hours on their most recent regular workday compared to that on a normal workday . the amount of the quality and quantity of productivity was measured on a scale from 0 ( nothing ) to 10 ( regular amount ) . the outcome productivity loss at work was classified into two categories : no productivity loss ( score = 10 ) and productivity loss at work ( score of 9 or lower ) . participants were asked how many days in the past 12 months they were not able to work due to health problems.22,23 those reporting sick leave were categorized into two categories : no sick leave ( 0 day ) and use of sick leave ( 1 day or more ) . systolic and diastolic blood pressure were measured by trained nurses using an automated digital sphygmomanometer ( hem-904 ; omron , kyoto , japan ) placed on the upper arm at heart height while the participant was seated , following 5 minutes of rest . hypertension was defined as antihypertensive medication use , systolic blood pressure of 130 mmhg or more , or diastolic blood pressure of 85 mmhg or more . dyslipidemia was defined as medication use , triglyceride level of 150 mg / dl or more , low - density lipoprotein cholesterol level of 140 mg / dl or more , or high - density lipoprotein cholesterol level of < 40 mg / dl . hyperglycemia was defined as blood glucose - lowering medication use or fasting glucose level of 110 mg / dl or more , as previously described.15 differences among normal lung function , mild al , and moderate - to - severe al were compared using an analysis of variance , the kruskal wallis test for continuous variables , or the chi - square test for categorical variables . the post hoc scheffe s test was used to assess the difference in characteristics according to lung function status . a multivariate logistic regression model adjusted for sex , age , body mass index ( bmi ) , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment was used to assess the relationship between severity of al and productivity loss at work as well as use of sick leave , with the odds ratios ( ors ) were estimated as measures of association with corresponding 95% confidence intervals ( 95% cis ) . adjustments were made according to sex , age , and bmi ( model 1 ) . in order to study the influence of lifestyle - related factors ( such as models 2 [ model 1 + smoking status ] , 3 [ model 2 + hypertension , hyperglycemia , and dyslipidemia ] , and 4 [ model 3 + sleep duration ] ) and work - related factors ( such as models 5 [ model 4 + work hours per day ] and 6 [ model 5 + workplace smoking environment ] ) , these factors were added separately to the basic statistical model ( model 1 ) describing the association between al severity and productivity loss at work or the presence of sick leave . all statistical analyses were performed using ibm spss statistics 22.0 software ( ibm corporation , armonk , ny , usa ) . whether the data showed normal distribution was assessed by shapiro wilk test . table 1 shows the study participants characteristics based on lung function status . of the 1,378 participants , 98 ( 7.1% ) had al , similar to the percentage reported by the nice study.24 the prevalence of al in this study population for the mild and moderate - to - severe gold stages of al was 3.5% ( n=48 ) and 3.6% ( n=50 ) , respectively . in this study , the prevalence of self - reported obstructive lung disease such as asthma and copd among participants with al was 23.5% ( n=23 ) and 1.0% ( n=1 ) , respectively . significant differences were seen between the normal lung function and mild al groups in terms of age and pack - years . significant differences were also seen between those with normal lung function and moderate - to - severe al in terms of age , weight , and pack - years . significant differences were seen in relation to sleep duration and workplace smoking environment . on the other hand , tables 2 and 3 show the ors for the quality and quantity of productivity loss at work with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the presence of the quality of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 1.97 , 95% ci : 1.103.52 , p=0.022 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.01 , 1.123.60 , and 0.019 ; 1.99 , 1.113.58 , and 0.021 ; 1.95 , 1.083.51 , and 0.027 ; 1.94 , 1.073.50 , and 0.028 ; and 2.04 , 1.123.71 , and 0.02 , respectively . there were no significant differences between normal lung function and mild al ( table 2 ) . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the quantity of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.16 , 95% ci : 1.213.87 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.17 , 1.213.90 , and 0.01 ; 2.16 , 1.203.89 , and 0.011 ; 2.11 , 1.173.81 , and 0.013 ; 2.10 , 1.163.80 , and 0.014 ; and 2.19 , 1.204.00 , and 0.011 , respectively . there were no significant differences between normal lung function and mild al ( table 3 ) . table 4 shows the ors for the use of sick leave with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , use of sick leave was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.45 , 95% ci : 1.244.86 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.40 , 1.214.76 , and 0.012 ; 2.43 , 1.224.85 , and 0.012 ; 2.50 , 1.255.00 , and 0.01 ; 2.51 , 1.255.02 , and 0.01 ; and 2.69 , 1.335.44 , and 0.006 , respectively . there were no significant differences between normal lung function and mild al ( table 4 ) . table 1 shows the study participants characteristics based on lung function status . of the 1,378 participants , 98 ( 7.1% ) had al , similar to the percentage reported by the nice study.24 the prevalence of al in this study population for the mild and moderate - to - severe gold stages of al was 3.5% ( n=48 ) and 3.6% ( n=50 ) , respectively . in this study , the prevalence of self - reported obstructive lung disease such as asthma and copd among participants with al was 23.5% ( n=23 ) and 1.0% ( n=1 ) , respectively . significant differences were seen between the normal lung function and mild al groups in terms of age and pack - years . significant differences were also seen between those with normal lung function and moderate - to - severe al in terms of age , weight , and pack - years . significant differences were seen in relation to sleep duration and workplace smoking environment . on the other hand , tables 2 and 3 show the ors for the quality and quantity of productivity loss at work with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the presence of the quality of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 1.97 , 95% ci : 1.103.52 , p=0.022 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.01 , 1.123.60 , and 0.019 ; 1.99 , 1.113.58 , and 0.021 ; 1.95 , 1.083.51 , and 0.027 ; 1.94 , 1.073.50 , and 0.028 ; and 2.04 , 1.123.71 , and 0.02 , respectively . there were no significant differences between normal lung function and mild al ( table 2 ) . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the quantity of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.16 , 95% ci : 1.213.87 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.17 , 1.213.90 , and 0.01 ; 2.16 , 1.203.89 , and 0.011 ; 2.11 , 1.173.81 , and 0.013 ; 2.10 , 1.163.80 , and 0.014 ; and 2.19 , 1.204.00 , and 0.011 , respectively . there were no significant differences between normal lung function and mild al ( table 3 ) . table 4 shows the ors for the use of sick leave with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , use of sick leave was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.45 , 95% ci : 1.244.86 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.40 , 1.214.76 , and 0.012 ; 2.43 , 1.224.85 , and 0.012 ; 2.50 , 1.255.00 , and 0.01 ; 2.51 , 1.255.02 , and 0.01 ; and 2.69 , 1.335.44 , and 0.006 , respectively . there were no significant differences between normal lung function and mild al ( table 4 ) . the main findings of this study are that productivity loss and use of sick leaves were significantly higher in participants with moderate - to - severe al . this study focused on the possible relationship of al severity with work productivity loss and sick leave and revealed that productivity loss at work and use of sick leave increased with al severity in japanese workers . in this study , we demonstrated that workers with moderate - to - severe al were more likely to report quality and quantity of productivity loss at work ( or = 2.04 , 95% ci : 1.123.71 , p=0.02 and or = 2.19 , 95% ci : 1.204.00 , p=0.011 , respectively ) and use of sick leave ( or = 2.69 , 95% ci : 1.335.44 , p=0.006 ) after adjusting for sex , age , bmi , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment . thus , these results suggested that increasing severity of al was associated with productivity loss at work and use of sick leave . previous research by robroek et al22 found that lifestyle - related factors , especially smoking and obesity , were associated with productivity loss at work and use of sick leave . comorbidities are major determinants of health status and health expenditure in patients with copd.1,25,26 patients with copd with comorbidities have poorer health outcomes than those without comorbidities.1,25,26 both insomnia and short sleep duration were independently associated with poor work ability.27 in this study , none of the participants demonstrated insomnia . health problems , as shown in the following paragraphs , might relate to work productivity . therefore , in this study , participants with respiratory diseases except for copd and asthma , mental disorders,28,29 allergic diseases , all kinds of cancer , cerebrovascular disease , brain tumor , ischemic heart disease , other cardiac diseases , collagen disease , mnire s disease , vertigo , and chronic headache were excluded from this study . mental disorders were independently associated with absenteeism and presenteeism.28,29 moreover , workplace smoking environment is related to absenteeism and productivity costs.30 therefore , these factors were added separately to the basic statistical model describing the association between al severity and productivity loss at work or use of sick leave . the underlying mechanisms of productivity loss at work in participants with al remain poorly understood at present . a previous report suggested a possible explanation of effects of health problems in terms of al on work productivity;8,9 participants with al might slow the pace at which they work and/or take more breaks ( quantity ) . in addition , they may be less careful and have to repeat work due to mistakes ( quality ) . watz et al9 also reported that significant limitations of physical activity are present in patients with copd from gold stage ii ( moderate al).8 thus , limitations of physical activity in participants with moderate - to - severe al might reflect work productivity . first , we did not employ reversibility testing since our institutional review board considered it unacceptable in the absence of a high suspicion of disease . for this reason , participants with al may have included some with a postbronchodilator fev1/fvc ratio > 70% . in this study , the prevalence of self - reported obstructive lung disease such as asthma and copd among participants with al was 23.5% ( n=23 ) and 1.0% ( n=1 ) , respectively . undiagnosed individuals may have had copd , and they may even have had asthma ; thus , we expressed it as al rather than copd . this limitation has also been reported in previous studies by oda et al.15 second , we used subjective single measures of productivity loss at work and use of sick leave according to the qq questionnaire.2123 variability of work ability , which is closely linked to measurement reliability , is an important issue in presenteeism studies . the quantity question of the qq method was associated with objective work output among floor layers ( r=0.48 ) . a disadvantage of this method is that productivity loss is assessed during the previous regular workday , and it does not take into account the expected fluctuations in productivity loss within workers across workdays . this limitation has also been reported in previous studies by robroek et al.22,23 other useful instruments have been developed to evaluate presenteeism , such as the work productivity and activity impairment questionnaire.31 however , it has not been concluded which instrument provides a better presenteeism estimate.32 third , this was a cross - sectional study , and the relatively small sample size for the group of al meant that comparisons against normal lung function had less statistical power . further studies are needed in a larger sample that includes subjects with a wider severity range of al . finally , this study was a single - center study performed with participants who were interested in this research project . , we consider this study to be worthwhile because it is the first to reveal the relationship of al severity with productivity loss at work and use of sick leave in japanese workers , as far as the authors know . this relationship remained significant even after adjusting for a range of potential confounders . even if the cause effect relationships are still unknown , early intervention in the participants with al at the workplace might be beneficial for promoting work ability . additional research is needed to clarify this aspect of presenteeism and absenteeism due to al .

backgroundthe aim of this study was to reveal the association between airflow limitation ( al ) severity and reduction with work productivity as well as use of sick leave among japanese workers.methodsthis cross - sectional study included 1,378 workers who underwent a lung function test during a health checkup at the japanese red cross kumamoto health care center . al was defined as forced expiratory volume in 1 second / forced vital capacity of < 0.7 . workers completed a questionnaire on productivity loss at work and sick leave . the quality and quantity of productivity loss at work were measured on a ten - point scale indicating how much work was actually performed on the previous workday . participants were asked how many days in the past 12 months they were unable to work because of health problems . logistic regression analysis was used to assess the associations between al severity and the quality and quantity of productivity loss at work as well as use of sick leave.resultscompared with workers without al , workers with moderate - to - severe al showed a significant productivity loss ( quality : odds ratio [ or ] = 2.04 , 95% confidence interval [ ci ] : 1.123.71 , p=0.02 and quantity : or = 2.19 , 95% ci : 1.204.00 , p=0.011 ) and use of sick leave ( or = 2.69 , 95% ci : 1.335.44 , p=0.006 ) after adjusting for sex , age , body mass index , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment.conclusional severity was significantly associated with work productivity loss and use of sick leave . our findings suggested that early intervention in the subjects with al at the workforce might be beneficial for promoting work ability .

Introduction Materials and methods Participants Lung function tests Productivity loss at work and sick leave Clinical information Statistical analyses Results Participant characteristics Productivity loss at work Sick leave Discussion Conclusion

few studies have examined the association between copd and work ability as well as use of sick leave.7,1014 employed adults with copd reported significantly lower quality of life and work productivity and increased health care resource utilization than employed adults without copd in the usa.12 workers diagnosed with copd had significantly higher levels of presenteeism and overall work impairment.12 furthermore , there are no data regarding the association between airflow limitation ( al ) severity and productivity at work or use of sick leave . we hypothesized that al could be an important factor associated with loss of productivity at work and use of sick leave . this study aimed to reveal the associations between al severity and productivity at work as well as use of sick leave among japanese workers . participants were employees who underwent medical health checkups at the japanese red cross kumamoto health care center and the medical health checkups included interview questionnaires , a physical examination , blood sampling , and spirometry , as previously described.1518 the interview questionnaires were conducted by a trained public health nurse to obtain data regarding medical history , smoking status , sleep duration , work hours per day , workplace smoking environment , productivity loss at work , and sickness absence . complete data on lung function tests , productivity loss at work , and sickness absence were obtained from 1,866 workers . additionally , participants with forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) > 70% and fev1 predicted < 80% ( n=126 ) were excluded from this study . subjects were divided according to lung function ( 1,280 had normal lung function , 48 had mild al , and 50 had moderate - to - severe al ) ( table 1 ) . our research protocol was approved by the human ethics committee of kumamoto university ( numbers 436 , 816 , and 870 ) and the japanese red cross kumamoto health care center . the subjects were divided into three groups : a control group ( normal lung function ) , gold stage i ( mild al ) , and gold stages ii the participants with normal lung function were defined as having a fev1/fvc > 70% and fev1 > productivity loss at work was measured according to the quality and quantity scale of the quantity and quality ( qq ) method,21 as described by robroek et al.22,23 participants were requested to indicate how much work they actually performed during regular hours on their most recent regular workday compared to that on a normal workday . the amount of the quality and quantity of productivity was measured on a scale from 0 ( nothing ) to 10 ( regular amount ) . the outcome productivity loss at work was classified into two categories : no productivity loss ( score = 10 ) and productivity loss at work ( score of 9 or lower ) . participants were asked how many days in the past 12 months they were not able to work due to health problems.22,23 those reporting sick leave were categorized into two categories : no sick leave ( 0 day ) and use of sick leave ( 1 day or more ) . differences among normal lung function , mild al , and moderate - to - severe al were compared using an analysis of variance , the kruskal wallis test for continuous variables , or the chi - square test for categorical variables . a multivariate logistic regression model adjusted for sex , age , body mass index ( bmi ) , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment was used to assess the relationship between severity of al and productivity loss at work as well as use of sick leave , with normal lung function as the reference . in order to study the influence of lifestyle - related factors ( such as models 2 [ model 1 + smoking status ] , 3 [ model 2 + hypertension , hyperglycemia , and dyslipidemia ] , and 4 [ model 3 + sleep duration ] ) and work - related factors ( such as models 5 [ model 4 + work hours per day ] and 6 [ model 5 + workplace smoking environment ] ) , these factors were added separately to the basic statistical model ( model 1 ) describing the association between al severity and productivity loss at work or the presence of sick leave . participants were employees who underwent medical health checkups at the japanese red cross kumamoto health care center and the medical health checkups included interview questionnaires , a physical examination , blood sampling , and spirometry , as previously described.1518 the interview questionnaires were conducted by a trained public health nurse to obtain data regarding medical history , smoking status , sleep duration , work hours per day , workplace smoking environment , productivity loss at work , and sickness absence . complete data on lung function tests , productivity loss at work , and sickness absence were obtained from 1,866 workers . additionally , participants with forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) > 70% and fev1 predicted < 80% ( n=126 ) were excluded from this study . subjects were divided according to lung function ( 1,280 had normal lung function , 48 had mild al , and 50 had moderate - to - severe al ) ( table 1 ) . our research protocol was approved by the human ethics committee of kumamoto university ( numbers 436 , 816 , and 870 ) and the japanese red cross kumamoto health care center . the criteria used for the al staging were also developed according to the gold guidelines and were as follows : stage i ( mild al ) : fev1/fvc < 70% and fev1 80% of predicted value ; stage ii ( moderate al ) : fev1/fvc < 70% and 50%fev1<80% of predicted value ; stage iii ( severe al ) : fev1/fvc < 70% and 30%fev1<50% of predicted value ; and stage iv ( very severe al ) : fev1/fvc the subjects were divided into three groups : a control group ( normal lung function ) , gold stage i ( mild al ) , and gold stages ii the participants with normal lung function were defined as having a fev1/fvc > 70% and fev1 > 80% of the predicted values . productivity loss at work was measured according to the quality and quantity scale of the quantity and quality ( qq ) method,21 as described by robroek et al.22,23 participants were requested to indicate how much work they actually performed during regular hours on their most recent regular workday compared to that on a normal workday . the amount of the quality and quantity of productivity was measured on a scale from 0 ( nothing ) to 10 ( regular amount ) . the outcome productivity loss at work was classified into two categories : no productivity loss ( score = 10 ) and productivity loss at work ( score of 9 or lower ) . participants were asked how many days in the past 12 months they were not able to work due to health problems.22,23 those reporting sick leave were categorized into two categories : no sick leave ( 0 day ) and use of sick leave ( 1 day or more ) . hyperglycemia was defined as blood glucose - lowering medication use or fasting glucose level of 110 mg / dl or more , as previously described.15 differences among normal lung function , mild al , and moderate - to - severe al were compared using an analysis of variance , the kruskal wallis test for continuous variables , or the chi - square test for categorical variables . the post hoc scheffe s test was used to assess the difference in characteristics according to lung function status . a multivariate logistic regression model adjusted for sex , age , body mass index ( bmi ) , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment was used to assess the relationship between severity of al and productivity loss at work as well as use of sick leave , with the odds ratios ( ors ) were estimated as measures of association with corresponding 95% confidence intervals ( 95% cis ) . in order to study the influence of lifestyle - related factors ( such as models 2 [ model 1 + smoking status ] , 3 [ model 2 + hypertension , hyperglycemia , and dyslipidemia ] , and 4 [ model 3 + sleep duration ] ) and work - related factors ( such as models 5 [ model 4 + work hours per day ] and 6 [ model 5 + workplace smoking environment ] ) , these factors were added separately to the basic statistical model ( model 1 ) describing the association between al severity and productivity loss at work or the presence of sick leave . of the 1,378 participants , 98 ( 7.1% ) had al , similar to the percentage reported by the nice study.24 the prevalence of al in this study population for the mild and moderate - to - severe gold stages of al was 3.5% ( n=48 ) and 3.6% ( n=50 ) , respectively . significant differences were also seen between those with normal lung function and moderate - to - severe al in terms of age , weight , and pack - years . on the other hand , tables 2 and 3 show the ors for the quality and quantity of productivity loss at work with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the presence of the quality of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 1.97 , 95% ci : 1.103.52 , p=0.022 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.01 , 1.123.60 , and 0.019 ; 1.99 , 1.113.58 , and 0.021 ; 1.95 , 1.083.51 , and 0.027 ; 1.94 , 1.073.50 , and 0.028 ; and 2.04 , 1.123.71 , and 0.02 , respectively . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the quantity of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.16 , 95% ci : 1.213.87 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.17 , 1.213.90 , and 0.01 ; 2.16 , 1.203.89 , and 0.011 ; 2.11 , 1.173.81 , and 0.013 ; 2.10 , 1.163.80 , and 0.014 ; and 2.19 , 1.204.00 , and 0.011 , respectively . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , use of sick leave was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.45 , 95% ci : 1.244.86 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.40 , 1.214.76 , and 0.012 ; 2.43 , 1.224.85 , and 0.012 ; 2.50 , 1.255.00 , and 0.01 ; 2.51 , 1.255.02 , and 0.01 ; and 2.69 , 1.335.44 , and 0.006 , respectively . of the 1,378 participants , 98 ( 7.1% ) had al , similar to the percentage reported by the nice study.24 the prevalence of al in this study population for the mild and moderate - to - severe gold stages of al was 3.5% ( n=48 ) and 3.6% ( n=50 ) , respectively . significant differences were also seen between those with normal lung function and moderate - to - severe al in terms of age , weight , and pack - years . on the other hand , tables 2 and 3 show the ors for the quality and quantity of productivity loss at work with normal lung function as the reference . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the presence of the quality of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 1.97 , 95% ci : 1.103.52 , p=0.022 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.01 , 1.123.60 , and 0.019 ; 1.99 , 1.113.58 , and 0.021 ; 1.95 , 1.083.51 , and 0.027 ; 1.94 , 1.073.50 , and 0.028 ; and 2.04 , 1.123.71 , and 0.02 , respectively . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , the quantity of productivity loss was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.16 , 95% ci : 1.213.87 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.17 , 1.213.90 , and 0.01 ; 2.16 , 1.203.89 , and 0.011 ; 2.11 , 1.173.81 , and 0.013 ; 2.10 , 1.163.80 , and 0.014 ; and 2.19 , 1.204.00 , and 0.011 , respectively . in logistic regression models adjusting for sex , age , and bmi ( model 1 ) , use of sick leave was higher in participants with moderate - to - severe al compared to those with normal lung function ( or = 2.45 , 95% ci : 1.244.86 , p=0.01 ) . in logistic regression models 2 ( model 1 + smoking status ) , 3 ( model 2 + hypertension , hyperglycemia , and dyslipidemia ) , 4 ( model 3 + sleep duration ) , 5 ( model 4 + work hours per day ) , and 6 ( model 5 + workplace smoking environment ) , ors , 95% ci , and p - values were 2.40 , 1.214.76 , and 0.012 ; 2.43 , 1.224.85 , and 0.012 ; 2.50 , 1.255.00 , and 0.01 ; 2.51 , 1.255.02 , and 0.01 ; and 2.69 , 1.335.44 , and 0.006 , respectively . the main findings of this study are that productivity loss and use of sick leaves were significantly higher in participants with moderate - to - severe al . this study focused on the possible relationship of al severity with work productivity loss and sick leave and revealed that productivity loss at work and use of sick leave increased with al severity in japanese workers . in this study , we demonstrated that workers with moderate - to - severe al were more likely to report quality and quantity of productivity loss at work ( or = 2.04 , 95% ci : 1.123.71 , p=0.02 and or = 2.19 , 95% ci : 1.204.00 , p=0.011 , respectively ) and use of sick leave ( or = 2.69 , 95% ci : 1.335.44 , p=0.006 ) after adjusting for sex , age , bmi , smoking status , hypertension , hyperglycemia , dyslipidemia , sleep duration , work hours per day , and workplace smoking environment . thus , these results suggested that increasing severity of al was associated with productivity loss at work and use of sick leave . previous research by robroek et al22 found that lifestyle - related factors , especially smoking and obesity , were associated with productivity loss at work and use of sick leave . comorbidities are major determinants of health status and health expenditure in patients with copd.1,25,26 patients with copd with comorbidities have poorer health outcomes than those without comorbidities.1,25,26 both insomnia and short sleep duration were independently associated with poor work ability.27 in this study , none of the participants demonstrated insomnia . mental disorders were independently associated with absenteeism and presenteeism.28,29 moreover , workplace smoking environment is related to absenteeism and productivity costs.30 therefore , these factors were added separately to the basic statistical model describing the association between al severity and productivity loss at work or use of sick leave . the underlying mechanisms of productivity loss at work in participants with al remain poorly understood at present . watz et al9 also reported that significant limitations of physical activity are present in patients with copd from gold stage ii ( moderate al).8 thus , limitations of physical activity in participants with moderate - to - severe al might reflect work productivity . this limitation has also been reported in previous studies by oda et al.15 second , we used subjective single measures of productivity loss at work and use of sick leave according to the qq questionnaire.2123 variability of work ability , which is closely linked to measurement reliability , is an important issue in presenteeism studies . this limitation has also been reported in previous studies by robroek et al.22,23 other useful instruments have been developed to evaluate presenteeism , such as the work productivity and activity impairment questionnaire.31 however , it has not been concluded which instrument provides a better presenteeism estimate.32 third , this was a cross - sectional study , and the relatively small sample size for the group of al meant that comparisons against normal lung function had less statistical power . , we consider this study to be worthwhile because it is the first to reveal the relationship of al severity with productivity loss at work and use of sick leave in japanese workers , as far as the authors know . even if the cause effect relationships are still unknown , early intervention in the participants with al at the workplace might be beneficial for promoting work ability .

according to the estimation of world health organisation , there are about 240 million people who are chronically infected with hepatitis b virus ( hbv ) . the chronic hbv infection is one of the major causes of hepatocellular carcinoma and liver cirrhosis . there is a large body of evidence for the essential role of cell - mediated immune responses for viral clearance in acute hbv infection . standard treatment regimens with pegylated interferon ( ifn)- and nucleoside / nucleotide analogs are used for therapy of chronic hepatitis b but are only partially successful . several recent studies indicated the possibility of stimulating specific immune responses against hbv in chronically infected patients . the recent approaches were summarized in previous reviews of our group and others and in the present issue [ 25 ] . a number of therapeutic vaccination trials using conventional hbv vaccines failed to demonstrate the effectiveness in terms of the induction of hbv - specific immune responses and suppression of hbv replication in chronic hbv carriers [ 4 , 5 ] . new approaches based on dna vaccines or anti - hbs antibody - hbs immune complex are now being tested in clinical trials [ 68 ] . as a principle recognized on the basis of available information , a combined strategy including antiviral treatment and immunomodulation will be needed to stimulate the full range of immune responses to achieve effective control over hbv infection . important aspects of the human hbv infection have been studied with a genetically closely related virus of hepadnaviridae , woodchuck hepatitis virus ( whv ) , which infects a north american rodent , the woodchuck . in the woodchuck model , combinations of antiviral treatment and therapeutic vaccinations led to the induction of specific t cell and b cell responses to whv antigens and sustained suppression of whv replication in some individual animals [ 2 , 4 , 9 , 10 ] . stimulation of innate immune responses may further improve the immunotherapeutic effect of combination strategies against the hepadnaviral infection . the significance of the innate immune response as a defense against microbial infections and its link to the adaptive immune responses has been recognized during the past years . toll - like receptors ( tlrs ) are a group of highly conserved molecules that play a critical role in the recognition of pathogen - associated molecular patterns ( pamps ) and in the activation of innate immune responses to infectious agents . tlrs are structurally characterized by an ectodomain composed of leucine - rich repeats for binding and recognition of pamps and a cytoplasmic domain homologous to the cytoplasmic region of the interleukin ( il)-1 receptor , known as the tir domain , for downstream signaling . tlr ligands are natural macromolecular components derived from pathogens and may be composed of lipids , lipopeptides , proteins , and nucleic acids . some synthetic small molecules could mimic tlr ligands and activate tlr - mediated cellular signaling . a subgroup within the tlr family including tlr3 , tlr7 , tlr8 , and tlr9 is localized in endosomes and recognizes nucleic acids such as viral dna or rna . the other subgroup of surface - expressed tlr1 , tlr2 , tlr4 , tlr5 , and tlr6 recognizes extracellular bacterial and fungal cell wall components , as well as some viral proteins [ 13 , 14 ] . binding of tlr agonists to their receptors initiates the activation of complex networks of intracellular signal transduction pathways to coordinate the inflammatory response . the important components of these signaling networks are the adaptor proteins and several protein kinases including erk , jnk , p38 map kinase , and pi-3 k kinase , and the transcription factors irf3/5/7 , nuclear factor kappa b ( nf-b ) , and ap-1 . pro - inflammatory cytokines , or co - stimulatory molecules , which are involved in antiviral responses [ 15 , 16 ] . the crucial adaptor proteins including myeloid differentiation primary - response protein 88 ( myd88 ) , used by nearly all tlrs except tlr3 , tir domain - containing adaptor protein ( tirap ) , tir domain - containing adaptor protein inducing interferon ( ifn)- ( trif ) , and trif - related adaptor molecule ( tram ) are recruited . tlr4 is unique among tlrs being able to activate two distinct signaling pathways , tirap / myd88 and tram / trif . the myd88-dependent pathway leads to the activation of downstream signal transduction involving il-1r - associated kinases ( iraks ) , tumor necrosis factor receptor ( tnfr)-associated factor 6 ( traf6 ) , transforming growth factor ( tgf)--activated kinase ( tak1 ) , and the inhibitor of nuclear factor-b ( ib ) kinase complex . through the nf-b and activating protein 1 ( ap1 ) , the myd88-dependent tlr activation results in the production of pro - inflammatory cytokines il-6 , il-10 , il-12 , and tnf-. in contrast , the trif - dependent pathway leads to the activation of ifn regulatory factors ( irfs ) and production of type i ifns [ 15 , 17 ] . exceptionally , plasmacytoid dendritic cells produce type i ifn after tlr7 and tlr9 activation via the myd88-irf7-dependent pathway . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines tlr signaling . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines there is accumulating evidence that the innate branch of the host immune system plays an important role in the control of hbv infection [ 1921 ] . the previous studies in chimpanzees and in patients showed that hbv infection does not lead to a measurable response involving type i ifns . investigated the transcriptome of the liver in three chimpanzees during the course of acute hbv infection . their analysis focused on two diverse groups of cellular genes : those in the early phase are associated with the innate immune response , and those in the late phase are associated with the adaptive immune response that terminates infection . they demonstrated that hbv does not induce any genes during entry and expansion , leading the authors to suggest that hbv is a stealth virus in the early phase of infection . by contrast , a large number of ifn--regulated genes are expressed in the liver during viral clearance . the upregulation of ifn--regulated genes in the liver results from the adaptive t cell response as specific t cells infiltrating the liver are major producers of ifn- . thus , hbv infection strongly differs from other viral infections like hcv infection in the early phase , as hcv induces a strong ifn- response in chimpanzees . measured type i ifn production in patients with acute hbv infection and found only a low level of type i ifn not higher than those found in healthy controls . the lack of early ifn response in vivo during acute hbv infection does not necessarily exclude the triggering of host ifn responses by hbv . hbv infection may be initiated only with few viral particles and may not induce a host response at the initial phase of infection that is measurable by gene array technology or cytokine detection . in addition , hbv may inhibit host ifn responses by specific mechanisms as described below . experimental data are available , indicating that hbv interacts with the host innate immune system but is able to inhibit host responses . it has been shown that hbv interacts with hepatic non - parenchymal cells ( npcs ) and induces the production of il-6 , though it is not clear how hepatic npcs sense hbv . within 3 h , these cells release inflammatory cytokines including il-1 , il-6 , il-8 , and tnf- without inducing an ifn response . il-6 is able in turn to inhibit the expression of hepatocyte nuclear factor ( hnf ) 1 and hnf 4 , two transcription factors essential for hbv gene expression and replication . however , relatively high doses of hbv particles are usually used in such experiments to induce measurable responses of host cells . ifn- has been identified as a major antiviral factor produced by npcs in response to tlr3 . . showed that tlr3 ligand poly i : c induces intrahepatic ifn- production and inhibits hbv replication by non - cytolytic mechanisms that either destabilize pregenomic ( pg)rna - containing capsids or prevent their assembly . tested the ability of different tlr ligands to inhibit hbv replication in the hbv transgenic mouse model . consistently , a single - dose injection of tlr3 , tlr4 , tlr5 , tlr7 , and tlr9 ligands suppressed hbv replication in the liver in an ifn-/-dependent manner . in a doxycycline ( dox)-inducible hbv replication system , ifn- pretreatment prevents the production of replication - competent pgrna - containing capsids but does not change the turnover rate of preformed hbv rna - containing capsids . apparently , the formation of replication - competent hbv capsids is one of the major targets of ifn - mediated antiviral actions . a great number of cellular ifn - stimulated genes ( isgs ) are activated by ifns and may inhibit the different steps of the hbv life cycle . a recent publication suggests that hbv covalently closed circular ( ccc ) dna could be degraded by the action of apobec3a and 3b cytidine deaminases . these findings partly explain the therapeutic effect of ifn- in patients with chronic hbv infection . ifn- is widely used to treat chronic hbv infection and can lead to sustained decrease of hbsag and virus clearance in about 30 % of chronically hbv - infected patients . in addition , type i ifns may modulate specific immune responses to hbv , resulting in hbe seroconversion or complete control of hbv infection . tlr3 activation of hepatic npcs could lead to ifn- production and hbv inhibition in in vitro [ 26 , 32 , 33 ] . upon poly i : c stimulation , hepatic npcs such as kupffer cells ( kc ) and liver sinusoid endothelial cells ( lsec ) release antiviral cytokines which are able to inhibit hbv replication in a co - culture model utilizing hbv - met cells that contains an integrated hbv genome . blocking with specific antibodies to type i and ii ifns identified ifn- as the major anti - hbv factor produced by poly i : c - treated npcs . while hbv dna replicative intermediates were efficiently suppressed , viral mrnas as well as secretion of hbsag and hbeag remained largely unchanged . importantly , screening of different tlr ligands demonstrated that hepatic npcs show a significant production of ifn- only in response to tlr3 stimulation ( and a lower extent to tlr4 stimulation , see below ) . therefore , tlr3-mediated response and ifn- production in the liver may contribute to the control of pathogens including hbv in a unique way . several studies suggest that hbv is able to inhibit pattern recognition receptor ( prr ) and ifn signaling . hbv surface and e antigen ( hbsag , hbeag ) and hbv particles could inhibit the activation of npcs by tlr3 ligands . co - culture of hepatic npcs with hbv - met cell supernatants , hbsag , hbeag as well as hbv virions results in abrogation of tlr - induced antiviral activity , correlating with decreased activation of irf3 , nf-b , and erk1/2 in npcs . our most recent data suggest that hbsag may trigger il10 production on hepatic cells and thereby attenuates the tlr3-mediated activation of npcs . this is consistent with the recent publication that hbsag induces tnf- and il-10 production by monocytes which leads to downregulation of tlr9 expression on pdcs , thereby inhibiting ifn- production by pdcs . at high amounts of hbv , in addition , many studies provide evidence that hbv polymerase may counteract the innate responses at two or more steps : ( 1 ) hbv polymerase is able to inhibit the irf3 activation by interacting with rna helicase ddx3 in hepatoma cells [ 37 , 38 ] ; ( 2 ) hbv polymerase is able to block ifn signaling by inhibition of pkc--mediated phosphorylation of stat 1 and importin - dependent translocation of stat 1/stat 2/irf9 complex [ 39 , 40 ] . hbx protein was reported to promote the decay of mitochondrial antiviral signaling protein ( mavs ) , the adaptor of rig - i , and mda5 receptors [ 41 , 42 ] . however , the relevance of these findings for the human hbv infection remains to be defined . tlr3-mediated functions are impaired in patients with chronic hbv infection and may recover partially under successful antiviral treatment . in the woodchuck model , pbmcs from animals with chronic whv infection show reduced responses to poly i : c stimulation . taken together , the interaction of hbv or the molecular components of hbv with the innate immune system is complex , leading both to activation and inhibition of host innate responses . figure 2 depicts the interaction of tlr3 signaling pathway with hbv in a schematic way.fig . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . antiviral isgs like mxa and ifit1/2 inhibit hbv replication at the transcriptional and posttranscriptional steps . . hbv polymerase could interfere with irf3 action and block the nuclear translocation of stat1/2 interaction of hbv and tlr3 . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . antiviral isgs like mxa and ifit1/2 inhibit hbv replication at the transcriptional and posttranscriptional steps . . hbv polymerase could interfere with irf3 action and block the nuclear translocation of stat1/2 unlike to tlr3 , tlr4 activation by lipopolysaccharide ( lps ) leads to low ifn- production only in kcs but not in lsecs and hepatocytes . however , tlr4-activated kcs release other yet undefined antiviral factors , inhibiting hbv replication in hbv - met cells . in contrast to tlr3 ligands , zhang et al . demonstrated that activation of cellular pathways by tlr4 ligands leads to inhibition of hepadnaviral replication . in the model of whv - infected primary hepatocytes ( pwhs ) , lps stimulation led to a pronounced reduction of whv replication intermediates without a significant ifn induction , while poly i : c transfection resulted in the ifn production and a highly increased expression of antiviral genes in pwhs , but only slight inhibitory effect on whv replication . lps was able to activate nf-b , mapk , and pi-3 k / akt pathways in pwhs . the inhibitors of mapk - erk and pi-3 k / akt pathways , but not those of ifn signaling pathways , block the antiviral effect of lps , indicating that ifn - independent pathways which activated by lps are able to downregulate hepadnaviral replication in hepatocytes . tlr2 and tlr4 share the cellular myd88-dependent signaling pathway in mammalian cells ( fig . 3 ) . consequently , tlr2 and tlr4 mediate the activation of the same signaling pathways downstream of myd88 , including nf-b , mapk , and pi-3 k / akt pathways . similarly , tlr2 is able to inhibit hbv or whv replication in human hepatoma cells or pwhs [ 46 , 47 ] . again , the antiviral action of tlr2 is dependent on the presence of adaptor molecules like tak1 , irak1/4 , and traf6 and the downstream mapk and pi-3 k / akt pathways . silencing of the expression of adaptor molecules or blocking the mapk and pi-3 k / akt pathways with chemical inhibitors significantly enhanced hbv replication . in the hbv transgenic mouse model , the injection of a single dose of tlr2 ligands reduced hbv replication in the liver but not as effective as ifn - inducing ligands . it was not examined whether tlr2 ligands also activate mapk and pi-3 k / akt pathways in vivo and thereby exert the antiviral action.fig . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines interaction of hbv and tlr2/4 . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines tlr2 activation and tlr4 activation lead to the production of pro - inflammatory cytokines il6 and tnf- in hepatic npcs and hepatocytes [ 32 , 45 , 47 , 48 ] . though the antiviral effect of tlr2 and tlr4 ligands does not directly depend on the production of pro - inflammatory cytokines , il6 and tnf- have been shown to inhibit hbv replication in primary hepatocytes [ 25 , 49 ] . xu et al . explored the tupaia model to investigate the effect of tnf- on hbv infection . stimulation of hbv - infected primary tupaia hepatocytes with recombinant tupaia tnf- led to viral suppression , while covalently closed circular dna and viral rna were still detectable , leading to the conclusion that tnf- may also contribute to control hbv infection . obviously , hbv developed measures to counteract the antiviral functions mediated by tlr2 . using hepatocytes and kcs isolated from liver biopsies of patients with chb , visvanathan et al . showed significantly decreased tlr2 expression on hepatocytes , kcs , and peripheral monocytes in patients with hbeag - positive chb in comparison with hbeag - negative chb and controls . hepatic cell lines harboring a recombinant baculovirus encoding hbv significantly reduced tnf- expression as well as phospho - p38 kinase expression in the presence of hbeag . in the absence of hbeag , hbv replication was associated with upregulation of the tlr2 pathway resulting in increased tnf- expression . hbeag was found to co - localize with toll / il-1 receptor ( tir)-containing proteins tram , mal , and tlr2 , interact with tir proteins mal and tram , and disrupt the homotypic tir tir interaction . consequently , hbeag suppressed tir - mediated activation of the inflammatory transcription factors , nf-b , and interferon- promoter activity . consistently , tlr2 expression was found to be significantly suppressed in pbmcs from chronically hbv - infected patients and in woodchuck liver tissue and pbmcs if chronically infected with whv [ 47 , 52 ] . previously , wu et al . demonstrated that hbv blocks the myd88 expression , the central adaptor molecule in tlr - mediated innate immune responses , by an antagonistic activity of the terminal protein ( tp ) domain of the hbv polymerase . it could be shown that hbv polymerase is able to block the nuclear translocation of stat 1 thus representing a general inhibitor of ifn signaling and ifn - inducible myd88 expression . activation of innate immunity is a prerequisite for proper adaptive immune responses . as an example , tlr2 is expressed widely such on antigen - presenting cells ( apc ) , endothelial and epithelial cells as well as on t - lymphocytes on which it acts as a costimulatory molecule . wu et al . found previously that tlr2 ligands could trigger the expression of costimulatory molecules on hepatic npcs . lsecs are unique organ - resident antigen - presenting cells capable of antigen cross - presentation and reported to prime nave cd8 + t cells to memory cells at non - inflammatory conditions . under certain conditions , recently , we examined functional maturation of lsecs by tlr ligand stimulation , demonstrating that pretreatment of lsecs with tlr1/2 ligand but not tlr3 and tlr4 ligands reverts their suppressive properties to induce specific t cell immunity . il-12 was identified to be one essential mediator for lsec - mediated cd8 + t cell immunity , which was produced at a low level but sustainably after tlr2 stimulation . our findings suggest that tlr2 activation has a great impact on t cell immunity in the liver and may be used to stimulate specific immune responses to persistent infection of hbv and hcv . on the other hand , wang et al . could show that hbsag inhibits tlr2-mediated stimulation of human pbmcs and il12 production . in the presence of hbsag , both pam3csk4-triggered il-12p40 mrna expression and il-12 production in phorbol 12-myristate 13-acetate ( pma)-differentiated thp-1 macrophages are reduced in a dose - dependent manner , while the production of il-1 , il-6 , il-8 , il-10 , and tnf- is not affected . the presence of hbsag inhibits the tlr2-mediated activation of nf-b and mapk signaling , by selective impairment of jnk-1/2 and c - jun phosphorylation . thus , hbsag likely interferes with the initiation of adaptive immune responses by selective inhibition of tlr2-stimulated il-12 production in monocytes . the findings mentioned above suggest that tlr ligands may be used for therapeutic approaches against chronic viral infection . however , only few trials using tlr ligands for therapies against viral infections have been carried out until now . cpg oligonucleotides ( odn ) , ligands of tlr9 , have been considered as promising candidates . a large number of new reagents that are potentially suitable as immunomodulators and therapeutics the sequence could be generated by modifications of cpg odn [ 58 , 59 ] . however , a candidate cpg odn has been tested in clinical trials and the woodchuck model for treatment of chronic hepatitis c and b but failed to show sufficient therapeutic effect if applied alone . recently , tlr7 ligand gs-9620 has been examined for its antiviral effect in the woodchuck and chimpanzee models . interestingly , a 4-week treatment with gs-9620 resulted in a sustained , marked reduction of serum whv dna and whv surface antigen ( whsag ) levels and in the induction of anti - whs antibody response , as well as a markedly decreased incidence of hepatocellular carcinoma in chronic whv - infected woodchucks . in chimpanzees , gs-9620 induced an increase of serum ifn- in a dose - dependent manner and triggered the isg expression in pbmcs and the liver . a reduction of hbv viral load and serum hbsag was observed in three chronically hbv - infected chimpanzees treated with gs-9620 . tlr7/8 ligands are promising drug candidates if their toxicity could be reduced to a tolerable range . a potential use of tlr ligands as adjuvants for therapeutic vaccines has been considered for long time . the rational design of specific tlr agonists may increase potency and tolerability of new adjuvants and provides the opportunity to meet the stringent safety criteria for new vaccine formulation . two improved adult hbv vaccines fendrix and supervax using tlr4 agonists as adjuvant are now on the market [ 63 , 64 ] . monophosphoryl lipid a ( mpla ) , a chemically modified derivative of the lipid a moiety of lps , is considerably less toxic but has similar immunostimulatory activity . mpla - formulated hepatitis b vaccines elicit protective anti - hbs antibody titers with only two injections instead of three . a class b cpg odn called 1018 iss in combination with recombinant hbsag has been tested in a phase iii clinical trial for persons older than 40 years of age . this vaccine formulation increases seroprotection rates to 100 % , compared with a rate of only 64 % in the alum - adjuvanted rhbsag group . such a vaccine formulation may be used for patients with impaired immune system , as it is more effective in the hypo - responsive population than conventional hbv vaccines . based on the current knowledge , tlr - mediated innate responses may not control hbv infection alone . though stimulation with tlr ligands reduces hbv replication in hepatocytes , the tlr - mediated antiviral action against hbv is far less efficient than those achieved by nucleoside analogs . the innate immune system is known to respond fast and aimed to slow down viral spread in primary infections ; thus , the link from innate to adaptive immune responses may be more important for the control of viral infection . recently , it was shown that poly i : c treatment leads to hbv clearance in hydrodynamic injection mouse model . in this model , hbv clearance required ifn- and ifn- , indicating a complex mode of poly i : c - triggered action . cxcr3 was also essential for hbv clearance after poly i : c injection , apparently responsible for the recruitment of t cells . other studies conducted by several groups have shown that tlr2 is expressed on activated and memory cd4 + and cd8 + t cells and serves as co - stimulatory molecule to enhance their proliferation , survival , and functions [ 7073 ] . tlr2 agonists stimulate activated t cells thus promoting their proliferation and differentiation in vitro and in vivo . consistently , tlr2 ligand pam3csk4 application in vivo with transferred tumor antigen ( ag)-specific cd8 + t cells results in enhanced therapeutic efficacy of these cd8 + t cells in tumor models [ 74 , 75 ] . moreover , covalent linkage of tlr2 ligand pam3csk4 or pam2csk4 with peptides representing cd8 + t cell or b cell epitopes efficiently primed respective specific cd8 + t cell or b cell immune responses in vivo [ 7679 ] . it was shown that tlr2 engagement on cd8 + t cells increased t - bet transcription in a myd88-akt - mtor- and protein kinase c - dependent manner . the molecular mechanisms underlying tlr2-mediated t cell proliferation and functional differentiation in hbv infection need in - depth analysis . thus , future research should not only investigate the direct antiviral effect of tlr - mediated action but also analyze and optimize the connection of innate and adaptive immune responses . in the specific case of tlr2 , it is desirable to identify specific markers expressed on cd8 + t cells after tlr2 stimulation . such markers may facilitate future analysis of cd8 + t cells in vitro and vivo and understanding the inhibitory action of hbv on tlr2 co - stimulation of cd8 + t cells . the significance of the innate immune response as a defense against microbial infections and its link to the adaptive immune responses has been recognized during the past years . toll - like receptors ( tlrs ) are a group of highly conserved molecules that play a critical role in the recognition of pathogen - associated molecular patterns ( pamps ) and in the activation of innate immune responses to infectious agents . tlrs are structurally characterized by an ectodomain composed of leucine - rich repeats for binding and recognition of pamps and a cytoplasmic domain homologous to the cytoplasmic region of the interleukin ( il)-1 receptor , known as the tir domain , for downstream signaling . tlr ligands are natural macromolecular components derived from pathogens and may be composed of lipids , lipopeptides , proteins , and nucleic acids . some synthetic small molecules could mimic tlr ligands and activate tlr - mediated cellular signaling . a subgroup within the tlr family including tlr3 , tlr7 , tlr8 , and tlr9 is localized in endosomes and recognizes nucleic acids such as viral dna or rna . the other subgroup of surface - expressed tlr1 , tlr2 , tlr4 , tlr5 , and tlr6 recognizes extracellular bacterial and fungal cell wall components , as well as some viral proteins [ 13 , 14 ] . binding of tlr agonists to their receptors initiates the activation of complex networks of intracellular signal transduction pathways to coordinate the inflammatory response . conformational changes and dimerization of tlrs occur upon binding with ligands . the important components of these signaling networks are the adaptor proteins and several protein kinases including erk , jnk , p38 map kinase , and pi-3 k kinase , and the transcription factors irf3/5/7 , nuclear factor kappa b ( nf-b ) , and ap-1 . the activation of these transcription factors leads to the induction of type i ifns , pro - inflammatory cytokines , or co - stimulatory molecules , which are involved in antiviral responses [ 15 , 16 ] . the crucial adaptor proteins including myeloid differentiation primary - response protein 88 ( myd88 ) , used by nearly all tlrs except tlr3 , tir domain - containing adaptor protein ( tirap ) , tir domain - containing adaptor protein inducing interferon ( ifn)- ( trif ) , and trif - related adaptor molecule ( tram ) are recruited . tlr4 is unique among tlrs being able to activate two distinct signaling pathways , tirap / myd88 and tram / trif . the myd88-dependent pathway leads to the activation of downstream signal transduction involving il-1r - associated kinases ( iraks ) , tumor necrosis factor receptor ( tnfr)-associated factor 6 ( traf6 ) , transforming growth factor ( tgf)--activated kinase ( tak1 ) , and the inhibitor of nuclear factor-b ( ib ) kinase complex . through the nf-b and activating protein 1 ( ap1 ) , the myd88-dependent tlr activation results in the production of pro - inflammatory cytokines il-6 , il-10 , il-12 , and tnf-. in contrast , the trif - dependent pathway leads to the activation of ifn regulatory factors ( irfs ) and production of type i ifns [ 15 , 17 ] . exceptionally , plasmacytoid dendritic cells produce type i ifn after tlr7 and tlr9 activation via the myd88-irf7-dependent pathway . 1tlr signaling . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines tlr signaling . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines there is accumulating evidence that the innate branch of the host immune system plays an important role in the control of hbv infection [ 1921 ] . the previous studies in chimpanzees and in patients showed that hbv infection does not lead to a measurable response involving type i ifns . investigated the transcriptome of the liver in three chimpanzees during the course of acute hbv infection . their analysis focused on two diverse groups of cellular genes : those in the early phase are associated with the innate immune response , and those in the late phase are associated with the adaptive immune response that terminates infection . they demonstrated that hbv does not induce any genes during entry and expansion , leading the authors to suggest that hbv is a by contrast , a large number of ifn--regulated genes are expressed in the liver during viral clearance . the upregulation of ifn--regulated genes in the liver results from the adaptive t cell response as specific t cells infiltrating the liver are major producers of ifn- . thus , hbv infection strongly differs from other viral infections like hcv infection in the early phase , as hcv induces a strong ifn- response in chimpanzees . measured type i ifn production in patients with acute hbv infection and found only a low level of type i ifn not higher than those found in healthy controls . the lack of early ifn response in vivo during acute hbv infection does not necessarily exclude the triggering of host ifn responses by hbv . hbv infection may be initiated only with few viral particles and may not induce a host response at the initial phase of infection that is measurable by gene array technology or cytokine detection . in addition , hbv may inhibit host ifn responses by specific mechanisms as described below . experimental data are available , indicating that hbv interacts with the host innate immune system but is able to inhibit host responses . it has been shown that hbv interacts with hepatic non - parenchymal cells ( npcs ) and induces the production of il-6 , though it is not clear how hepatic npcs sense hbv . within 3 h , these cells release inflammatory cytokines including il-1 , il-6 , il-8 , and tnf- without inducing an ifn response . il-6 is able in turn to inhibit the expression of hepatocyte nuclear factor ( hnf ) 1 and hnf 4 , two transcription factors essential for hbv gene expression and replication . however , relatively high doses of hbv particles are usually used in such experiments to induce measurable responses of host cells . ifn- has been identified as a major antiviral factor produced by npcs in response to tlr3 . . showed that tlr3 ligand poly i : c induces intrahepatic ifn- production and inhibits hbv replication by non - cytolytic mechanisms that either destabilize pregenomic ( pg)rna - containing capsids or prevent their assembly . tested the ability of different tlr ligands to inhibit hbv replication in the hbv transgenic mouse model . consistently , a single - dose injection of tlr3 , tlr4 , tlr5 , tlr7 , and tlr9 ligands suppressed hbv replication in the liver in an ifn-/-dependent manner . in a doxycycline ( dox)-inducible hbv replication system , ifn- pretreatment prevents the production of replication - competent pgrna - containing capsids but does not change the turnover rate of preformed hbv rna - containing capsids . apparently , the formation of replication - competent hbv capsids is one of the major targets of ifn - mediated antiviral actions . a great number of cellular ifn - stimulated genes ( isgs ) are activated by ifns and may inhibit the different steps of the hbv life cycle . a recent publication suggests that hbv covalently closed circular ( ccc ) dna could be degraded by the action of apobec3a and 3b cytidine deaminases . these findings partly explain the therapeutic effect of ifn- in patients with chronic hbv infection . ifn- is widely used to treat chronic hbv infection and can lead to sustained decrease of hbsag and virus clearance in about 30 % of chronically hbv - infected patients . in addition , type i ifns may modulate specific immune responses to hbv , resulting in hbe seroconversion or complete control of hbv infection . tlr3 activation of hepatic npcs could lead to ifn- production and hbv inhibition in in vitro [ 26 , 32 , 33 ] . upon poly i : c stimulation , hepatic npcs such as kupffer cells ( kc ) and liver sinusoid endothelial cells ( lsec ) release antiviral cytokines which are able to inhibit hbv replication in a co - culture model utilizing hbv - met cells that contains an integrated hbv genome . blocking with specific antibodies to type i and ii ifns identified ifn- as the major anti - hbv factor produced by poly i : c - treated npcs . while hbv dna replicative intermediates were efficiently suppressed , viral mrnas as well as secretion of hbsag and hbeag remained largely unchanged . importantly , screening of different tlr ligands demonstrated that hepatic npcs show a significant production of ifn- only in response to tlr3 stimulation ( and a lower extent to tlr4 stimulation , see below ) . therefore , tlr3-mediated response and ifn- production in the liver may contribute to the control of pathogens including hbv in a unique way . several studies suggest that hbv is able to inhibit pattern recognition receptor ( prr ) and ifn signaling . hbv surface and e antigen ( hbsag , hbeag ) and hbv particles could inhibit the activation of npcs by tlr3 ligands . co - culture of hepatic npcs with hbv - met cell supernatants , hbsag , hbeag as well as hbv virions results in abrogation of tlr - induced antiviral activity , correlating with decreased activation of irf3 , nf-b , and erk1/2 in npcs . our most recent data suggest that hbsag may trigger il10 production on hepatic cells and thereby attenuates the tlr3-mediated activation of npcs . this is consistent with the recent publication that hbsag induces tnf- and il-10 production by monocytes which leads to downregulation of tlr9 expression on pdcs , thereby inhibiting ifn- production by pdcs . at high amounts of hbv , in addition , many studies provide evidence that hbv polymerase may counteract the innate responses at two or more steps : ( 1 ) hbv polymerase is able to inhibit the irf3 activation by interacting with rna helicase ddx3 in hepatoma cells [ 37 , 38 ] ; ( 2 ) hbv polymerase is able to block ifn signaling by inhibition of pkc--mediated phosphorylation of stat 1 and importin - dependent translocation of stat 1/stat 2/irf9 complex [ 39 , 40 ] . hbx protein was reported to promote the decay of mitochondrial antiviral signaling protein ( mavs ) , the adaptor of rig - i , and mda5 receptors [ 41 , 42 ] . however , the relevance of these findings for the human hbv infection remains to be defined . tlr3-mediated functions are impaired in patients with chronic hbv infection and may recover partially under successful antiviral treatment . in the woodchuck model , pbmcs from animals with chronic whv infection show reduced responses to poly i : c stimulation . taken together , the interaction of hbv or the molecular components of hbv with the innate immune system is complex , leading both to activation and inhibition of host innate responses . figure 2 depicts the interaction of tlr3 signaling pathway with hbv in a schematic way.fig . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . antiviral isgs like mxa and ifit1/2 inhibit hbv replication at the transcriptional and posttranscriptional steps . . hbv polymerase could interfere with irf3 action and block the nuclear translocation of stat1/2 interaction of hbv and tlr3 . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . antiviral isgs like mxa and ifit1/2 inhibit hbv replication at the transcriptional and posttranscriptional steps . unlike to tlr3 , tlr4 activation by lipopolysaccharide ( lps ) leads to low ifn- production only in kcs but not in lsecs and hepatocytes . however , tlr4-activated kcs release other yet undefined antiviral factors , inhibiting hbv replication in hbv - met cells . in contrast to tlr3 ligands , zhang et al . demonstrated that activation of cellular pathways by tlr4 ligands leads to inhibition of hepadnaviral replication . in the model of whv - infected primary hepatocytes ( pwhs ) , lps stimulation led to a pronounced reduction of whv replication intermediates without a significant ifn induction , while poly i : c transfection resulted in the ifn production and a highly increased expression of antiviral genes in pwhs , but only slight inhibitory effect on whv replication . lps was able to activate nf-b , mapk , and pi-3 k / akt pathways in pwhs . the inhibitors of mapk - erk and pi-3 k / akt pathways , but not those of ifn signaling pathways , block the antiviral effect of lps , indicating that ifn - independent pathways which activated by lps are able to downregulate hepadnaviral replication in hepatocytes . tlr2 and tlr4 share the cellular myd88-dependent signaling pathway in mammalian cells ( fig . 3 ) . consequently , tlr2 and tlr4 mediate the activation of the same signaling pathways downstream of myd88 , including nf-b , mapk , and pi-3 k / akt pathways . similarly , tlr2 is able to inhibit hbv or whv replication in human hepatoma cells or pwhs [ 46 , 47 ] . again , the antiviral action of tlr2 is dependent on the presence of adaptor molecules like tak1 , irak1/4 , and traf6 and the downstream mapk and pi-3 k / akt pathways . silencing of the expression of adaptor molecules or blocking the mapk and pi-3 k / akt pathways with chemical inhibitors significantly enhanced hbv replication . in the hbv transgenic mouse model , the injection of a single dose of tlr2 ligands reduced hbv replication in the liver but not as effective as ifn - inducing ligands . it was not examined whether tlr2 ligands also activate mapk and pi-3 k / akt pathways in vivo and thereby exert the antiviral action.fig . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines interaction of hbv and tlr2/4 . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines tlr2 activation and tlr4 activation lead to the production of pro - inflammatory cytokines il6 and tnf- in hepatic npcs and hepatocytes [ 32 , 45 , 47 , 48 ] . though the antiviral effect of tlr2 and tlr4 ligands does not directly depend on the production of pro - inflammatory cytokines , il6 and tnf- have been shown to inhibit hbv replication in primary hepatocytes [ 25 , 49 ] . xu et al . explored the tupaia model to investigate the effect of tnf- on hbv infection . stimulation of hbv - infected primary tupaia hepatocytes with recombinant tupaia tnf- led to viral suppression , while covalently closed circular dna and viral rna were still detectable , leading to the conclusion that tnf- may also contribute to control hbv infection . obviously , hbv developed measures to counteract the antiviral functions mediated by tlr2 . using hepatocytes and kcs isolated from liver biopsies of patients with chb , visvanathan et al . showed significantly decreased tlr2 expression on hepatocytes , kcs , and peripheral monocytes in patients with hbeag - positive chb in comparison with hbeag - negative chb and controls . hepatic cell lines harboring a recombinant baculovirus encoding hbv significantly reduced tnf- expression as well as phospho - p38 kinase expression in the presence of hbeag . in the absence of hbeag , hbv replication was associated with upregulation of the tlr2 pathway resulting in increased tnf- expression . hbeag was found to co - localize with toll / il-1 receptor ( tir)-containing proteins tram , mal , and tlr2 , interact with tir proteins mal and tram , and disrupt the homotypic tir tir interaction . consequently , hbeag suppressed tir - mediated activation of the inflammatory transcription factors , nf-b , and interferon- promoter activity . consistently , tlr2 expression was found to be significantly suppressed in pbmcs from chronically hbv - infected patients and in woodchuck liver tissue and pbmcs if chronically infected with whv [ 47 , 52 ] . previously , wu et al . demonstrated that hbv blocks the myd88 expression , the central adaptor molecule in tlr - mediated innate immune responses , by an antagonistic activity of the terminal protein ( tp ) domain of the hbv polymerase . it could be shown that hbv polymerase is able to block the nuclear translocation of stat 1 thus representing a general inhibitor of ifn signaling and ifn - inducible myd88 expression . activation of innate immunity is a prerequisite for proper adaptive immune responses . as an example , tlr2 is expressed widely such on antigen - presenting cells ( apc ) , endothelial and epithelial cells as well as on t - lymphocytes on which it acts as a costimulatory molecule . wu et al . found previously that tlr2 ligands could trigger the expression of costimulatory molecules on hepatic npcs . lsecs are unique organ - resident antigen - presenting cells capable of antigen cross - presentation and reported to prime nave cd8 + t cells to memory cells at non - inflammatory conditions . under certain conditions , recently , we examined functional maturation of lsecs by tlr ligand stimulation , demonstrating that pretreatment of lsecs with tlr1/2 ligand but not tlr3 and tlr4 ligands reverts their suppressive properties to induce specific t cell immunity . il-12 was identified to be one essential mediator for lsec - mediated cd8 + t cell immunity , which was produced at a low level but sustainably after tlr2 stimulation . our findings suggest that tlr2 activation has a great impact on t cell immunity in the liver and may be used to stimulate specific immune responses to persistent infection of hbv and hcv . on the other hand , wang et al . could show that hbsag inhibits tlr2-mediated stimulation of human pbmcs and il12 production . in the presence of hbsag , both pam3csk4-triggered il-12p40 mrna expression and il-12 production in phorbol 12-myristate 13-acetate ( pma)-differentiated thp-1 macrophages are reduced in a dose - dependent manner , while the production of il-1 , il-6 , il-8 , il-10 , and tnf- is not affected . the presence of hbsag inhibits the tlr2-mediated activation of nf-b and mapk signaling , by selective impairment of jnk-1/2 and c - jun phosphorylation . thus , hbsag likely interferes with the initiation of adaptive immune responses by selective inhibition of tlr2-stimulated il-12 production in monocytes . the findings mentioned above suggest that tlr ligands may be used for therapeutic approaches against chronic viral infection . however , only few trials using tlr ligands for therapies against viral infections have been carried out until now . cpg oligonucleotides ( odn ) , ligands of tlr9 , have been considered as promising candidates . a large number of new reagents that are potentially suitable as immunomodulators and therapeutics the sequence could be generated by modifications of cpg odn [ 58 , 59 ] . however , a candidate cpg odn has been tested in clinical trials and the woodchuck model for treatment of chronic hepatitis c and b but failed to show sufficient therapeutic effect if applied alone . recently , tlr7 ligand gs-9620 has been examined for its antiviral effect in the woodchuck and chimpanzee models . interestingly , a 4-week treatment with gs-9620 resulted in a sustained , marked reduction of serum whv dna and whv surface antigen ( whsag ) levels and in the induction of anti - whs antibody response , as well as a markedly decreased incidence of hepatocellular carcinoma in chronic whv - infected woodchucks . in chimpanzees , gs-9620 induced an increase of serum ifn- in a dose - dependent manner and triggered the isg expression in pbmcs and the liver . a reduction of hbv viral load and serum hbsag was observed in three chronically hbv - infected chimpanzees treated with gs-9620 . tlr7/8 ligands are promising drug candidates if their toxicity could be reduced to a tolerable range . a potential use of tlr ligands as adjuvants for therapeutic vaccines has been considered for long time . the rational design of specific tlr agonists may increase potency and tolerability of new adjuvants and provides the opportunity to meet the stringent safety criteria for new vaccine formulation . two improved adult hbv vaccines fendrix and supervax using tlr4 agonists as adjuvant are now on the market [ 63 , 64 ] . monophosphoryl lipid a ( mpla ) , a chemically modified derivative of the lipid a moiety of lps , is considerably less toxic but has similar immunostimulatory activity . mpla - formulated hepatitis b vaccines elicit protective anti - hbs antibody titers with only two injections instead of three . a class b cpg odn called 1018 iss in combination with recombinant hbsag has been tested in a phase iii clinical trial for persons older than 40 years of age . this vaccine formulation increases seroprotection rates to 100 % , compared with a rate of only 64 % in the alum - adjuvanted rhbsag group . such a vaccine formulation may be used for patients with impaired immune system , as it is more effective in the hypo - responsive population than conventional hbv vaccines . based on the current knowledge , tlr - mediated innate responses may not control hbv infection alone . though stimulation with tlr ligands reduces hbv replication in hepatocytes , the tlr - mediated antiviral action against hbv is far less efficient than those achieved by nucleoside analogs . the innate immune system is known to respond fast and aimed to slow down viral spread in primary infections ; thus , the link from innate to adaptive immune responses may be more important for the control of viral infection . recently , it was shown that poly i : c treatment leads to hbv clearance in hydrodynamic injection mouse model . in this model , hbv clearance required ifn- and ifn- , indicating a complex mode of poly i : c - triggered action . cxcr3 was also essential for hbv clearance after poly i : c injection , apparently responsible for the recruitment of t cells . other studies conducted by several groups have shown that tlr2 is expressed on activated and memory cd4 + and cd8 + t cells and serves as co - stimulatory molecule to enhance their proliferation , survival , and functions [ 7073 ] . tlr2 agonists stimulate activated t cells thus promoting their proliferation and differentiation in vitro and in vivo . consistently , tlr2 ligand pam3csk4 application in vivo with transferred tumor antigen ( ag)-specific cd8 + t cells results in enhanced therapeutic efficacy of these cd8 + t cells in tumor models [ 74 , 75 ] . moreover , covalent linkage of tlr2 ligand pam3csk4 or pam2csk4 with peptides representing cd8 + t cell or b cell epitopes efficiently primed respective specific cd8 + t cell or b cell immune responses in vivo [ 7679 ] . it was shown that tlr2 engagement on cd8 + t cells increased t - bet transcription in a myd88-akt - mtor- and protein kinase c - dependent manner . the molecular mechanisms underlying tlr2-mediated t cell proliferation and functional differentiation in hbv infection need in - depth analysis . thus , future research should not only investigate the direct antiviral effect of tlr - mediated action but also analyze and optimize the connection of innate and adaptive immune responses . in the specific case of tlr2 , it is desirable to identify specific markers expressed on cd8 + t cells after tlr2 stimulation . such markers may facilitate future analysis of cd8 + t cells in vitro and vivo and understanding the inhibitory action of hbv on tlr2 co - stimulation of cd8 + t cells .

the role of adaptive immune responses in the control of hepatitis b virus ( hbv ) infection is well accepted . the contribution of innate immune responses to the viral control is recognized but yet not fully understood . toll - like receptors ( tlrs ) sense pathogen - associated molecule patterns and activate antiviral mechanisms including the intracellular antiviral pathways and the production of antiviral effectors like interferons ( ifns ) and pro - inflammatory cytokines . activation of the tlr3 pathway and the production of ifn- represent one of the major mechanisms leading to the suppression of hbv replication in the liver , as shown in different in vitro and in vivo models . tlr4 signaling and tlr2 signaling result in the activation of intracellular pathways including mapk and pi-3 k / akt in hepatocytes and reduce hbv replication in an ifn - independent manner . hbv is able to counteract the actions of tlr3 and tlr2/4 through downregulation of tlr expression and attenuation of the cellular signaling pathways . thus , tlr ligands are promising candidates as immunomodulators and therapeutics for the treatment of chronic hbv infection . specific antiviral treatment against hbv could recover the tlr functions in chronic hbv infection and increase the effectiveness of therapeutic approaches based on tlr activation .

Introduction Toll-like receptor (TLR) system Recognition of HBV by host cells TLR3 and HBV TLR2/4 and HBV Therapeutic approaches Perspectives

according to the estimation of world health organisation , there are about 240 million people who are chronically infected with hepatitis b virus ( hbv ) . the chronic hbv infection is one of the major causes of hepatocellular carcinoma and liver cirrhosis . a number of therapeutic vaccination trials using conventional hbv vaccines failed to demonstrate the effectiveness in terms of the induction of hbv - specific immune responses and suppression of hbv replication in chronic hbv carriers [ 4 , 5 ] . as a principle recognized on the basis of available information , a combined strategy including antiviral treatment and immunomodulation will be needed to stimulate the full range of immune responses to achieve effective control over hbv infection . in the woodchuck model , combinations of antiviral treatment and therapeutic vaccinations led to the induction of specific t cell and b cell responses to whv antigens and sustained suppression of whv replication in some individual animals [ 2 , 4 , 9 , 10 ] . the significance of the innate immune response as a defense against microbial infections and its link to the adaptive immune responses has been recognized during the past years . toll - like receptors ( tlrs ) are a group of highly conserved molecules that play a critical role in the recognition of pathogen - associated molecular patterns ( pamps ) and in the activation of innate immune responses to infectious agents . binding of tlr agonists to their receptors initiates the activation of complex networks of intracellular signal transduction pathways to coordinate the inflammatory response . the myd88-dependent pathway leads to the activation of downstream signal transduction involving il-1r - associated kinases ( iraks ) , tumor necrosis factor receptor ( tnfr)-associated factor 6 ( traf6 ) , transforming growth factor ( tgf)--activated kinase ( tak1 ) , and the inhibitor of nuclear factor-b ( ib ) kinase complex . through the nf-b and activating protein 1 ( ap1 ) , the myd88-dependent tlr activation results in the production of pro - inflammatory cytokines il-6 , il-10 , il-12 , and tnf-. in contrast , the trif - dependent pathway leads to the activation of ifn regulatory factors ( irfs ) and production of type i ifns [ 15 , 17 ] . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines tlr signaling . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines there is accumulating evidence that the innate branch of the host immune system plays an important role in the control of hbv infection [ 1921 ] . thus , hbv infection strongly differs from other viral infections like hcv infection in the early phase , as hcv induces a strong ifn- response in chimpanzees . consistently , a single - dose injection of tlr3 , tlr4 , tlr5 , tlr7 , and tlr9 ligands suppressed hbv replication in the liver in an ifn-/-dependent manner . apparently , the formation of replication - competent hbv capsids is one of the major targets of ifn - mediated antiviral actions . in addition , type i ifns may modulate specific immune responses to hbv , resulting in hbe seroconversion or complete control of hbv infection . therefore , tlr3-mediated response and ifn- production in the liver may contribute to the control of pathogens including hbv in a unique way . several studies suggest that hbv is able to inhibit pattern recognition receptor ( prr ) and ifn signaling . tlr3-mediated functions are impaired in patients with chronic hbv infection and may recover partially under successful antiviral treatment . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . lps was able to activate nf-b , mapk , and pi-3 k / akt pathways in pwhs . the inhibitors of mapk - erk and pi-3 k / akt pathways , but not those of ifn signaling pathways , block the antiviral effect of lps , indicating that ifn - independent pathways which activated by lps are able to downregulate hepadnaviral replication in hepatocytes . consequently , tlr2 and tlr4 mediate the activation of the same signaling pathways downstream of myd88 , including nf-b , mapk , and pi-3 k / akt pathways . again , the antiviral action of tlr2 is dependent on the presence of adaptor molecules like tak1 , irak1/4 , and traf6 and the downstream mapk and pi-3 k / akt pathways . silencing of the expression of adaptor molecules or blocking the mapk and pi-3 k / akt pathways with chemical inhibitors significantly enhanced hbv replication . in the hbv transgenic mouse model , the injection of a single dose of tlr2 ligands reduced hbv replication in the liver but not as effective as ifn - inducing ligands . it was not examined whether tlr2 ligands also activate mapk and pi-3 k / akt pathways in vivo and thereby exert the antiviral action.fig . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines interaction of hbv and tlr2/4 . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines tlr2 activation and tlr4 activation lead to the production of pro - inflammatory cytokines il6 and tnf- in hepatic npcs and hepatocytes [ 32 , 45 , 47 , 48 ] . though the antiviral effect of tlr2 and tlr4 ligands does not directly depend on the production of pro - inflammatory cytokines , il6 and tnf- have been shown to inhibit hbv replication in primary hepatocytes [ 25 , 49 ] . activation of innate immunity is a prerequisite for proper adaptive immune responses . our findings suggest that tlr2 activation has a great impact on t cell immunity in the liver and may be used to stimulate specific immune responses to persistent infection of hbv and hcv . in the presence of hbsag , both pam3csk4-triggered il-12p40 mrna expression and il-12 production in phorbol 12-myristate 13-acetate ( pma)-differentiated thp-1 macrophages are reduced in a dose - dependent manner , while the production of il-1 , il-6 , il-8 , il-10 , and tnf- is not affected . thus , hbsag likely interferes with the initiation of adaptive immune responses by selective inhibition of tlr2-stimulated il-12 production in monocytes . interestingly , a 4-week treatment with gs-9620 resulted in a sustained , marked reduction of serum whv dna and whv surface antigen ( whsag ) levels and in the induction of anti - whs antibody response , as well as a markedly decreased incidence of hepatocellular carcinoma in chronic whv - infected woodchucks . though stimulation with tlr ligands reduces hbv replication in hepatocytes , the tlr - mediated antiviral action against hbv is far less efficient than those achieved by nucleoside analogs . the innate immune system is known to respond fast and aimed to slow down viral spread in primary infections ; thus , the link from innate to adaptive immune responses may be more important for the control of viral infection . tlr2 agonists stimulate activated t cells thus promoting their proliferation and differentiation in vitro and in vivo . thus , future research should not only investigate the direct antiviral effect of tlr - mediated action but also analyze and optimize the connection of innate and adaptive immune responses . the significance of the innate immune response as a defense against microbial infections and its link to the adaptive immune responses has been recognized during the past years . toll - like receptors ( tlrs ) are a group of highly conserved molecules that play a critical role in the recognition of pathogen - associated molecular patterns ( pamps ) and in the activation of innate immune responses to infectious agents . the activation of these transcription factors leads to the induction of type i ifns , pro - inflammatory cytokines , or co - stimulatory molecules , which are involved in antiviral responses [ 15 , 16 ] . the myd88-dependent pathway leads to the activation of downstream signal transduction involving il-1r - associated kinases ( iraks ) , tumor necrosis factor receptor ( tnfr)-associated factor 6 ( traf6 ) , transforming growth factor ( tgf)--activated kinase ( tak1 ) , and the inhibitor of nuclear factor-b ( ib ) kinase complex . through the nf-b and activating protein 1 ( ap1 ) , the myd88-dependent tlr activation results in the production of pro - inflammatory cytokines il-6 , il-10 , il-12 , and tnf-. in contrast , the trif - dependent pathway leads to the activation of ifn regulatory factors ( irfs ) and production of type i ifns [ 15 , 17 ] . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines tlr signaling . upon the activation of tlrs by their respective ligands , the adaptor molecules myd88 , tirap , trif , and tram are recruited and further activate the kinases tak1 , mapks , traf3 , tbk1 , and ikks , resulting in nuclear translocation of transcriptions factors ap-1 , nf-b , irf3 , or irf7 , and subsequent transcription of ifns and pro - inflammatory cytokines there is accumulating evidence that the innate branch of the host immune system plays an important role in the control of hbv infection [ 1921 ] . thus , hbv infection strongly differs from other viral infections like hcv infection in the early phase , as hcv induces a strong ifn- response in chimpanzees . tested the ability of different tlr ligands to inhibit hbv replication in the hbv transgenic mouse model . consistently , a single - dose injection of tlr3 , tlr4 , tlr5 , tlr7 , and tlr9 ligands suppressed hbv replication in the liver in an ifn-/-dependent manner . apparently , the formation of replication - competent hbv capsids is one of the major targets of ifn - mediated antiviral actions . in addition , type i ifns may modulate specific immune responses to hbv , resulting in hbe seroconversion or complete control of hbv infection . upon poly i : c stimulation , hepatic npcs such as kupffer cells ( kc ) and liver sinusoid endothelial cells ( lsec ) release antiviral cytokines which are able to inhibit hbv replication in a co - culture model utilizing hbv - met cells that contains an integrated hbv genome . therefore , tlr3-mediated response and ifn- production in the liver may contribute to the control of pathogens including hbv in a unique way . several studies suggest that hbv is able to inhibit pattern recognition receptor ( prr ) and ifn signaling . at high amounts of hbv , in addition , many studies provide evidence that hbv polymerase may counteract the innate responses at two or more steps : ( 1 ) hbv polymerase is able to inhibit the irf3 activation by interacting with rna helicase ddx3 in hepatoma cells [ 37 , 38 ] ; ( 2 ) hbv polymerase is able to block ifn signaling by inhibition of pkc--mediated phosphorylation of stat 1 and importin - dependent translocation of stat 1/stat 2/irf9 complex [ 39 , 40 ] . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . tlr3 activation in hepatic npcs leads to the production of ifn- and subsequently the upregulation of isgs in hepatocytes . lps was able to activate nf-b , mapk , and pi-3 k / akt pathways in pwhs . the inhibitors of mapk - erk and pi-3 k / akt pathways , but not those of ifn signaling pathways , block the antiviral effect of lps , indicating that ifn - independent pathways which activated by lps are able to downregulate hepadnaviral replication in hepatocytes . consequently , tlr2 and tlr4 mediate the activation of the same signaling pathways downstream of myd88 , including nf-b , mapk , and pi-3 k / akt pathways . again , the antiviral action of tlr2 is dependent on the presence of adaptor molecules like tak1 , irak1/4 , and traf6 and the downstream mapk and pi-3 k / akt pathways . silencing of the expression of adaptor molecules or blocking the mapk and pi-3 k / akt pathways with chemical inhibitors significantly enhanced hbv replication . in the hbv transgenic mouse model , the injection of a single dose of tlr2 ligands reduced hbv replication in the liver but not as effective as ifn - inducing ligands . it was not examined whether tlr2 ligands also activate mapk and pi-3 k / akt pathways in vivo and thereby exert the antiviral action.fig . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines interaction of hbv and tlr2/4 . tlr2/4 activation in hepatocytes inhibits hbv replication in an ifn - independent manner but requires the participation of intracellular signaling pathways like mapk pathway . tlr4 stimulation in kcs leads to the production of ifn- and an unknown antiviral effector which inhibits hbv replication in hepatocytes . hbsag and hbeag are able to block tlr2 signaling at different steps , preventing the production of pro - inflammatory cytokines tlr2 activation and tlr4 activation lead to the production of pro - inflammatory cytokines il6 and tnf- in hepatic npcs and hepatocytes [ 32 , 45 , 47 , 48 ] . though the antiviral effect of tlr2 and tlr4 ligands does not directly depend on the production of pro - inflammatory cytokines , il6 and tnf- have been shown to inhibit hbv replication in primary hepatocytes [ 25 , 49 ] . stimulation of hbv - infected primary tupaia hepatocytes with recombinant tupaia tnf- led to viral suppression , while covalently closed circular dna and viral rna were still detectable , leading to the conclusion that tnf- may also contribute to control hbv infection . it could be shown that hbv polymerase is able to block the nuclear translocation of stat 1 thus representing a general inhibitor of ifn signaling and ifn - inducible myd88 expression . activation of innate immunity is a prerequisite for proper adaptive immune responses . our findings suggest that tlr2 activation has a great impact on t cell immunity in the liver and may be used to stimulate specific immune responses to persistent infection of hbv and hcv . in the presence of hbsag , both pam3csk4-triggered il-12p40 mrna expression and il-12 production in phorbol 12-myristate 13-acetate ( pma)-differentiated thp-1 macrophages are reduced in a dose - dependent manner , while the production of il-1 , il-6 , il-8 , il-10 , and tnf- is not affected . thus , hbsag likely interferes with the initiation of adaptive immune responses by selective inhibition of tlr2-stimulated il-12 production in monocytes . interestingly , a 4-week treatment with gs-9620 resulted in a sustained , marked reduction of serum whv dna and whv surface antigen ( whsag ) levels and in the induction of anti - whs antibody response , as well as a markedly decreased incidence of hepatocellular carcinoma in chronic whv - infected woodchucks . though stimulation with tlr ligands reduces hbv replication in hepatocytes , the tlr - mediated antiviral action against hbv is far less efficient than those achieved by nucleoside analogs . the innate immune system is known to respond fast and aimed to slow down viral spread in primary infections ; thus , the link from innate to adaptive immune responses may be more important for the control of viral infection . tlr2 agonists stimulate activated t cells thus promoting their proliferation and differentiation in vitro and in vivo . thus , future research should not only investigate the direct antiviral effect of tlr - mediated action but also analyze and optimize the connection of innate and adaptive immune responses .

the title of this paper builds on the famous fable of jean de la fontaine ( 16211695 ) the oak and the reed in which he compares the two plants facing the natural elements ( table 2 ) . the moral of the fable is that the oak remains immovable while the reed bends into the wind , but when the wind becomes stronger , the oak is uprooted while the flexible reed survives . this metaphor illustrates the hypothesis we develop to explain part of the gender paradox noted by allard and robine as a result of a large national survey on centenarians : women are more prevalent in old age but in poorer overall health . it leads to an impossible challenge : if you want to become a centenarian , be a woman , you 're more likely to achieve the breakthrough but once you get there , be a man , as you will be in better shape . comparing the responses of the reeds to the wind with the adaptation of elderly to the inherent frailty process that progresses with age , we defend the hypothesis that men , as oaks , have the capacity to cope with the challenges of old age until a breaking point , whereupon they die , while women , as reeds , are more flexible and can adapt and survive . in order to illustrate our hypothesis , we put into perspective various observations related to the french oldest old population coming from three different studies : in the search of the secret of centenarians ( 19902000 ) ( a la recherche du secret des centenaires ( siecle and ipsen foundation ) ) , european challenge for healthy ageing ( echa , 2003 - 2004 ) , and genetics of healthy aging ( geha , 20042010 ) . the observation of the gender paradox was made over a ten - year period by allard and robine based upon a study of 3500 centenarians alive in france in 1990 . indeed , the ipsen survey ( 19902000 ) followed 800 centenarians , 95 men and 705 women , including mme jeanne calment ( a women ! ) , the oldest person who ever existed and who lived until 122 years old . women were more numerous than men in the centenarian population in 1990 , and it is still the case today . the figures ( table 1 ) show that women in old age are more numerous than men , with a sex - ratio ( i.e. , number of women divided by the number of men ) strongly increasing with age . moreover , women live longer according to french national institute of statistics ( insee ) : life expectancy at birth , in 2010 , was 84.8 years for women and 78.1 for men ( bilan dmographique 2010 , insee premire 1332january 2011 ) . the longevity gap was 6.7 years between men and women . beyond the french context , when we look at the living supercentenarians ( people having reached the age of 110 years ) around the world in the gerontology research group database ( as of january 17 , 2011 http://www.grg.org/adams/e.htm ) , we observe that there are 79 women for 4 men , a sex - ratio of 19.8 . therefore , women live longer and are more numerous than men , and this situation becomes more pronounced with increasing age . if health , that is , the absence of disease or good functionality , contributes to longevity , then logically old women should be in better health than old men , that is , have less disease and disability . on the contrary , allard and robine showed that in the three standard dimensions of functional abilities , physical ( strength , mobility , agility ) , sensory ( vision and hearing ) and cognitive ( memory , attention , mental performance , and affect ) , men aged over 100 years have better performance than women . first , let us summarize the results of the ipsen survey , the first french national survey on centenarians . a thorough standardized clinical examination was performed by the general practitioner ( gp ) of the centenarians . according to this medical examination , 65.2% of men were evaluated as having a good or very good health status , while only 56.5% of women were this healthy while 10.7% of women were evaluated as having a poor or very poor health while only 3.4% of men were at this bottom end of the health spectrum . regarding vision , 35.1% of men had good or very good vision , while only 27.19% of women were in a similar condition . centenarians totally blind or with bad vision were 22.0% for women and 19.1% for men . unlike the other data , the centenarians who had good or very good hearing were more numerous in the female population , 28.2% against 21.1% in the male population . in the ipsen survey , the cognitive health of the centenarians was tested using the 10 questions of the pfeiffer test . this test is a screening test , similar to the mmse ( though shorter ) for possible cognitive impairments and dementia . men who made no errors numbered 39.1% while women numbered only 12.9% . since this study , no comparable survey on centenarians has been run in france , but we can examine the french data of two european studies : european challenge for healthy aging ( echa ) and genetics of healthy aging ( geha ) , regarding activity limitations and cognitive impairments . the use of the mini - mental state examination ( mmse ) resulted in an average score of 17.6 for men and 10.7 for women . regarding the activities of daily living ( adl ) , 30.1% of men displayed no activity limitation while women numbered only 11.8% in this case . similarly , while only 20.4% of men had difficulties with 4 or more adl activities ( the adl evaluated were feeding , dressing , transferring , toileting , and bathing ) , 48% of women showed difficulties . the average age of the 201 french male participants was 92.7 years old , and the average age of the 461 french female participants was 93.4 years . regarding the adl , men without difficulties reached 66.2% , while women numbered only 46.4% . men having difficulties with 4 or more adl activities numbered 9.5% , while 16.2% of women showed difficulties . even if we do not have specific data for the centenarians , 754,000 people were affected by dementia in france in 2010 , 72% of them were women and 28% men , according to the last estimations . the data presented here on health differences between men and women could be discussed in terms of data collection methodology or representativeness of the samples ; however , they are sufficient to illustrate our main point , that is , women live longer than men and they are more numerous in old age but men are in better health . there are numerous studies in many countries confirming the above observations , and there are many hypotheses to explain the two components of these observations as we will explain below . facts come from descriptive sciences , demography , epidemiology , and other fields , and from both the social sciences and the biological sciences . they can be linked with the whole life course of the subjects : we know that men are more affected by violent death ( war , murder , work - related , traffic accidents , suicide , etc . ) , and their behaviour patterns expose them to risk factors such as excessive alcohol intake , tobacco and other drug consumption , or other risk raking behaviour . many hypotheses come from biological sciences : of course , physiologies of men and women are different . genetic , hormonal , and phenotypical differences , such as body size , are well known . other hypotheses come from psychological and social sciences : behaviours , education , and social roles are obviously gender - related ; social inequalities between men and women during childhood and work life could lead to differing social and medical support , and so forth . box 1 lists some of the proposed hypotheses suggesting the complexity of the explanations of the differentials in health and longevity between genders . eventually the survival advantage for women results in a larger percentage of women reaching older ages . for all age groups , more women survive , but despite living longer , they display more comorbid conditions . the mechanisms reported in box 1 could explain the higher rates of disability among women . however , it is also possible that , for any morbidity level , men remain more active , outwardly displaying less disability , ignoring pain , discomfort , and risk , and this behaviour pattern contributes to a shortening of their life . it is this last aspect of the gender gap that we aim to explore in this paper from an anthropological perspective . although some reviews are available on the gender gap ( as briefly referred to in box 1 ) , there are far fewer that attempt to disentangle the relative contribution of the various biological and social factors and even fewer that examine this issue for the oldest old . our goal is not to dissect the utility of the above hypotheses , but to suggest another one , based upon gender - linked behaviour patterns of the oldest old , which can contribute to partially explain the gender paradox in healthy survival among the oldest old . for this purpose , we used qualitative data from comprehensive and semistructured interviews of centenarians held between 2003 and 2007 . balard met more than 100 centenarians or near centenarians ( people at least 95 years old ) while undertaking doctoral dissertation research under the framework of the echa and geha studies . he led comprehensive talks , in order to collect alternative points of view on the centenarians ' daily lives , to be compared with the pictures painted by the epidemiologic questionnaires . the comprehensive talks were used to explore and examine research participants ' concerns . in addition to these comprehensive talks , balard performed in - depth interviews with a group of 14 key informants who were followed for 4 years and were subjected to 5 to 10 in - depth interviews each . in this context , the word informant means a person able to bear witness to his or her group , society , and culture . the key informants were chosen according to their ability to communicate and their comfort in interacting . even if people with dementia were not a priori excluded , people in advanced stages of dementia were not interviewed for practical and ethical reasons . most of the interviews were face - to - face talks to avoid interference between the elderly and his / her family circle . the interviews were as close as possible to informal talks , without using a real interview guide but trying to go with the flow and to follow the recurring concerns evoked by the centenarians during the comprehensive talks . as bourdieu recommended , it is better to listen to than to question . , the qualitative interviews should provide to the researcher an access to the mental representations of the interviewees , to understand the meaning of respondents ' experiences and life worlds . as charmaz put it : qualitative interviewing provides an open - ended , in - depth exploration of an aspect of life about which the interviewee has substantial experience , often combined with considerable insight . in our research , the particularity of our key informants was to be one of the oldest old , that is , a centenarian ( or near centenarian ) . in this context , the main themes of the interviewees were their life stories , current daily lives , thoughts , representations of old age , sense of purpose in life ( raison d'tre ) , assessment of the quality of their lives , and strategies to cope with frailty . our posture was consistent with the advice of goffman to subjectize yourself ( to enter the subjectivity of the speaker ) . the goal was to translate the thoughts or , in lapantine`s words , the representations of the speaker , obtained from interviews centred on the interviewee 's conceptions , reasoning , and subjective logic . centenarians were considered as meaning makers . to facilitate the words of the informants , the interviewer opted to introduce himself as a student who meets the elderly to learn from their experience of life . this introduction turned out to be highly facilitating because it produced a dissymmetrical relationship in which the informant was in a position of superiority . the elderly was positioned as a teacher , imparting knowledge to a novice , thus preventing him / her from doubting statements given or perceiving the interview as a test , which may cause anxiety . in accordance with qualitative research , the coding and analysis of the interviews were based on the meaning node obtained by the categories given by the informants ' discourses . as a result of which , the usual theoretical frameworks on quality of life ( psychological indices , life satisfaction , subjective well - being , etc . ) and successful aging based on health and autonomy were not used . someone once asked me what he had to do to get old . do not do anything , it comes . old age comes without being noticed ( mme emilia , 98 years old ) . ( for ethical reasons , the names of the informants have been changed . ) physicians , families and all the others know a lot of things thanks to the intelligence of their brain and sometimes thanks to the intelligence of their heart . but we , we live inside old age . we feel old age and feeling is much more than knowledge . the analysis of the interviews revealed that the definitions of aging ( vieillissement ) and old age ( vieillesse ) , given by the oldest old , did not really differ between men and women . while avoiding a deep discussion of ethnolinguistic concepts , we nonetheless should clarify one point . in french , there are two words , vieux ( old ) and g ( aged ) , which could be translated as old person . the oldest old , and probably most people accept to be designated as g but refuse the word vieux which seems to be a taboo for them . in their representation , g refers to the aging process while vieux refers to an identity . when they are talking about their experience , our key informants explained that they feel they are aging but they are not old . concerning the aging process the first and probably the most important one is the ability to walk . for the oldest old so the consciousness of the beginning of the aging process appears when they feel the first difficulties walking . thereafter , when they have to reduce the distance they used to walk , or when they have to sit down several times during their usual walk , they consider that they are further in their aging process . in their representations , the one who can not walk is the old one ( le vieux ) that 's why m. pierre ( 98 years old ) could say when talking about the other residents of his nursing home here , things have changed , more and more old ones ( vieux ) entered , you know people in wheelchairs . the second symptom that the oldest old consider as a marker of their aging process is the feeling of fatigue . they explain fatigue is at first occasional and then appears when they do usual activities : the mere presence of many people around them , and worse if they are engaged in discussion with them , causes fatigue . just before becoming old ( vieux ) the third symptom the oldest old consider as a marker of the aging process is the feeling of vulnerability that they express by saying now i have to be careful with everything . depending on the informants , having to be careful could focus on weather : i have to be careful not to get cold . i never go out when it rains or it is windy ( m. lon ) . some of the informants express their vulnerability related to others : now , i have to be careful , if someone , if somebody attacks me , i could not defend myself ( mme marie , 96 years old ) . the fourth and last symptom that oldest old recognize as markers of their aging process they talk about the decrease of their senses : today , i have difficulties to see ; they admit being always impaired by small health problems while they were not used to be so in the past . through these different symptoms , the oldest old admit they are aging but they defend themselves against being labelled old ( vieux ) . the old one ( le vieux ) is not aging , he is at the end of the aging process . in their representations , the old one is a human being living under a death sentence . according to the representation of the oldest old , the old one ( le vieux ) is first and foremost the one who is useless . in accordance with the social values of his / her generation , the oldest old consider that the social utility and the sense of life of human beings are in work and family . m. aim , 95 years old said now i am old ( vieux ) , i am unfit , useless another marker of old age for the oldest old is the feeling of not being listened to by their entourage . mme emilia ( 99 years old ) said the old ones ( vieux ) are the ones who are no more listened to . for the oldest old , being listened to is a real mark of social utility because as mme anna ( 96 years old ) says a lot of them consider that today their social role is to pass on their experience to young people . in this view , those who can not pass on their experience are useless and old ( vieux ) . we have already explained that the old one ( le vieux ) is the one who can not walk , he / she is also the one who lost one 's head . those who are demented are like the old ones ( le vieux ) , they are marginalized and socially disqualified . the last qualifier used by the informants to describe the old one is close to the concept of dependence but it is expressed differently by the oldest old . as mme anna said you are old when you are at the mercy of everyone . everybody depends on other persons but being at the mercy of somebody means to be at risk to lose control of one 's life , to be forced or manipulated . in the representation of the aging process expressed by the oldest old and in their ideas of old age , we find many elements of a cross - cultural definition of age already put forward by anthropologists [ 1721 ] . health , safety , functioning ( mobility , capacity to act ) , and individual productivity ( cf . productive if it creates societal value , whether or not it is reimbursed ) are some elements that many societies include in their definitions of aging . aging implies a frailty process , according to linda fried , a physiologic state of increased vulnerability to stressors that results from decrease of physiologic reserves , and even dysregulation , of multiple physiologic systems . according to our observations , frailty is not only physiological . in old age , frailty is also social , cultural , and psychological , involving the sense of identity . indeed , the oldest old explain they suffer from the lag between their time and the society of today . they also suffer from a break in their identity , a sense of diminished self or what levi - strauss called when he turned 90 years old the feeling of being a broken hologram ( reconstitution of memories of roger - pol droit published in le monde 29/01/1999 . to apply this concept to our informants , the real self is the frail old man or woman they see in the mirror and feel when confronting the challenges that come with aged bodies while the virtual self is the representation they have of themselves , which still preserves a living idea of the whole and a continuity with the past ( almost a timeless identity ) . it is in this context that we hypothesize that men and women are not equal in their ability to face the various symptoms and related challenges of old age and dimensions of frailty . we hypothesise that , while men try to fight against the stigma of old age until death , women try to find a way to accept it and continue to live on . through these gender - specific adaptive strategies , the gap between the ( in levi - strauss terminology ) is more easily bridged for most of the oldest old women of this generation when compared to the men . according to the concept of generational habitus suggested by mauger and built on from the work of mannheim and bourdieu , we hypothesize that the differences in behaviour between men and women , even at the oldest ages , are coming from a generational mentality , that is , the typical ways in which people think and their attitudes toward life . the concept of generational habitus is close to the idea of the existence of contemporaneous connections between individuals . the generation creates a rather narrow circle of individuals who despite the diversity of the other factors at play , are linked into a whole by the fact that they lived the same major events and changes during their receptivity period . mauger used the concepts of mental tools and habit - shaping force to illustrate how people are influenced by their generational membership . over the life course , the main receptivity period is during childhood , when people acquire the gender stereotypes of their culture . of course , the way of thinking , being , and doing evolves over the whole life course , but these cultural landmarks regularly resurface , especially when identity , sense of self , and self - esteem are threatened , as in old age , when gradual or sudden decline in physical capacity , loss of control over one 's environment , or other age - related challenges emerge . in order to illustrate the influence of the generational habitus for the behaviour patterns of the oldest old , we can evoke the community of thought which exists among our informants . indeed , it appears that our informants share many values despite their difference in social status ( occupation , level of education , and income ) . for them , they have a very different conception of care than their children do . for them , care is part of the traditional system of mutual help and supportive relationships that they have known in their village or neighbourhood . in this view , care should not be a professional task and/or something that is paid for , except for specific medical care services . they consider that it is the role of their nearby family or close relatives to help them with daily tasks and provide support in old age . they can not accept that they should pay for cleaning and cooking whereas they have ( or they think they have ) previously helped others in the past . this generational habitus still influences them in their daily choices , values , and thinking . the analysis of the interviews showed a clear gap between the representation of the social role of men and women for our informants . the large majority of people born during the first two decades of the 20th century in france were country folk even if some of them spent their adulthood in towns . as showed by segalen , this rural environment was characterised by an important distinction between men and women concerning social identity , social role , and production functions . these findings are close to the work of guttman who describes men as breadwinners , active , and dominant , and women as homemakers who provide emotional support and accept dependency as part of their social role , that is confined mainly to the domestic sphere . in this context , segalen showed that despite the appearance of male domination , there was mutual support , solidarity , complicity , and reciprocity between men and women . lebra referred to the exchange of such mutual obligations as reciprocal dependency where there exists a reciprocity or exchange of dependency . others have recognized that despite the appearance of subjugation and economic dependency in relation to their husbands , women wield considerable power and status within the domestic sphere that accompanies their role monopoly as full time homemaker . feminist anthropological approaches have analyzed these social exchanges within the context of a public and private dichotomy where men wield political power as the ortner elaborated on this theme when she published a landmark article likening female to male as nature is to culture . this theory dealt with the perception that men are the upholders of culture whereas women are more associated with nature . women are seen as closer to nature in reference to three dimensions : ( 1 ) women 's bodies are seen as more natural since they are more involved with childbirth and childrearing ; ( 2 ) the social roles of women that intertwine with child rearing are viewed as closer to nature , specifically confining women to the domestic realm ; ( 3 ) social perceptions of the female psyche portray woman as closer to nature . of course , the power , status , and roles of men and women vary between cultures and time periods . moreover , men and women have multiple overlapping and contextualized identities based upon gender , age , ethnicity , class , regional , and other differences that evolve over the life course . following his theory , the traditional gender role is stronger during what he called the parental imperative ( i.e. , to ensure the survival of children and the handing down of the social values . once the children have reached adulthood , the parents could express the other - gender side of their personality ) . this phenomenon seems to be due to the softening of the traditional gendered roles that occurs with the aging process . men become less authoritarian and more sensitive , whereas women accept less to be dominated and become more powerful . guttman used the image of the warrior who became a peaceful chief , or that of the virile older woman . many anthropologists have shown that during adulthood , and especially after menopause , women 's social roles , status , and identity change . for example in gabon , according to bikoma , the non - menstruating woman takes part in strictly masculine demonstrations . many works have described how women 's status changes , often rising as they grow older [ 35 , 36 ] . thus , it appears that the gender differences in behaviour change over the life course , most often appearing as a softening of the traditional gender characteristics . the child of one of our informants confirms today it is different but when i was a child , my father was terrifying . does very old age cause an increase in the androgyny process observed in middle - life or is there a specific moment in the life course in which the traditional gender role reappears ? we consider that the answer is significantly different between traditional societies and postindustrial ones , such as the france of today . the finding of singleton that in some societies , when you get older , you see better ( the author refers to the mystical and magical abilities often attributed to the oldest old in traditional societies ) and you are better seen does not seem to hold in our postindustrial societies . indeed , the image of very old age is quite negative in france . it conveys the ideas of illness , handicap , and senility . as we explained earlier , the oldest old worry about losing their identity as well as becoming an old person ( un vieux ) . it is possible that , facing this unknown and stressful event that represents very old age , one important adaptational strategy consists in taking refuge in what they consider the most important things ( i.e. , what they value most ) in their life and identity . men try to show that they are still strong enough , helpful ( useful ) , cognitively aware and intact , and able to make decisions for themselves , while women concentrate on issues such as being emotionally close to their family members , seeing them happy in life , and maintaining other important human relationships . thus , it appears that the components of the social identity of women are less challenged by the frailty process , offering them more possibilities to adapt to the losses associated with the aging process when compared to men . frailty implies to be no longer able to be the same while feeling the necessity to be oneself intgration et exclusion des personnes ges dans les dcisions publiques et prives , paris , 10 et 11 septembre 2007 ) . frailty implies to be no longer able to be the same while feeling the necessity to be oneself ( journe d'tudes internationales : l'ge et le pouvoir en question . intgration et exclusion des personnes ges dans les dcisions publiques et prives , paris , 10 et 11 septembre 2007 ) . the interviews with the male informants reveal that preserving their virtual self is one of the leitmotives of their dialogues and a constancy of their behaviour . their self - identity is based on their past roles and statuses as worker , breadwinner , and head of family . ( exceptionally diligent and productive ) . m. aim with my horse , i worked every day from sunrise to sunset , i worked for me and for the others too . when i was 86 , i used to prune vines 5 or 6 hours a day . physical strength and physical endurance are two important dimensions of the self - identity they make efforts to lay claim to . others have insisted on their skill at work like m. henri ( 96 years old ) who explained that he made a piece out of wood to replace a part of a tank engine during world war ii . they did their best to work as long as possible even if they were officially retired . for them , it is vitally important to prove that they are still valid , competent , autonomous , and able . with an interlocutor , their self - presentation leans towards self - staging or presenting themselves in the light of their ideal self - image . when they became unable to go on with their work , they tried to find activities in which they could continue to prove they were competent and useful like gardening , or repairing and improving things around the home . within their family , the men of this generation are used to being in authority and to be listened to , even if these characteristics weaken over time and with age . during the interviews , they insisted on showing that their opinion is still listened to and respected by their children to whom they continue to give advice . to survive in old age , the men in our study seemed to choose to stay immovable . they did not like to deal with their new and vulnerable self - identity and refused to be compared to the old ones ( les vieux ) . however , there comes a time when the gap between their real self and the image they have of themselves is too large to continue to self - stage . thus , when walking becomes too difficult , when it is impossible to preserve a useful activity in conformity to their identity , when they have the feeling that they are losing their authority because they are not able to manage daily affairs , men at these oldest old ages express a kind of surrender . we have already quoted the sentence of m. aim : now i am old ( vieux ) , i am unfit , useless m. lon , who was a very positive centenarian during the first interviews , confessed 3 months before his death : now i get to the end i am too old . the events that precipitate the surrender and probably the in addition to the emotional suffering , they appear helpless without the person devoted to take care of their daily personal needs . usually oriented to outdoor activities , they found it extremely difficult to accept that they must now stay home . when they felt they were losing control over their environment , their self - sufficiency , and perhaps most importantly their continuity in self , the oldest men seem to prefer to give up and die rather than accept a state of disability and/or the idea of further gradual decline . to use the terminology of evert et al . therefore , as long as they can delay significant physical limitations and severe disability , they have the will to continue to keep going and literally stay alive . the behaviour of oldest old men is highlighted by their efforts to remain physically and mentally strong and active . this may contribute to explain their better physical and cognitive functional health status , compared to women , but also their tendency to take more risks and their excess mortality . because they are giving such significance to their functionality and physical performance , the oldest men make a great deal of effort to walk even if it is dangerous for them and this risk - taking behaviour may lead to falls and fatal events . m. aim 's daughter explained that the neighbour found her father lying in his garden after a fall on three separate occasions . two of our male informants explained that they thought about committing suicide ; the first because he could not endure the thought of himself as degraded and useless , and the second because he was obliged to leave his home to enter a nursing home . for instance , at age 85 and over , the suicide mortality rate in france is 6.5 times higher for men than for women . we saw that men can train themselves more than women to perform various physical tasks . we also wonder whether they are not tempted to exaggerate when reporting on their health status and functional abilities . do they try to bias reality when reporting their self - perceived health or adl abilities ? cognitive tests or physical tests , such as the hand grip test , are a priori objective tests . they delay or seem to delay the old one ( le vieux ) stigma until a breaking point . this behaviour may partly explain why they are less numerous among older ages and why they live shorter lives . possibly , the stronger selection effect that occurs for men reinforces their behaviour and the feeling that they are in better health . compared to other men of their generation , they are the survivors , the chosen ones . this relative appreciation may result in a self - appraisal that they were the most robust and/or able of their birth cohort , it may be that they literally could not see themselves otherwise . although playing their major social role within the domestic sphere , contrary to their husbands , they were never the ultimate authority , even in their own home . when they were children , they lived under their father 's authority and then , as a spouse , under their husband 's authority . thus , to be in authority is not something cultural , it is not a habit . in this generation , many women have worked as housewives . their family role was firstly to take care of others , housework , childrearing , and help their husband in his activity . in their life , taking care of others is what better illustrates their self identity . then , when they were mother and wife , they were responsible for the health and the blossoming of their children and husband . most of them were also helping their husband in his work , taking care of several tasks . later , these women were in charge of their old parents or step - parents . the representations attached to their identity were neither physical strength , nor being in authority , but the ability to listen to and understand the needs of others , to be a person full of maternal affection for others . the activities linked to their social identity were very often cooking , doing the washing or other household tasks , and childrearing . m. aim 's daughter explains the death of my mother raised the issue of my father being alone . she has to prepare his medicine , read for him ( very painful ) , especially to serve the meal for him . the daughter explains that her mother sacrificed herself for her husband . referring to this point we notice that this could partly explain why the protective effect of marriage in france is only valid for men in this generation . mme emilia ( 99 years old ) confirms that in her generation the role of women was dedicated to support other family members : when i was a child , i helped my mother i had to take care of my young brothers and sisters [ ] i do n't want to live with my children because i know what taking care of an old person implies , i have done that for my father and my mother - in - law . according to cool , we note that there is continuity in women 's core social role of housewife for this generation and we agree that women seem to show an ability to adapt to discontinuity and/or conflicting roles throughout the life course . indeed , if the collected material shows a general continuity in the social role of the women of our sample , it also shows that they had to face many discontinuities , upheavals and challenges over the life course . we hypothesize that their greater flexibility and ability to adapt is partially explained by their generational habitus shaping them as good girl and exemplary spouse . as segalen expressed it : authority to men and power to women . women have learned not to fight against their environment but to adapt to it and to find the best ways to live in it . when they grew old , women did not feel the need to fight against old age stigma as men did . physical strength , being in authority , and an identity based on their ability to be the head of the family are not female prerogatives for this generation . we observed that women more easily accepted having difficulties with some activities , such as gardening , walking outside the house , and could more easily give up such activities . we also observed that women were not emotionally injured when their children made decisions for them . they explained that as an old person , the most important thing for them was to see their children and grandchildren . as mme emilia , and many oldest - old women explained now , i let myself live . what she means is that she chose not to fight against old age but to live with it . confronted by a number of losses , such as memory or mobility , mme germaine says i do not care about that today ; it is not the most important what really matters is to see my children and their families in good health . women seem to have a high ability to accept not being able to fully control their life , and their body , their fate . when they fail in the mmse test , they appear less uncomfortable with this than men do . they do not consider that their identity and pride are at stake . in conclusion , women seem to have a better ability to accept and live with the various symptoms of old age and frailty . therefore , even if they accumulate health problems , chronic degenerative diseases , functional limitations , and activity restrictions , even if they are no longer performing as they once did , they can survive . referring to evert et al not focusing on their losses , they are able to continue to live without experiencing a large gap between their virtual identity and their real identity . this behaviour was reported by all six oldest old female informants in our study . we can also notice that , while the majority of the men in the french sample of the geha study did their best to be successful with the physical and cognitive tests that were proposed , women seemed not to consider them so important . it is possible that for the women in geha , the interaction and the presence of someone to communicate with was more important than the tests per se and could partially contribute to explaining why they were less successful in the tests when compared to men . we insisted on the generational habitus that influences the gender behaviour in old age and that we observed in the current cohorts of the oldest old people . being much less self - centred than men , women of these cohorts found a way to relativize their frailty and live with it , instead of ignoring or concealing it as men seemed to do . the main part of these gender - based behavioural or adaptational differences in regard to frailty and old age possibly came from the generational habitus which constructed the physiological reserve , psychological attitudes , ways of thinking , values , and social behaviour of the oldest old men and women in a very different manner . men and women of these birth cohorts have grown up , lived , and grown old as completely different social beings . their gender - specific behaviour in old age continues to influence their longevity and quality of life in different ways . however , looking at the current homogenization of male and female social roles and behaviours ( especially health - related behaviors ) , we can postulate that the gender gaps observed today among centenarians may decrease over time . more recent cohorts of women have partially adopted risky behaviours , such as smoking or drinking alcohol , which were the prerogative of men in the past , and increasing participation in what was once considered exclusively male occupations ( transport , military , law enforcement , etc . ) may expose women to more violent deaths due to accidents , homicide , and/or suicide . indeed , in line with decreasing polarization of gender roles , the size of women 's life expectancy advantage over men has been steadily shrinking in most developed nations ( some exceptions such as japan exist ) for the past few decades . moreover , men in developed nations seem to be paying more attention to their health in recent years when compared to men of previous generations , with recent gains in male life expectancy ( in comparison to female ) appearing to be related more to an acceleration of progress for males , rather than a worsening of health situation for women . decreasing male deaths from cancer ( especially lung ) and cardiovascular disease are the major mortality - related contributors [ 61 , 62 ] . in this context , the gender gap in longevity will likely continue to decrease in the future as more women take up smoking and other health risk behaviors . but will this new generational habitus change the gender differences for the oldest old ? the 200,000 centenarians who will live in france in 2060 , according to the latest population forecasts , will have a generational habitus constructed in the postwar period , shaped by social , cultural , technological , economic , and behavioural forces far different than that of today 's centenarians . but will the behaviour of men and women be more alike as they enter the ranks of the oldest old ? of course only time will tell . the gender gap in longevity and disability will likely remain in a state of flux for some time due to the continual shifts in the social , economic , and behavioural dynamics that determine health and longevity for both women and men . french people , who were born at the beginning of the 20th century ( i.e. , before 1915 ) , display a generational habitus which clearly juxtaposes men and women in terms of social identity , family role , and sense of purpose in life . this habitus still influences many gender - linked behaviours and adaptational strategies , even at the oldest ages . we suggest that these differences in behaviour favour functional health for men and longevity for women . the generational habitus influences the representation of the raison d'tre for men and women . raison d'tre , for these cohorts , is more self - centric and oak - like explaining why , when the gap between their real and virtual identity and self - image as the provider and pillar of their family and community becomes too wide and difficult to sustain , they find it nearly impossible to go on . on the contrary , the female raison d'tre , for the same cohort , is based more on their status in relation to others , in particular as givers of care , even if it is reduced to their mere presence next to those they love . they are more flexible , adaptive , and reed - like ; therefore , even if more severely disabled than men , women can continue to survive . quote from claude - levi strauss on the occasion of his 90th birthday(note that he continued on until he reached 100 years old , a centenarian , as did most of the subjects in this study . ) montaigne dit que la vieillesse nous diminue chaque jour et nous entame de telle sorte que , quand la mort survient , elle n'emporte plus qu'un quart d'homme ou un demi - homme . montaigne est mort 59 ans , et ne pouvait avoir ide de l'extrme vieillesse o je me trouve aujourd'hui . je ne pensais pas atteindre et qui constitue une des plus curieuses surprises de mon existence , j'ai le sentiment d'tre comme un hologramme bris . cet hologramme ne possde plus son unit entire et cependant , comme tout hologramme , chaque partie restante conserve une image et une reprsentation complte du tout . [ ] ainsi y - a - t - il aujourd'hui pour moi un moi rel , qui n'est plus qu'un quart ou la moiti d'un homme , et un moi virtuel , qui conserve encore vive une ide du tout . le moi virtuel dresse un projet de livre , commence d'en organiser les chapitres et dit au moi rel : et le moi rel , qui ne peut plus , dit au moi virtuel : c'est ton affaire . je vous suis trs reconnaissant d'avoir , pour quelques instants , grce votre prsence aujourd'hui et votre amiti , fait cesser ce dialogue en permettant un moment ces deux moi de concider de nouveau . je sais bien que le moi rel continue de fondre jusqu' la dissolution ultime , mais je vous suis reconnaissant de m'avoir tendu la main , me donnant ainsi le sentiment , pour un instant , qu'il en est autrement.montaigne says that old age diminishes us so that , when death arrives , it claims only a quarter or a half a man . montaigne died at fifty - nine and surely had no idea of the extreme old age , in which i find myself today . having lived to a ripe old age which i never expected to attain , and which is one of the strangest surprises i have experienced , i feel like a shattered hologram . this hologram no longer possesses its entire unity , yet , as with any hologram , each surviving shard preserves an image and full representation of the whole.so today for me there is a real self , which is but a quarter or half a man , and a virtual self , which preserves a living idea of the whole . the virtual self is planning a new book and beginning to organize its chapters , and to the real self it says , but the real self , which can not carry on , says to the virtual self , that 's your problem . my life nowadays is defined by this very strange dialogue.i am very grateful to you whose presence here today and whose friendship have for a short time silenced this dialogue and allowed these two selves to coincide again briefly . i know full well that the real self will continue to melt away until the moment of final dissolution , but i thank you for reaching out and for an instant giving me the sense that it might not be so . ( note that he continued on until he reached 100 years old , a centenarian , as did most of the subjects in this study . ) montaigne dit que la vieillesse nous diminue chaque jour et nous entame de telle sorte que , quand la mort survient , elle n'emporte plus qu'un quart d'homme ou un demi - homme . montaigne est mort 59 ans , et ne pouvait avoir ide de l'extrme vieillesse o je me trouve aujourd'hui . dans ce grand ge que je ne pensais pas atteindre et qui constitue une des plus curieuses surprises de mon existence , j'ai le sentiment d'tre comme un hologramme bris . cet hologramme ne possde plus son unit entire et cependant , comme tout hologramme , chaque partie restante conserve une image et une reprsentation complte du tout . [ ] ainsi y - a - t - il aujourd'hui pour moi un moi rel , qui n'est plus qu'un quart ou la moiti d'un homme , et un moi virtuel , qui conserve encore vive une ide du tout . le moi virtuel dresse un projet de livre , commence d'en organiser les chapitres et dit au moi rel : et le moi rel , qui ne peut plus , dit au moi virtuel : ma vie se droule prsent dans ce dialogue trs trange . [ ] je vous suis trs reconnaissant d'avoir , pour quelques instants , grce votre prsence aujourd'hui et votre amiti , fait cesser ce dialogue en permettant un moment ces deux moi de concider de nouveau . je sais bien que le moi rel continue de fondre jusqu' la dissolution ultime , mais je vous suis reconnaissant de m'avoir tendu la main , me donnant ainsi le sentiment , pour un instant , qu'il en est autrement . montaigne says that old age diminishes us so that , when death arrives , it claims only a quarter or a half a man . montaigne died at fifty - nine and surely had no idea of the extreme old age , in which i find myself today . having lived to a ripe old age which i never expected to attain , and which is one of the strangest surprises i have experienced , i feel like a shattered hologram . this hologram no longer possesses its entire unity , yet , as with any hologram , each surviving shard preserves an image and full representation of the whole . so today for me there is a real self , which is but a quarter or half a man , and a virtual self , which preserves a living idea of the whole . the virtual self is planning a new book and beginning to organize its chapters , and to the real self it says , but the real self , which can not carry on , says to the virtual self , that 's your problem . i am very grateful to you whose presence here today and whose friendship have for a short time silenced this dialogue and allowed these two selves to coincide again briefly . i know full well that the real self will continue to melt away until the moment of final dissolution , but i thank you for reaching out and for an instant giving me the sense that it might not be so . claude levi - strauss speaking extemporaneously to friends assembled to celebrate his 90th birthday ( january 1999 ) .

since the 1990s , several studies involving french centenarians have shown a gender paradox in old age . even if women are more numerous in old age and live longer than men , men are in better physical and cognitive health , are higher functioning , and have superior vision . if better health should lead to a longer life , why are men not living longer than women ? this paper proposes a hypothesis based on the differences in the generational habitus between men and women who were born at the beginning of the 20th century . the concept of generational habitus combines the generation theory of mannheim with the habitus concept of bourdieu based on the observation that there exists a way of being , thinking , and doing for each generation . we hypothesized that this habitus still influences many gender - linked behaviours in old age . men , as oaks , seem able to delay the afflictions of old age until a breaking point , while women , as reeds , seem able to survive despite an accumulation of health deficits .

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

the title of this paper builds on the famous fable of jean de la fontaine ( 16211695 ) the oak and the reed in which he compares the two plants facing the natural elements ( table 2 ) . this metaphor illustrates the hypothesis we develop to explain part of the gender paradox noted by allard and robine as a result of a large national survey on centenarians : women are more prevalent in old age but in poorer overall health . it leads to an impossible challenge : if you want to become a centenarian , be a woman , you 're more likely to achieve the breakthrough but once you get there , be a man , as you will be in better shape . comparing the responses of the reeds to the wind with the adaptation of elderly to the inherent frailty process that progresses with age , we defend the hypothesis that men , as oaks , have the capacity to cope with the challenges of old age until a breaking point , whereupon they die , while women , as reeds , are more flexible and can adapt and survive . in order to illustrate our hypothesis , we put into perspective various observations related to the french oldest old population coming from three different studies : in the search of the secret of centenarians ( 19902000 ) ( a la recherche du secret des centenaires ( siecle and ipsen foundation ) ) , european challenge for healthy ageing ( echa , 2003 - 2004 ) , and genetics of healthy aging ( geha , 20042010 ) . the observation of the gender paradox was made over a ten - year period by allard and robine based upon a study of 3500 centenarians alive in france in 1990 . women were more numerous than men in the centenarian population in 1990 , and it is still the case today . the figures ( table 1 ) show that women in old age are more numerous than men , with a sex - ratio ( i.e. the longevity gap was 6.7 years between men and women . beyond the french context , when we look at the living supercentenarians ( people having reached the age of 110 years ) around the world in the gerontology research group database ( as of january 17 , 2011 http://www.grg.org/adams/e.htm ) , we observe that there are 79 women for 4 men , a sex - ratio of 19.8 . therefore , women live longer and are more numerous than men , and this situation becomes more pronounced with increasing age . if health , that is , the absence of disease or good functionality , contributes to longevity , then logically old women should be in better health than old men , that is , have less disease and disability . on the contrary , allard and robine showed that in the three standard dimensions of functional abilities , physical ( strength , mobility , agility ) , sensory ( vision and hearing ) and cognitive ( memory , attention , mental performance , and affect ) , men aged over 100 years have better performance than women . according to this medical examination , 65.2% of men were evaluated as having a good or very good health status , while only 56.5% of women were this healthy while 10.7% of women were evaluated as having a poor or very poor health while only 3.4% of men were at this bottom end of the health spectrum . unlike the other data , the centenarians who had good or very good hearing were more numerous in the female population , 28.2% against 21.1% in the male population . in the ipsen survey , the cognitive health of the centenarians was tested using the 10 questions of the pfeiffer test . the use of the mini - mental state examination ( mmse ) resulted in an average score of 17.6 for men and 10.7 for women . similarly , while only 20.4% of men had difficulties with 4 or more adl activities ( the adl evaluated were feeding , dressing , transferring , toileting , and bathing ) , 48% of women showed difficulties . regarding the adl , men without difficulties reached 66.2% , while women numbered only 46.4% . the data presented here on health differences between men and women could be discussed in terms of data collection methodology or representativeness of the samples ; however , they are sufficient to illustrate our main point , that is , women live longer than men and they are more numerous in old age but men are in better health . they can be linked with the whole life course of the subjects : we know that men are more affected by violent death ( war , murder , work - related , traffic accidents , suicide , etc . ) many hypotheses come from biological sciences : of course , physiologies of men and women are different . other hypotheses come from psychological and social sciences : behaviours , education , and social roles are obviously gender - related ; social inequalities between men and women during childhood and work life could lead to differing social and medical support , and so forth . however , it is also possible that , for any morbidity level , men remain more active , outwardly displaying less disability , ignoring pain , discomfort , and risk , and this behaviour pattern contributes to a shortening of their life . it is this last aspect of the gender gap that we aim to explore in this paper from an anthropological perspective . although some reviews are available on the gender gap ( as briefly referred to in box 1 ) , there are far fewer that attempt to disentangle the relative contribution of the various biological and social factors and even fewer that examine this issue for the oldest old . our goal is not to dissect the utility of the above hypotheses , but to suggest another one , based upon gender - linked behaviour patterns of the oldest old , which can contribute to partially explain the gender paradox in healthy survival among the oldest old . he led comprehensive talks , in order to collect alternative points of view on the centenarians ' daily lives , to be compared with the pictures painted by the epidemiologic questionnaires . in this context , the word informant means a person able to bear witness to his or her group , society , and culture . in this context , the main themes of the interviewees were their life stories , current daily lives , thoughts , representations of old age , sense of purpose in life ( raison d'tre ) , assessment of the quality of their lives , and strategies to cope with frailty . the goal was to translate the thoughts or , in lapantine`s words , the representations of the speaker , obtained from interviews centred on the interviewee 's conceptions , reasoning , and subjective logic . in accordance with qualitative research , the coding and analysis of the interviews were based on the meaning node obtained by the categories given by the informants ' discourses . we feel old age and feeling is much more than knowledge . the analysis of the interviews revealed that the definitions of aging ( vieillissement ) and old age ( vieillesse ) , given by the oldest old , did not really differ between men and women . for the oldest old so the consciousness of the beginning of the aging process appears when they feel the first difficulties walking . the old one ( le vieux ) is not aging , he is at the end of the aging process . in accordance with the social values of his / her generation , the oldest old consider that the social utility and the sense of life of human beings are in work and family . m. aim , 95 years old said now i am old ( vieux ) , i am unfit , useless another marker of old age for the oldest old is the feeling of not being listened to by their entourage . the last qualifier used by the informants to describe the old one is close to the concept of dependence but it is expressed differently by the oldest old . everybody depends on other persons but being at the mercy of somebody means to be at risk to lose control of one 's life , to be forced or manipulated . in the representation of the aging process expressed by the oldest old and in their ideas of old age , we find many elements of a cross - cultural definition of age already put forward by anthropologists [ 1721 ] . health , safety , functioning ( mobility , capacity to act ) , and individual productivity ( cf . in old age , frailty is also social , cultural , and psychological , involving the sense of identity . to apply this concept to our informants , the real self is the frail old man or woman they see in the mirror and feel when confronting the challenges that come with aged bodies while the virtual self is the representation they have of themselves , which still preserves a living idea of the whole and a continuity with the past ( almost a timeless identity ) . it is in this context that we hypothesize that men and women are not equal in their ability to face the various symptoms and related challenges of old age and dimensions of frailty . we hypothesise that , while men try to fight against the stigma of old age until death , women try to find a way to accept it and continue to live on . through these gender - specific adaptive strategies , the gap between the ( in levi - strauss terminology ) is more easily bridged for most of the oldest old women of this generation when compared to the men . according to the concept of generational habitus suggested by mauger and built on from the work of mannheim and bourdieu , we hypothesize that the differences in behaviour between men and women , even at the oldest ages , are coming from a generational mentality , that is , the typical ways in which people think and their attitudes toward life . the concept of generational habitus is close to the idea of the existence of contemporaneous connections between individuals . the generation creates a rather narrow circle of individuals who despite the diversity of the other factors at play , are linked into a whole by the fact that they lived the same major events and changes during their receptivity period . of course , the way of thinking , being , and doing evolves over the whole life course , but these cultural landmarks regularly resurface , especially when identity , sense of self , and self - esteem are threatened , as in old age , when gradual or sudden decline in physical capacity , loss of control over one 's environment , or other age - related challenges emerge . in order to illustrate the influence of the generational habitus for the behaviour patterns of the oldest old , we can evoke the community of thought which exists among our informants . they consider that it is the role of their nearby family or close relatives to help them with daily tasks and provide support in old age . this generational habitus still influences them in their daily choices , values , and thinking . the analysis of the interviews showed a clear gap between the representation of the social role of men and women for our informants . the large majority of people born during the first two decades of the 20th century in france were country folk even if some of them spent their adulthood in towns . as showed by segalen , this rural environment was characterised by an important distinction between men and women concerning social identity , social role , and production functions . these findings are close to the work of guttman who describes men as breadwinners , active , and dominant , and women as homemakers who provide emotional support and accept dependency as part of their social role , that is confined mainly to the domestic sphere . in this context , segalen showed that despite the appearance of male domination , there was mutual support , solidarity , complicity , and reciprocity between men and women . lebra referred to the exchange of such mutual obligations as reciprocal dependency where there exists a reciprocity or exchange of dependency . this theory dealt with the perception that men are the upholders of culture whereas women are more associated with nature . women are seen as closer to nature in reference to three dimensions : ( 1 ) women 's bodies are seen as more natural since they are more involved with childbirth and childrearing ; ( 2 ) the social roles of women that intertwine with child rearing are viewed as closer to nature , specifically confining women to the domestic realm ; ( 3 ) social perceptions of the female psyche portray woman as closer to nature . of course , the power , status , and roles of men and women vary between cultures and time periods . moreover , men and women have multiple overlapping and contextualized identities based upon gender , age , ethnicity , class , regional , and other differences that evolve over the life course . this phenomenon seems to be due to the softening of the traditional gendered roles that occurs with the aging process . does very old age cause an increase in the androgyny process observed in middle - life or is there a specific moment in the life course in which the traditional gender role reappears ? men try to show that they are still strong enough , helpful ( useful ) , cognitively aware and intact , and able to make decisions for themselves , while women concentrate on issues such as being emotionally close to their family members , seeing them happy in life , and maintaining other important human relationships . thus , it appears that the components of the social identity of women are less challenged by the frailty process , offering them more possibilities to adapt to the losses associated with the aging process when compared to men . the interviews with the male informants reveal that preserving their virtual self is one of the leitmotives of their dialogues and a constancy of their behaviour . their self - identity is based on their past roles and statuses as worker , breadwinner , and head of family . to survive in old age , the men in our study seemed to choose to stay immovable . thus , when walking becomes too difficult , when it is impossible to preserve a useful activity in conformity to their identity , when they have the feeling that they are losing their authority because they are not able to manage daily affairs , men at these oldest old ages express a kind of surrender . this may contribute to explain their better physical and cognitive functional health status , compared to women , but also their tendency to take more risks and their excess mortality . because they are giving such significance to their functionality and physical performance , the oldest men make a great deal of effort to walk even if it is dangerous for them and this risk - taking behaviour may lead to falls and fatal events . they delay or seem to delay the old one ( le vieux ) stigma until a breaking point . possibly , the stronger selection effect that occurs for men reinforces their behaviour and the feeling that they are in better health . indeed , if the collected material shows a general continuity in the social role of the women of our sample , it also shows that they had to face many discontinuities , upheavals and challenges over the life course . physical strength , being in authority , and an identity based on their ability to be the head of the family are not female prerogatives for this generation . women seem to have a high ability to accept not being able to fully control their life , and their body , their fate . when they fail in the mmse test , they appear less uncomfortable with this than men do . in conclusion , women seem to have a better ability to accept and live with the various symptoms of old age and frailty . we can also notice that , while the majority of the men in the french sample of the geha study did their best to be successful with the physical and cognitive tests that were proposed , women seemed not to consider them so important . we insisted on the generational habitus that influences the gender behaviour in old age and that we observed in the current cohorts of the oldest old people . being much less self - centred than men , women of these cohorts found a way to relativize their frailty and live with it , instead of ignoring or concealing it as men seemed to do . the main part of these gender - based behavioural or adaptational differences in regard to frailty and old age possibly came from the generational habitus which constructed the physiological reserve , psychological attitudes , ways of thinking , values , and social behaviour of the oldest old men and women in a very different manner . men and women of these birth cohorts have grown up , lived , and grown old as completely different social beings . their gender - specific behaviour in old age continues to influence their longevity and quality of life in different ways . more recent cohorts of women have partially adopted risky behaviours , such as smoking or drinking alcohol , which were the prerogative of men in the past , and increasing participation in what was once considered exclusively male occupations ( transport , military , law enforcement , etc . ) the 200,000 centenarians who will live in france in 2060 , according to the latest population forecasts , will have a generational habitus constructed in the postwar period , shaped by social , cultural , technological , economic , and behavioural forces far different than that of today 's centenarians . but will the behaviour of men and women be more alike as they enter the ranks of the oldest old ? the gender gap in longevity and disability will likely remain in a state of flux for some time due to the continual shifts in the social , economic , and behavioural dynamics that determine health and longevity for both women and men . french people , who were born at the beginning of the 20th century ( i.e. , before 1915 ) , display a generational habitus which clearly juxtaposes men and women in terms of social identity , family role , and sense of purpose in life . this habitus still influences many gender - linked behaviours and adaptational strategies , even at the oldest ages . we suggest that these differences in behaviour favour functional health for men and longevity for women . the generational habitus influences the representation of the raison d'tre for men and women . on the contrary , the female raison d'tre , for the same cohort , is based more on their status in relation to others , in particular as givers of care , even if it is reduced to their mere presence next to those they love . they are more flexible , adaptive , and reed - like ; therefore , even if more severely disabled than men , women can continue to survive . quote from claude - levi strauss on the occasion of his 90th birthday(note that he continued on until he reached 100 years old , a centenarian , as did most of the subjects in this study . ) montaigne died at fifty - nine and surely had no idea of the extreme old age , in which i find myself today . having lived to a ripe old age which i never expected to attain , and which is one of the strangest surprises i have experienced , i feel like a shattered hologram . this hologram no longer possesses its entire unity , yet , as with any hologram , each surviving shard preserves an image and full representation of the whole.so today for me there is a real self , which is but a quarter or half a man , and a virtual self , which preserves a living idea of the whole . ( note that he continued on until he reached 100 years old , a centenarian , as did most of the subjects in this study . ) montaigne died at fifty - nine and surely had no idea of the extreme old age , in which i find myself today . having lived to a ripe old age which i never expected to attain , and which is one of the strangest surprises i have experienced , i feel like a shattered hologram . this hologram no longer possesses its entire unity , yet , as with any hologram , each surviving shard preserves an image and full representation of the whole . so today for me there is a real self , which is but a quarter or half a man , and a virtual self , which preserves a living idea of the whole .

incretinrelated drugs include glucagonlike peptide1 ( glp1 ) receptor agonists and dipeptidyl peptidase4 ( dpp4 ) inhibitors , which suppress glucagon secretion1 , confer a cell protective effect3 and do not alter bodyweight6 . the aforementioned effects have not been observed with conventional drugs , and these drugs are expected to provide an entirely new treatment option for diabetes . liraglutide , a glp1 receptor agonist , was approved in europe in june 2009 and in japan and the usa in january 2010 . in the phase iii clinical trial of the liraglutide effect and action in diabetes ( lead ) study , the safety and effectiveness of liraglutide , as a monotherapy or in various combination therapies with oral hypoglycemic agents ( ohas ) , was evaluated8 . in both studies furthermore , effects such as weight loss , decrease in systolic blood pressure , improved pancreatic cell function and improved cardiovascular markers have been confirmed . in the clinical development program for liraglutide in japan one study compared the efficacy and safety of liraglutide monotherapy and glibenclamide monotherapy15 , and the other compared the efficacy and safety of liraglutide combination therapy with sulfonylurea drugs , and sulfonylurea drug monotherapy17 . liraglutide has excellent hypoglycemic effects and can be used in hyperglycemic patients , who have maintained some insulin secretion capacity in response to hyperglycemia . from a longterm perspective , liraglutide should be aggressively used shortly after diabetes onset because of its protective effects on pancreatic cells that could prevent their dysfunction3 . in the present study , we compared liraglutide effects between the following two groups : ( i ) drugnave patients or those with a relatively short diabetic history who switched from ohas ; and ( ii ) those with a measureable diabetic progression who switched from insulin . in addition , the suitable timing of liraglutide administration in patients with type 2 diabetes in japan was evaluated . however , insulin induces hypoglycemia and weight gain because of its fat accumulation effect19 . because liraglutide treatment can reduce bodyweight6 and is associated with reduced hypoglycemia , weight loss and a lower risk of hypoglycemia are expected on switching to liraglutide from insulin . to establish standards for liraglutide therapy , we compared background characteristics of both a successful and failed group of patients who switched to liraglutide from insulin . patients with type 2 diabetes who visited to osaka red cross hospital ( osaka , japan ) between june 2010 and august 2011 , and started liraglutide treatment were observed in the present study . type 2 diabetic patients treated with diet therapy with or without ohas and/or insulin , had glycated hemoglobin ( hba1c ) concentrations > 5.5 and < 15.8% , were aged between 24 and 87 years , and had bodyweight > 40 kg were included in the present study . patients were excluded if they had detectable glutamic acid decarboxylase antibody , impaired hepatic function , significant cardiovascular disease ( heart failure , coronary artery disease or uncontrolled hypertension ) or nonstabilized proliferative retinopathy . before liraglutide administration , 82 patients were treated with insulin ( 64 patients with multiple daily injection and 18 with single basal injections ) , 128 patients were treated with ohas , including sulfonylurea ( 62 patients ) , biguanide ( 70 patients ) , thiazolidinedione ( 30 patients ) , alphaglucosidase inhibitor ( 11 patients ) , phenylalanine derivative ( 7 patients ) and dpp4 inhibitor ( 14 patients ) . in outpatients , the initial liraglutide dose was 0.3 mg , which was increased to 0.6 mg after 1 week . in hospitalized patients , after resolving glucose toxicity with intensive insulin therapy , the initial liraglutide dose was 0.3 mg , which was increased to 0.6 mg after 3 days . insulin was discontinued on liraglutide administration , whereas ohas were tapered or added based on each physician 's recommendation . responders included patients with improvement in hba1c or glycosylated albumin ( glya ) levels measured during a 4month observation period , or those with hba1c concentrations < 6.5% after switching to liraglutide from insulin . the present study was approved by the relevant ethics committee and was carried out in accordance with the declaration of helsinki . statistical analyses were carried out using the statview 5.0 software ( sas institute , inc . , differences in glycemic control improvement and changes in bodyweight between the drugnave / previous oha group and the group that switched from insulin was analyzed by anova . the present study was approved by the relevant ethics committee and was carried out in accordance with the declaration of helsinki . statistical analyses were carried out using the statview 5.0 software ( sas institute , inc . , cary , nc , usa ) . differences between groups were assessed using paired or unpaired twotailed student 's ttests . differences in glycemic control improvement and changes in bodyweight between the drugnave / previous oha group and the group that switched from insulin was analyzed by anova . patients included 74 males and 81 females ( n = 155 ) , and their characteristics are shown in table 1 . compared with the drugnave / previous oha group , the group that switched from insulin showed longer diabetes history , lower body mass index ( bmi ) values and lower cpeptide immunoreactivity ( cpr ) levels , suggesting diabetes progression . improvement in blood glucose levels was confirmed in 122 of the 155 patients , and 37 of 122 ( 30.3% ) and 60 of 122 ( 49.2% ) patients achieved hba1c levels of < 6.5 and < 7.0% , respectively . of these 122 patients , 87 were treated with ohas , including sulfonylurea in 49 , biguanide in 35 , thiazolidinedione in 13 , alphaglucosidase inhibitor in four and phenylalanine derivative in four . liraglutide was discontinued because of its sideeffects in 13 patients , and no improvement was observed in the remaining 20 patients . sideeffects included nausea ( n = 6 ) , discomfort ( n = 3 ) , dizziness ( n = 1 ) , anorexia ( n = 1 ) , abdominal distension ( n = 1 ) and stomach ache ( n = 1 ) . significant hypoglycemia was not detected in any patients during this study . in the drugnave group , improved blood glucose levels were observed in all nine patients ( 100% ) . in the group that switched from ohas to liraglutide , 56 of 64 patients ( 88% ) found liraglutide to be effective , 3 ( 5% ) discontinued liraglutide because of its sideeffects and 5 ( 7% ) discontinued liraglutide because of no improvement in glycemic control . in the group that switched to liraglutide from insulin , 57 of 82 patients ( 70% ) found liraglutide effective , 10 ( 12% ) discontinued liraglutide because of sideeffects and 15 ( 18% ) discontinued liraglutide because they showed no improvement in glycemic control ( figure 1 ) . gray and black bars indicate the group of nonresponders that discontinued liraglutide because glycemic control did not improve and the group that discontinued liraglutide because of sideeffects , respectively . all values are expressed as mean sem ( n = 155 ) . * p < 0.05 , * * p < 0.01 vs group of drug nave ; p < 0.05 , p < 0.01 vs group of previous oral hypoglycemic agents ( ohas ) . bmi , body mass index ; cpr , cpeptide immunoreactivity ; dm , diabetes mellitus ; glya , glycosylated albumin ; hba1c , glycated hemoglobin . we compared the effect on glycemic control of responders divided according to the pretreatment status ( before liraglutide administration ) . compared with the group that switched from insulin ( n = 57 ) , the group of drugnave / previous ohas ( n = 65 ) showed no difference in age , but the diabetes history tended to be shorter ( 11.6 1.0 vs 13.5 1.2 years ; p = 0.11 ) , there was a high proportion of hospitalized patients ( 53.8 vs 21.1% ) , high bmi values ( 29.8 0.7 vs 25.8 0.7 kg / m ; p < 0.01 ) , higher fasting cpr levels ( 2.5 0.2 vs 1.8 0.1 ng / ml ; p < 0.01 ) , higher maximum cpr levels ( 5.1 0.3 vs 3.8 0.2 ng / ml ; p < 0.01 ) , higher delta cpr levels before and after breakfast ( 2.5 0.3 vs 1.9 0.2 ng / ml ; p < 0.05 ) , and higher urine cpr levels ( 93.2 8.0 vs 71.7 6.4 g / day ; p < 0.05 ) , respectively ( table 2 ) . during the 4month observation period , improvement in hba1c levels was observed in the drugnave / previous oha group ( from 9.1 0.2 to 7.2 0.2% ) and in the group that switched from insulin ( from 8.6 0.2 to 7.8 0.2% ) . considerable and significant improvements were observed in both groups , and the extent of improvement was significant in the former ( figure 2a , upper ) . the improvement in glya levels was also observed in the drugnave / previous ohas group ( from 24.1 0.9 to 18.7 0.6% ) and in the group that switched from insulin ( from 24.3 0.9 to 22.0 0.7% ) . considerable and significant improvements were observed in both groups , and the extent of improvement was significant in the former ( figure 2a , lower ) . with respect to bodyweight , a reduction of 4.5 0.6% was observed in the group of drugnave / previous ohas , with a reduction of 5.1 0.8% in the group that switched from insulin ; a significant improvement was observed in both groups , and the extent was comparable between these two groups without significance ( figure 2b ) . the drugnave / previous oha group showed a higher proportion of hospitalized patients compared with those who switched from insulin ( 53.8 vs 21.1% ) ; therefore , hospitalization might influence the effects of liraglutide treatment . we then divided patients into subgroups of hospitalized patients and outpatients , and compared the effects of liraglutide treatment in each . in hospitalized patients , substantial improvement was observed in hba1c levels in the drugnave / previous oha group ( from 9.8 0.4 to 7.3 0.3% ) and in the group that switched from insulin ( from 10.0 0.6 to 7.8 0.5% ) . a significant improvement was observed in both groups , and the extent of improvement was comparable between the two without significance . with respect to bodyweight , a 6.6 1.8% reduction was observed in the drugnave / previous oha group , and a 6.2 1.3% reduction was observed in the group that switched from insulin ; a significant improvement was observed in both , but not significant between the two ( figure 2c , upper ) . meanwhile , in the outpatient group , the improvement in hba1c levels was observed in the drugnave / previous oha group ( from 8.2 0.2 to 7.1 0.2% ) , and in the group that switched from insulin ( from 8.2 0.2 to 7.8 0.2% ) . a significant improvement was observed in both groups , and the extent was significant in the former . with respect to bodyweight , a 6.6 1.3% reduction was observed in the drugnave / previous oha group , with a 6.2 1.3% reduction in the group that switched from insulin ; a significant improvement was observed in both groups , but was not significant between the two ( figure 2c , lower ) . ( a ) the improvement in glycated hemoglobin ( hba1c ) levels in the group of drugnave / previous oral hypoglycemic agents ( ohas ; round ) and in the group that switched from insulin ( triangle ) in the 4month observation period ( upper panel ) . the improvement in glycosylated albumin ( glya ) levels in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period ( lower panel ) . ( b ) the improvement in percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period . ( c ) the improvement in hba1c levels and percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period in hospitalized patients ( upper panel ) . the improvement in hba1c levels and percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period in outpatients ( lower panel ) . the comparison was carried out by paired twotailed student 's ttest and repeated anova tests . * p < 0.05 , * * p < 0.01 versus responders of drug nave / previous oral hypoglycemic agents ( ohas ) ; p < 0.05 , p < 0.01 versus responders that switched from insulin . bmi , body mass index ; cpr , cpeptide immunoreactivity ; dm , diabetes mellitus ; glya , glycosylated albumin ; hba1c , glycated hemoglobin . in the group that changed from ohas to liraglutide , eight of 64 patients ( 12.5% ) found liraglutide to be ineffective ( figure 1 ) . we carefully carried out a detailed comparison of background factors between the responders and nonresponders of the drugnave / previous oha group ; however , no significant differences were observed between the two ( table 2 , left column ) . next , we carried out a study on the group that switched to liraglutide from insulin . as shown in ( figure 1 ) , 57 of 82 patients ( 70% ) showed improved glycemic control in the group that switched from insulin ; in the remaining 25 patients ( 30% ) , no improvement was observed or liraglutide was discontinued because of sideeffects . compared with the nonresponders , the responders were younger ( 60.3 1.8 vs 69.4 2.1 years ; p < 0.01 ) , had a shorter history of diabetes ( 13.5 1.2 vs 20.2 2.0 years ; p < 0.01 ) , had higher fasting cpr levels ( 1.8 0.1 vs 1.2 0.2 ng / ml ; p < 0.01 ) , higher maximum cpr levels ( 3.8 0.2 vs 3.0 0.3 ng / ml ; p < 0.01 ) , higher delta cpr levels before and after breakfast ( 1.9 0.2 vs 1.1 0.1 ng / ml ; p < 0.01 ) , higher urine cpr levels ( 71.7 6.4 vs 39.6 6.0 g / day ; p < 0.01 ) , and used fewer insulin units per day ( 22.0 1.7 vs 32.6 4.4 u / day ; p < 0.05 ) , respectively ( table 2 , right column ) . we focused on individual cpr levels in blood and urine for both responders and nonresponders ( figure 3a ) . from this viewpoint , switching to liraglutide from insulin might succeed if fasting cpr was > 2.2 ng / ml , if delta cpr before and after breakfast was > 1.6 ng / ml , and if urine cpr was > 70 g / day . we also focused on the daily insulin dosage ( u / day ) and age with respect to switching to liraglutide from insulin ( figure 3b ) . in elderly patients . for patients aged 3070 years , switching to liraglutide from insulin was effective for those using insulin approximately 20 u / day , although some cases were unsuccessful despite using smaller doses ( approximately 10 u / day ) ; most cases were unsuccessful in switching if the amount of insulin per day exceeded 40 u / day , except in some younger patients . therefore , when switching to liraglutide from insulin , although several cases were observed with some exceptions , a cautious decision with confirming endogenous insulin secretion capacity , age and daily insulin usage should be obtained . ( cpr ; left panel ) , delta cpr before and after breakfast ( middle panel ) , and urine cpr ( right panel ) levels between responders and nonresponders . investigation and comparison of background factors between responders ( white circles ) and nonresponders ( gray circles , no improvement of glycemic conrol ; black circles , sideeffects ) after switching to liraglutide from insulin . the vertical axis represents the amount of daily insulin use before switching to liraglutide , and the horizontal axis represents the age . type 2 diabetes is a progressive disease , and its pathogenesis includes insulin resistance and insulin secretory dysfunction , which is recognized before disease onset . a decrease by half in pancreatic cell function has been reported on diagnosis20 , and a 20% decrease at the stage of impaired glucose tolerance21 . furthermore , compared with the western population , the japanese population is more prone to insulin secretion failure caused by pancreatic cell dysfunction22 . dpp4 inhibitors and glp1 receptor agonists , which are incretinrelated drugs , have a protective effect on pancreatic cells3 . given the longterm perspective , pancreatic cell dysfunction in diabetic patients can be prevented by relatively early intensive therapy with these agents when postprandial hyperglycemia becomes detectable . in phase iii liraglutide clinical trials in japan , greater improvement in glycemic control was observed in drugnave patients compared with those who switched from oral agents15 . however , the difference between the liraglutide effects with respect to pretreatment status , including insulin and disease stage , has not been previously reported . here , we examined and compared the liraglutide effects in type 2 diabetic patients having a relatively shorter diabetic history and were drugnave or previously treated with ohas , and patients who switched from insulin . compared with the group that switched from insulin , the drugnave / previous oha group showed no differences in age , but their diabetes history tended to be shorter , with higher bmi values , higher cpr levels in blood and urine , and particularly , a more pronounced improvement in blood glucose levels with a higher percentage of responders . from point of this view , liraglutide administration in early diabetic patients who have retained some endogenous insulin secretory capacity are more effective . randomized clinical trials of incretinrelated drugs including liraglutide should be carried out , and by accumulating evidence on optimal hyperglycemia management , a diabetes treatment algorithm should be proposed in the future . because insulin exerts a potent hypoglycemic action , a significant risk for hypoglycemia is often observed . insulin induces fat accumulation in the peripheral adipose tissue by inhibiting lipolysis through hormonesensitive lipases23 , leading to weight gain19 . insulin exerts an anorexic effect on the central nervous system24 ; however , insulintreated diabetic patients often eat too much for the fear or experience of hypoglycemia attack , which can also lead to weight gain25 . diabetic patients are often obese , which is frequently complicated by metabolic syndrome with hypertension or dyslipidemia26 . therefore , by using insulin for glycemic control , blood glucose levels per se improve , but weight gain could increase accompanied with hyperlipidemia and hypertension . for example , higher insulin doses in the veteran affairs diabetes trial were associated with weight gain as well as hypoglycemia , a predictor of cardiovascular mortality and macrovascular outcomes27 . liraglutide treatment has been shown to be associated with reduced hypoglycemia , and it promotes weight loss6 by reducing appetite through the suppression of eliminating gastric contents28 or by acting on the central nervous system29 . therefore , switching to liraglutide from insulin reduces hypoglycemia risk , and alleviates dyslipidemia and hypertension because of weight loss . furthermore , except for basal supported oral therapy , insulin is often injected twice ( in morning and evening before meals ) or four times in basalbolus therapy . because liraglutide has a halflife of 13 h , only one injection per day is required , thus relieving patients from multiple daily insulin injections . therefore , the disadvantages of insulin , such as the risk of hypoglycemia , weight gain and the necessity of multiple injections might be overcome by switching to liraglutide in certain cases . however , liraglutide is not a substitute for insulin . in japan , during the 4 months ( 11 june 2010 to 7 october 2010 ) since the approval of liraglutide , four cases of diabetic ketoacidosis and 16 cases of hyperglycemia have been reported . in 17 out of 20 hyperglycemic cases , the condition resulted after switching to liraglutide from insulin . therefore , liraglutide should not be considered a substitute for insulin , and the insulindependent status should be determined to gauge whether liraglutide treatment is appropriate before administration . some facilities carry out glucagon load testing to evaluate endogenous insulin secretion ; however , the criterion has not yet been clearly defined . to determine the standard criterion for switching to liraglutide from insulin , we carried out a study on the group that switched to liraglutide from insulin , and carried out a detailed comparison of background factors between the responders and nonresponders . consequently , switching to liraglutide from insulin might succeed if fasting cpr is > 2.2 ng / ml , delta cpr before and after breakfast is > 1.6 ng / ml , and urine cpr is > 70 g / day . several cases were successfully treated even with low cpr levels ; however , in such cases , insulin was used to avoid glucose toxicity , which was believed to lower cpr levels . conversely , the unsuccessful cases were found to show relatively high fasting cpr levels that were complicated by reduced renal function caused by diabetic nephropathy , as decreased cpr clearance might increase apparent cpr levels . by carefully omitting these cases , cpr values might be more accurately delineated . in type 2 diabetic patients , the elderly frequently experience progressive exhaustion of pancreatic cells , but younger people have relatively functional pancreatic cells30 . the capacity of liraglutide to assist with pancreatic cell proliferation has also been reported to decline with age31 . we then focused on the age and daily insulin dosage with respect to switching to liraglutide from insulin . aged 3070 years , most found that switching to liraglutide was effective if their daily insulin dosage was approximately 20 u / day ; most cases were unsuccessful in switching if the amount of insulin per day exceeded 40 u / day , except in some younger patients . therefore , switching to liraglutide from insulin might succeed if the patient is not elderly , and if a smaller insulin dose is used . no other reports have described liraglutide use in patients who switched from insulin , and only one report described switching to exenatide from insulin32 . in that study consequently , 18 patients ( 62% ) successfully achieved glycemic control , whereas the remaining 11 ( 38% ) were unsuccessful . however , responders were defined if a hba1c increase was < 0.5% , and the content of that paper has also received critical comments33 . several exceptions were observed in the present study . in switching from insulin , some patients found liraglutide to be ineffective despite using less insulin ( approximately 10 u / day ) . liraglutide was ineffective in some drugnave / previous oha patients , although their cpr levels were high , suggesting that endogenous insulin secretory capacity was well preserved . we could not clearly explain the reason , but can only speculate that the patients ' habits ( exercise , tv viewing , smoking and eating habits ) during the present study might have directly worsened their glycemic control . in the lead test , daily liraglutide usage was 1.2 or 1.8 mg , and dosedependent effects were confirmed8 . in japan , the magnitude of hba1c reduction with liraglutide in the present study ( a 1.3% reduction in hba1c using 0.6 mg of liraglutide in 4 months ) was comparable with that reported in japanese patients34 ( a 1.6% reduction in hba1c using 0.6 mg of liraglutide over 14 weeks ) and in nonjapanese patients35 ( a 0.7% reduction in hba1c using 0.6 mg of liraglutide over 12 weeks ) . in the present study , we used a fixed amount of daily liraglutide ; that is , 0.6 mg , during the observation period . improvement in glycemic control was not observed in 20 of 155 patients , and achievement of hba1c < 7.0% was not observed in approximately half of the patients . the maximum dose of liraglutide is 0.9 mg in japan , and some patients believed that the drug effect would increase if 0.9 mg was used , which should be investigated in the future . in conclusion , the present study showed that liraglutide improved blood glucose levels , which was more pronounced in the drugnave / previous oha group compared with the group that switched from insulin . in addition , cpr levels , age and the daily insulin dosage were considered important factors when deciding to switch to liraglutide from insulin . based on these findings , we can establish standards for liraglutide treatment that are appropriate for type 2 diabetic patients .

abstractaims / introductionliraglutide , a glucagonlike peptide1 receptor agonist , is expected to provide a new treatment option for diabetes . however , the suitable timing of liraglutide administration in type 2 diabetic patients has not yet been clarified.materials and methodswe reviewed type 2 diabetic patients ( n = 155 ) who visited the osaka red cross hospital for glycemic control , with administration of liraglutide at a dose of 0.6 mg ( average glycated hemoglobin [ hba1c ] level , 8.7 0.1% ) . the effect of liraglutide based on the pretreatment status was compared . we also analyzed the background factors of both a successful and failed group of patients who switched to liraglutide from insulin.resultsan improvement in blood glucose levels was confirmed in 122 of 155 patients . during the 4month observation period , the improvement in hba1c levels was significantly greater in the group of drugnave / previous oral hypoglycemic agent ( 9.1 0.2 to 7.2 0.2% ) than that in the group switching from insulin ( 8.6 0.2 to 7.8 0.2% ) . in addition , cpeptide immunoreactivity levels ( fasting > 2.2 ng / ml ; delta > 1.6 ng / ml ; urine > 70 g / day ) , younger age and a smaller number of insulin units used per day were considered important when deciding on switching to liraglutide from insulin.conclusionsliraglutide was more effective in patients who had not been treated previously or received oral hypoglycemic agents than in patients switching from insulin . with respect to switching to liraglutide from insulin , the most important factors to be considered were cpeptide immunoreactivity levels , age , and the number of insulin units used per day .

Introduction Materials and Methods Ethics Statistical Analyses Results Discussion

the aforementioned effects have not been observed with conventional drugs , and these drugs are expected to provide an entirely new treatment option for diabetes . liraglutide , a glp1 receptor agonist , was approved in europe in june 2009 and in japan and the usa in january 2010 . in the phase iii clinical trial of the liraglutide effect and action in diabetes ( lead ) study , the safety and effectiveness of liraglutide , as a monotherapy or in various combination therapies with oral hypoglycemic agents ( ohas ) , was evaluated8 . in the clinical development program for liraglutide in japan one study compared the efficacy and safety of liraglutide monotherapy and glibenclamide monotherapy15 , and the other compared the efficacy and safety of liraglutide combination therapy with sulfonylurea drugs , and sulfonylurea drug monotherapy17 . in addition , the suitable timing of liraglutide administration in patients with type 2 diabetes in japan was evaluated . because liraglutide treatment can reduce bodyweight6 and is associated with reduced hypoglycemia , weight loss and a lower risk of hypoglycemia are expected on switching to liraglutide from insulin . to establish standards for liraglutide therapy , we compared background characteristics of both a successful and failed group of patients who switched to liraglutide from insulin . patients with type 2 diabetes who visited to osaka red cross hospital ( osaka , japan ) between june 2010 and august 2011 , and started liraglutide treatment were observed in the present study . type 2 diabetic patients treated with diet therapy with or without ohas and/or insulin , had glycated hemoglobin ( hba1c ) concentrations > 5.5 and < 15.8% , were aged between 24 and 87 years , and had bodyweight > 40 kg were included in the present study . in outpatients , the initial liraglutide dose was 0.3 mg , which was increased to 0.6 mg after 1 week . responders included patients with improvement in hba1c or glycosylated albumin ( glya ) levels measured during a 4month observation period , or those with hba1c concentrations < 6.5% after switching to liraglutide from insulin . , differences in glycemic control improvement and changes in bodyweight between the drugnave / previous oha group and the group that switched from insulin was analyzed by anova . differences in glycemic control improvement and changes in bodyweight between the drugnave / previous oha group and the group that switched from insulin was analyzed by anova . patients included 74 males and 81 females ( n = 155 ) , and their characteristics are shown in table 1 . compared with the drugnave / previous oha group , the group that switched from insulin showed longer diabetes history , lower body mass index ( bmi ) values and lower cpeptide immunoreactivity ( cpr ) levels , suggesting diabetes progression . improvement in blood glucose levels was confirmed in 122 of the 155 patients , and 37 of 122 ( 30.3% ) and 60 of 122 ( 49.2% ) patients achieved hba1c levels of < 6.5 and < 7.0% , respectively . in the drugnave group , improved blood glucose levels were observed in all nine patients ( 100% ) . in the group that switched from ohas to liraglutide , 56 of 64 patients ( 88% ) found liraglutide to be effective , 3 ( 5% ) discontinued liraglutide because of its sideeffects and 5 ( 7% ) discontinued liraglutide because of no improvement in glycemic control . in the group that switched to liraglutide from insulin , 57 of 82 patients ( 70% ) found liraglutide effective , 10 ( 12% ) discontinued liraglutide because of sideeffects and 15 ( 18% ) discontinued liraglutide because they showed no improvement in glycemic control ( figure 1 ) . gray and black bars indicate the group of nonresponders that discontinued liraglutide because glycemic control did not improve and the group that discontinued liraglutide because of sideeffects , respectively . all values are expressed as mean sem ( n = 155 ) . * p < 0.05 , * * p < 0.01 vs group of drug nave ; p < 0.05 , p < 0.01 vs group of previous oral hypoglycemic agents ( ohas ) . we compared the effect on glycemic control of responders divided according to the pretreatment status ( before liraglutide administration ) . compared with the group that switched from insulin ( n = 57 ) , the group of drugnave / previous ohas ( n = 65 ) showed no difference in age , but the diabetes history tended to be shorter ( 11.6 1.0 vs 13.5 1.2 years ; p = 0.11 ) , there was a high proportion of hospitalized patients ( 53.8 vs 21.1% ) , high bmi values ( 29.8 0.7 vs 25.8 0.7 kg / m ; p < 0.01 ) , higher fasting cpr levels ( 2.5 0.2 vs 1.8 0.1 ng / ml ; p < 0.01 ) , higher maximum cpr levels ( 5.1 0.3 vs 3.8 0.2 ng / ml ; p < 0.01 ) , higher delta cpr levels before and after breakfast ( 2.5 0.3 vs 1.9 0.2 ng / ml ; p < 0.05 ) , and higher urine cpr levels ( 93.2 8.0 vs 71.7 6.4 g / day ; p < 0.05 ) , respectively ( table 2 ) . during the 4month observation period , improvement in hba1c levels was observed in the drugnave / previous oha group ( from 9.1 0.2 to 7.2 0.2% ) and in the group that switched from insulin ( from 8.6 0.2 to 7.8 0.2% ) . the improvement in glya levels was also observed in the drugnave / previous ohas group ( from 24.1 0.9 to 18.7 0.6% ) and in the group that switched from insulin ( from 24.3 0.9 to 22.0 0.7% ) . with respect to bodyweight , a reduction of 4.5 0.6% was observed in the group of drugnave / previous ohas , with a reduction of 5.1 0.8% in the group that switched from insulin ; a significant improvement was observed in both groups , and the extent was comparable between these two groups without significance ( figure 2b ) . the drugnave / previous oha group showed a higher proportion of hospitalized patients compared with those who switched from insulin ( 53.8 vs 21.1% ) ; therefore , hospitalization might influence the effects of liraglutide treatment . in hospitalized patients , substantial improvement was observed in hba1c levels in the drugnave / previous oha group ( from 9.8 0.4 to 7.3 0.3% ) and in the group that switched from insulin ( from 10.0 0.6 to 7.8 0.5% ) . with respect to bodyweight , a 6.6 1.8% reduction was observed in the drugnave / previous oha group , and a 6.2 1.3% reduction was observed in the group that switched from insulin ; a significant improvement was observed in both , but not significant between the two ( figure 2c , upper ) . meanwhile , in the outpatient group , the improvement in hba1c levels was observed in the drugnave / previous oha group ( from 8.2 0.2 to 7.1 0.2% ) , and in the group that switched from insulin ( from 8.2 0.2 to 7.8 0.2% ) . with respect to bodyweight , a 6.6 1.3% reduction was observed in the drugnave / previous oha group , with a 6.2 1.3% reduction in the group that switched from insulin ; a significant improvement was observed in both groups , but was not significant between the two ( figure 2c , lower ) . ( a ) the improvement in glycated hemoglobin ( hba1c ) levels in the group of drugnave / previous oral hypoglycemic agents ( ohas ; round ) and in the group that switched from insulin ( triangle ) in the 4month observation period ( upper panel ) . the improvement in glycosylated albumin ( glya ) levels in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period ( lower panel ) . ( b ) the improvement in percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period . ( c ) the improvement in hba1c levels and percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period in hospitalized patients ( upper panel ) . the improvement in hba1c levels and percent bodyweight in the group of drugnave / previous ohas ( round ) and in the group that switched from insulin ( triangle ) in the 4month observation period in outpatients ( lower panel ) . * p < 0.05 , * * p < 0.01 versus responders of drug nave / previous oral hypoglycemic agents ( ohas ) ; p < 0.05 , p < 0.01 versus responders that switched from insulin . bmi , body mass index ; cpr , cpeptide immunoreactivity ; dm , diabetes mellitus ; glya , glycosylated albumin ; hba1c , glycated hemoglobin . in the group that changed from ohas to liraglutide , eight of 64 patients ( 12.5% ) found liraglutide to be ineffective ( figure 1 ) . we carefully carried out a detailed comparison of background factors between the responders and nonresponders of the drugnave / previous oha group ; however , no significant differences were observed between the two ( table 2 , left column ) . next , we carried out a study on the group that switched to liraglutide from insulin . as shown in ( figure 1 ) , 57 of 82 patients ( 70% ) showed improved glycemic control in the group that switched from insulin ; in the remaining 25 patients ( 30% ) , no improvement was observed or liraglutide was discontinued because of sideeffects . compared with the nonresponders , the responders were younger ( 60.3 1.8 vs 69.4 2.1 years ; p < 0.01 ) , had a shorter history of diabetes ( 13.5 1.2 vs 20.2 2.0 years ; p < 0.01 ) , had higher fasting cpr levels ( 1.8 0.1 vs 1.2 0.2 ng / ml ; p < 0.01 ) , higher maximum cpr levels ( 3.8 0.2 vs 3.0 0.3 ng / ml ; p < 0.01 ) , higher delta cpr levels before and after breakfast ( 1.9 0.2 vs 1.1 0.1 ng / ml ; p < 0.01 ) , higher urine cpr levels ( 71.7 6.4 vs 39.6 6.0 g / day ; p < 0.01 ) , and used fewer insulin units per day ( 22.0 1.7 vs 32.6 4.4 u / day ; p < 0.05 ) , respectively ( table 2 , right column ) . from this viewpoint , switching to liraglutide from insulin might succeed if fasting cpr was > 2.2 ng / ml , if delta cpr before and after breakfast was > 1.6 ng / ml , and if urine cpr was > 70 g / day . we also focused on the daily insulin dosage ( u / day ) and age with respect to switching to liraglutide from insulin ( figure 3b ) . for patients aged 3070 years , switching to liraglutide from insulin was effective for those using insulin approximately 20 u / day , although some cases were unsuccessful despite using smaller doses ( approximately 10 u / day ) ; most cases were unsuccessful in switching if the amount of insulin per day exceeded 40 u / day , except in some younger patients . therefore , when switching to liraglutide from insulin , although several cases were observed with some exceptions , a cautious decision with confirming endogenous insulin secretion capacity , age and daily insulin usage should be obtained . investigation and comparison of background factors between responders ( white circles ) and nonresponders ( gray circles , no improvement of glycemic conrol ; black circles , sideeffects ) after switching to liraglutide from insulin . the vertical axis represents the amount of daily insulin use before switching to liraglutide , and the horizontal axis represents the age . however , the difference between the liraglutide effects with respect to pretreatment status , including insulin and disease stage , has not been previously reported . here , we examined and compared the liraglutide effects in type 2 diabetic patients having a relatively shorter diabetic history and were drugnave or previously treated with ohas , and patients who switched from insulin . compared with the group that switched from insulin , the drugnave / previous oha group showed no differences in age , but their diabetes history tended to be shorter , with higher bmi values , higher cpr levels in blood and urine , and particularly , a more pronounced improvement in blood glucose levels with a higher percentage of responders . from point of this view , liraglutide administration in early diabetic patients who have retained some endogenous insulin secretory capacity are more effective . therefore , by using insulin for glycemic control , blood glucose levels per se improve , but weight gain could increase accompanied with hyperlipidemia and hypertension . therefore , switching to liraglutide from insulin reduces hypoglycemia risk , and alleviates dyslipidemia and hypertension because of weight loss . therefore , the disadvantages of insulin , such as the risk of hypoglycemia , weight gain and the necessity of multiple injections might be overcome by switching to liraglutide in certain cases . in 17 out of 20 hyperglycemic cases , the condition resulted after switching to liraglutide from insulin . therefore , liraglutide should not be considered a substitute for insulin , and the insulindependent status should be determined to gauge whether liraglutide treatment is appropriate before administration . some facilities carry out glucagon load testing to evaluate endogenous insulin secretion ; however , the criterion has not yet been clearly defined . to determine the standard criterion for switching to liraglutide from insulin , we carried out a study on the group that switched to liraglutide from insulin , and carried out a detailed comparison of background factors between the responders and nonresponders . consequently , switching to liraglutide from insulin might succeed if fasting cpr is > 2.2 ng / ml , delta cpr before and after breakfast is > 1.6 ng / ml , and urine cpr is > 70 g / day . in type 2 diabetic patients , the elderly frequently experience progressive exhaustion of pancreatic cells , but younger people have relatively functional pancreatic cells30 . we then focused on the age and daily insulin dosage with respect to switching to liraglutide from insulin . aged 3070 years , most found that switching to liraglutide was effective if their daily insulin dosage was approximately 20 u / day ; most cases were unsuccessful in switching if the amount of insulin per day exceeded 40 u / day , except in some younger patients . therefore , switching to liraglutide from insulin might succeed if the patient is not elderly , and if a smaller insulin dose is used . no other reports have described liraglutide use in patients who switched from insulin , and only one report described switching to exenatide from insulin32 . in switching from insulin , some patients found liraglutide to be ineffective despite using less insulin ( approximately 10 u / day ) . in japan , the magnitude of hba1c reduction with liraglutide in the present study ( a 1.3% reduction in hba1c using 0.6 mg of liraglutide in 4 months ) was comparable with that reported in japanese patients34 ( a 1.6% reduction in hba1c using 0.6 mg of liraglutide over 14 weeks ) and in nonjapanese patients35 ( a 0.7% reduction in hba1c using 0.6 mg of liraglutide over 12 weeks ) . in the present study , we used a fixed amount of daily liraglutide ; that is , 0.6 mg , during the observation period . improvement in glycemic control was not observed in 20 of 155 patients , and achievement of hba1c < 7.0% was not observed in approximately half of the patients . the maximum dose of liraglutide is 0.9 mg in japan , and some patients believed that the drug effect would increase if 0.9 mg was used , which should be investigated in the future . in conclusion , the present study showed that liraglutide improved blood glucose levels , which was more pronounced in the drugnave / previous oha group compared with the group that switched from insulin . in addition , cpr levels , age and the daily insulin dosage were considered important factors when deciding to switch to liraglutide from insulin . based on these findings , we can establish standards for liraglutide treatment that are appropriate for type 2 diabetic patients .

sweden is gradually changing to a religiously and culturally diverse as well as multiethnic society and the swedish population includes 19% with a foreign background . according to the swedish national board of health and welfare people with a foreign background are more likely to be disadvantaged when it comes to socioeconomic situation and health . however , health care is publicly funded and available to everyone . childhood cancer care includes very advanced medical , supportive , and nursing care , and in sweden it is centralized to six childhood cancer care centres . a holistic care of the sick child in the context of its family has always been important in childhood cancer care in sweden and family - centred care is a pillar in paediatric care . family - centred care not only includes family involvement in the care of the child but views the family as the recipient of care as well and this should be planned to benefit the whole family . parents report that the quality of care is of great importance and is reflected in the creation of trust . furthermore , rapport between parents and staff , particularly nurses , is very important for the interaction to be perceived as positive by parents but also for effective sharing of the care of the child . parents want to be involved in the care of their child but parental involvement is influenced by support , professionalism , work environment , and responsibility ; responsibility in terms of keeping track of and checking the child 's treatment . routines for involving parents in care have been found to be better in paediatric oncology units in sweden compared to other units at children 's hospitals but there are barriers to this involvement that need to be overcome if parents are to be successfully involved in care . linguistic , cultural , and religious differences as well as organizational obstacles hinder the development of rapport and caring relationships and thus also parental involvement in care . in instances where providers and recipients of health care do not speak the same language , developing an effective caring relationship can be impeded and an increase of the risk of serious medical events in paediatric care has also been found . in paediatric oncology , parents are expected to be increasingly involved in the care of their child but the experience of foreign - born parents is not well known in this context . the purpose of this study was to gain knowledge about foreign - born parents ' main concern and their experiences of the situation , including their child 's illness and treatment , and social interactions in the context of paediatric oncology care and to explain how this was dealt with . in this qualitative study , a classical grounded theory approach [ 1416 ] was chosen to discover how participants deal with their social situation and interactions ; resolving their main concern . in accordance with the method [ 1416 ] , sampling was guided by the purpose of the study ; aiming for easily accessible people with personal experience and knowledge about the subject . the first author and a care coordination nurse identified potential participants , at one paediatric oncology centre , through a list of newly diagnosed patients . inclusion criteria were as follows : presence of the parent at the hospital during the child 's admissions . the child was still undergoing treatment at the childhood cancer care unit . a care coordination nurse , who was not involved either in the research project or in the patients ' direct care , invited potential participants to participate in the study and three declined . the care coordinator nurse also gave them written information in swedish or arabic about the aim and the procedure of the study . also parents from other language groups were invited to participate and interpreters were offered , however , no written information was available in any other languages . after that the first author contacted parents , who consented verbally to participate , to decide on the date , time , and place for the interview . this resulted in 11 parents ( 10 mothers , 1 father ) ; demographic data of the participants are presented in table 1 . they varied in length ; 65120 minutes ( median 91 minutes ) with interpreter and 30170 minutes ( median 74 minutes ) without interpreter . the interviews took the form of semistructured conversations where the first author invited participants to tell about their experiences of their child 's illness and treatment . an independent authorized translator translated the questionnaire and the interview guide into arabic , after that an interpreter translated them back to swedish and the two versions were compared to identify differences . the swedish language was used in all interviews but interpreters of own mother tongues were offered to all participants . relating to participants ' preferences , all but four of the interviews were conducted without interpreters , with the rest facilitated by the same female arabic - speaking authorized healthcare interpreter . triangular seating and consecutive translation were used during the interviews and the interpreter was informed of the aim of the study . field notes were taken directly after each interview and all were audio recorded and transcribed . field notes and transcribed interviews were coded using the qualitative data analysis software nvivo 2.0 as a tool . open coding line - by - line was used until the core and related categories had emerged . during coding , codes were grouped together into concepts , which in classical grounded theory is the naming of a pattern of behaviour . consistent with grounded theory , when the core concept emerged , selective coding was conducted . this was combined with theoretical sampling , thus data was collected , coded , and analysed jointly to enable the process of deciding what data to collect , based on the selected codes , and this modified probing questions used in subsequent interviews [ 1416 ] . the second author validated the codes and concepts by listening to the recordings independently , as well as by reading transcripts and field notes . also literature was used as data in what is termed constant comparison in grounded theory . further , theoretical coding included the exploration of potential theoretical codes that could integrate concepts , in this study approaches is an example of a theoretical code . memos were written throughout the analysis ; these are notes about codes , concepts , and their relationships to each other . most of the analyses were performed using swedish but concepts were also named in english . ethical approval was obtained from the regional ethical review board . before the interviews , the voluntary nature of participation and the right to withdraw at any time were emphasized . to protect confidentiality of participants , in the context of childhood cancer care , the main concern of foreign - born parents is to make it through an uncertain situation of extreme emotional burden and stress by struggling on . foreign - born parents often feel in a position of powerless dependence in relation to the child 's illness and also in relation to healthcare staff . protecting family interests is one aspect of struggling on , which encompasses the engagement in information monitoring . protecting family interests is how parents interact with healthcare staff , which includes cooperation , contesting , and reluctant resigning . the concepts are presented below and are outlined in figure 1 . being dependent on others , for care and information , results in perceived powerlessness . the powerlessness is not a matter of complete incapacity but rather a reflection of not being in control . as parents enter the healthcare system , often with a feeling of being exposed , in need of help and powerless in relation to the child 's illness , the interaction with healthcare staff can accentuate or lessen these feelings . discrimination and suspicion of discrimination accentuate the feeling of powerless - dependence as well as frustration . furthermore , powerless dependence is influenced by the opportunity for parents to present their point of view and share opinions with healthcare staff and by being listened to . this is influenced by the social status of the parent : i feel that in sweden [ people have the attitude toward me that ] -you do not speak plain swedish , you speak with an accent , what do you know ? ' ( mother from europe of a child with leukaemia . ) additionally , linguistic obstacles negatively influence attempts to present opinions and thereby accentuate the feeling of powerless dependence . the greater the language barrier , the more likely is the feeling of powerless dependence : i did n't have such a great role [ in the treatment decision for my sick child ] because i ca n't speak the language so i could n't exercise my role ( mother from greater middle east of a child with brain tumour . ) however , this can be overcome if parents perceive that they are listened to and that their views matter in decisions about the child 's care and treatment . thus , the more the vulnerable person experience that their views matter and that they are listened to , the less the level of felt powerless - dependence . information monitoring assists parents in gaining some control over the situation and increasing the possibility that family interests will be protected . the type of information sought includes health - related information about the child 's diagnosis , prognosis , the child 's present condition such as blood values , and the available treatment options . the latter include treatment alternatives , side - effects , self - help , and seeking a second opinion . information is sought from different sources , including physicians , nursing staff , other parents , the sick child , the literature , and the internet . for some parents , the level is low because they are already satisfied with , or overwhelmed by , the information at hand , whereas others have a very high level of information seeking and monitoring , as exemplified by we have received so much information ; we have as much as we need and more to . ( mother from europe of a child with a solid tumour . ) you have to ask each individual doctor , what do you think and what would you do and in that way you can get many different opinions and then you have to think and come up with a joint decision , the best . ( mother from europe of a child with a solid tumour . ) when satisfied with the information provided by healthcare personnel , parents are less likely to actively seek information . conversely , disaffection with the information makes it more likely that parents will engage in high level information seeking behaviour to protect family interests . the protection of family interests includes protecting parents ' own interests and principally those of the sick child . parents interact with healthcare staff in such a way as to protect family interests and to achieve the best possible care for the child through cooperation , contesting , and reluctant resigning . families enter the healthcare system with different levels of trust and with different expectations . where there is disparity between these expectations and the possibilities of the delivery of care , parents are more likely to actively protect family interests . the approaches used vary with personality , individual preferences , and perceived levels of powerless - dependence . they will also vary for the same parent , who will fluctuate between different approaches , depending on the situation and conditions . all parents use different approaches at different times and can use two approaches simultaneously for different issues or different staff members . conditions that influence the choice of approaches include the way the family is considered by the staff , attitudes among staff , treatment alternatives , prognosis of the disease , and the family 's previous experiences . sometimes these approaches arise as a consequence of interactions with healthcare staff , for example , the establishment of rapport might lead to a trustful cooperation . at other times approaches are deliberate efforts to influence decisions and manoeuvre the care situation and can be considered strategic . parents try to stay on good terms with the staff and create good relationships to obtain the best possible care for their child while protecting family interests . one parent said the moment when i have given my child into the doctor 's hands ) cooperation is negotiated , particularly in relation to the balancing of responsibilities between parents and staff in relation to the care of the child . this negotiation can be open or closed ; the latter is achieved with subtle , implicit messages , or even assumptions of role divisions . when subtle messages are misunderstood or not perceived at all , this could lead to conflict in the negotiation . at times , parents entrust responsibilities and decisions to the staff , as exemplified by the following quote : then it will be fine , if you think that it is the best , then we are on we say : okay , then we are consenting or what it is called . ( mother from europe of a child with leukaemia . ) cooperation is used by all parents , but the level of trust varies . the higher the level of trust , the better the conditions for cooperation . cooperation is motivated by the relationship with healthcare staff and by the wish to protect family interests but can also be partly motivated by , or be a consequence of , powerless dependence in an attempt to decrease vulnerability . when trust is lacking or care expectations are not met , cooperation changes to contesting to protect family interests . contesting is an approach to protect family interests and often includes persuasive communicating , that is , providing convincing information in a persistent way , sometimes with emotional display and threats . convincing information often concerns needs of the child that parents are trying to convince healthcare staff about , for example , the need to be cared for at a specialist unit or the need for extra recourses or support . contesting is often linked to a strong feeling of powerless dependence and sometimes also a mistrust of the healthcare staff or healthcare system . occasionally , contesting is a consequence of a perceived wrongdoing by healthcare staff ; at other times , it is a strategic approach to influence and manoeuvre healthcare staff in care decisions . when parents suspect that their interests are being wrongfully ignored , for instance , because of racism and prejudice , they will try to contest for their rights : i have said outright to the nurses what i think . ( mother from greater middle east of a child with leukaemia . ) for many , contesting starts as they try to gain access to care if this is denied , for example , before a serious disease is confirmed , or if they believe that their rights are not being acknowledged : we were very badly treated actually sometimes you can see as a foreigner that it is a little bit unfair then we came there for the third time in a week and her father said that you have to shout at them , you have to tell them that they should examine your daughter properly . then i said , you must come too and say it yourself , you 're a man , maybe they will listen . contesting is situation dependent and where there is a lack of mutual trust parents are more likely to respond to healthcare staff by contesting . when trusting relations have not been developed , because , for example , of personalities , experience of wrong treatment or obstacles to transcultural caring relationships , parents will try to remain in control by contesting in every possible way to protect family interests : ( mother from europe of a child with leukaemia . ) even in trusting relationships , if family interests are threatened in any way , then parents are likely to engage in contesting to achieve their goals . once goals have been realized , parents attempt to resume trustful cooperation but this can be very difficult to achieve when staff perceive contesting as negative . if this reaction persists , then parents are even more likely to engage in contesting as a response to healthcare staff , thereby creating a causal loop , where parents and staff react to each other in ways that make negative reactions more likely and the reestablishment of trustful cooperation more doubtful . if parents fail to protect their family interests through a combination of cooperation and contesting , then they are likely to resign themselves to the situation through reluctant resigning . reluctant resigning is an approach to protect family interests and is more likely when parents feel powerless dependence . contesting is used when there is some hope of influencing decisions whereas reluctant resigning is often triggered by a critical juncture , when parents feel unable to influence what is happening or decisions being made without consultation . simply , they feel disempowered and unable to do anything about it except to accept the situation as it is , or risk permanently alienating staff and becoming exhausted in the process . both i and my husband were very irritated by them [ physicians ] they tried to persuade us and tell us how we should do this . the only thing that i could say then was this ; if you do not have that experience this is not a guinea pig who you can just try and try on , so to say , so if there is anywhere else where he could get a better treatment . it 's a pity ; he 's just a small child , to have to expose him to this . that was the only thing that i could say i felt like a butchered bird . in this classical grounded theory study with foreign - born parents in paediatric cancer care , struggling on includes information - monitoring such as health information seeking , avoiding and controlling behaviours . different patterns of information - monitoring ( miller , 1980 in ) and information - seeking behaviours have previously been described . it is of great importance to keep parents sufficiently informed , especially because , within paediatric oncology , parents are not currently entirely satisfied with the amount and timing of information [ 20 , 21 ] . furthermore , lack of information to parents in paediatric cancer care leads to a feeling of being unwelcome and abandoned . parents and patients often receive an insufficient level and amount of information , leading to conflict in care . even though information giving has been a constant subject of nursing research for many years , insufficient information giving continues to be an issue as evidenced in this study . information monitoring is influenced by psychosocial , cultural , and sociodemographic factors , such as ethnicity ( johnson , 1997 in ) and it might be expected that there would be a low level of information seeking in this group of foreign - born parents . however , education and social roles also influence information seeking and the findings in the present study that information monitoring is important to protecting family interests might be explained by the fact that participants had a rather high level of education and also by taking into account that they are parents advocating on behalf of their sick children and not themselves . in a previous study , parents did not view their interactions with nurses as collaborative which is considered the ideal relationship between parents and staff for working together in the care of the child . in the context of the present study , cooperation is an approach for sharing and negotiating the care of the child between parents and staff and thus could form the basis for collaboration . this study suggests that contextual demands and feelings of powerless dependence of the interviewed parents are the primary reasons for contesting . failure to attain trust in transcultural caring relationships further hinder the development of such relationships . also , differences in expectations result in conflicts between parents and healthcare staff in the context of paediatric oncology . thus , careful inquiry needs to be made into what parents ' expectations are and how they can benefit from assistance in assessing the possibilities and realities of the healthcare system , to better support and protect them from disappointment and the risk of ending up in reluctant resigning . contesting might not be an efficient way to protect family interests in care because of the potential risk of alienating staff and the subsequent negative impact on relations and also because healthcare staff prefer trustful cooperation and the development of caring relationships [ 11 , 26 ] . used by nurses as they try to communicate their visualization of subtle changes in patients to physicians in a way that is credible [ 27 , 28 ] . parents ' expressions of anger are often interpreted as a reaction to multiple factors , such as frustration , powerlessness , and suspicions of racism . if contesting includes the expression of negative emotions , it can also have negative consequences for the development of relationships with healthcare staff and has been associated with higher levels of distress in parents . moreover , norberg et al . argue that the expression of negative emotions can have negative consequences for communication with healthcare staff . language proficiency has previously been identified as a social marker to justify the exclusion and negative categorization of people with a foreign accent or nonspeakers of the local language . this experience is also evident in several of the quotes of participants in the present study , where lack of proficiency in the swedish language becomes an obstacle in protecting family interests in health care . however , one could argue in accordance with johnstone and kanitsaki that the problem is the inability of the healthcare system to meet the needs of people lacking proficiency in the local language , categorized as institutional prejudice , leading to discrimination . foreign - born parents are often excluded from research because of methodological difficulties related to language differences . not talking the same language leads to a need to use an interpreter which could be considered a limitation , as findings might be influenced by the interpreter 's assumptions and by losing some information in interpretation . however , the voice of foreign - born parents needs to be heard and this is one step from the context of paediatric cancer care in sweden . suggest five criteria for rigor in cross - cultural nursing research : cultural relevance , contextuality , appropriateness , mutual respect , and flexibility . even though this is not a cross - cultural study in the sense of comparing different cultures , these criteria are still relevant . the inductive approach of this study ensures several of these criteria are met including cultural relevance as it aims to study what is of importance to the parents ' interviewed ; flexibility to research question ; respect in allowing participants to tell of their own experience . with regard to contextuality , the researchers were well aware of the situation in paediatric cancer care and had a broad understanding of the situation of foreign - born people in sweden . in the current study , the appropriateness with regard to communication and translation process could be considered fairly good because of the availability of interpreters and the back translation of the written information into arabic , however , the written information was not made available in any other languages than arabic and swedish . the approaches identified in this study can provide a better understanding and anticipation of parents ' approaches to protecting family interests and healthcare staff can adopt strategies to facilitate cooperation . for example , a more frequent use of interpreters could facilitate trustful cooperation as well as persuasive communicating . however , there are potential difficulties with using interpreters , including the risk of triadic relationships which can hinder the caring relationship , loss of information control and information compacting , as well as errors in interpretation . further research is needed on institutional prejudice and the inability of the healthcare system to meet the needs of people lacking proficiency in the local language . implementation research would also be desirable as to enable healthcare staff to use available tools and services to bridge obstacles to communication and at the same time gain knowledge of the social processes . furthermore , healthcare staff need to listen to foreign - born parents and deal with their concerns seriously to prevent powerless - dependence . healthcare staff also need to properly assess foreign - born parents ' expectations of the healthcare system and to further empower them ; ensuring that the best interests of their child and family are upheld . furthermore , it is of great importance to find out what amount and type of information parents want and to give that information in a congruent way . finally , trust is key to preventing reluctant resigning and for a trustful cooperation in the common fight against childhood cancer .

sweden 's population is gradually changing to become more multiethnic and diverse and that applies also for recipients of health care , including childhood cancer care . a holistic view on the sick child in the context of its family has always been a cornerstone in childhood cancer care in sweden . the purpose of this study was to gain knowledge about the experiences and main concern of foreign - born parents in the context of paediatric cancer care . interviews were performed with eleven foreign - born parents and data were analysed using a classic grounded theory approach . foreign - born parents often feel in a position of powerless dependence , but family interests are protected in their approaches to interaction with healthcare staff , through cooperation , contesting , and reluctant resigning . healthcare staff need to listen to foreign - born parents and deal with their concerns seriously to prevent powerless - dependence and work for trustful cooperation in the common fight against childhood cancer .

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

sweden is gradually changing to a religiously and culturally diverse as well as multiethnic society and the swedish population includes 19% with a foreign background . according to the swedish national board of health and welfare people with a foreign background are more likely to be disadvantaged when it comes to socioeconomic situation and health . however , health care is publicly funded and available to everyone . childhood cancer care includes very advanced medical , supportive , and nursing care , and in sweden it is centralized to six childhood cancer care centres . a holistic care of the sick child in the context of its family has always been important in childhood cancer care in sweden and family - centred care is a pillar in paediatric care . family - centred care not only includes family involvement in the care of the child but views the family as the recipient of care as well and this should be planned to benefit the whole family . parents report that the quality of care is of great importance and is reflected in the creation of trust . furthermore , rapport between parents and staff , particularly nurses , is very important for the interaction to be perceived as positive by parents but also for effective sharing of the care of the child . parents want to be involved in the care of their child but parental involvement is influenced by support , professionalism , work environment , and responsibility ; responsibility in terms of keeping track of and checking the child 's treatment . routines for involving parents in care have been found to be better in paediatric oncology units in sweden compared to other units at children 's hospitals but there are barriers to this involvement that need to be overcome if parents are to be successfully involved in care . linguistic , cultural , and religious differences as well as organizational obstacles hinder the development of rapport and caring relationships and thus also parental involvement in care . in instances where providers and recipients of health care do not speak the same language , developing an effective caring relationship can be impeded and an increase of the risk of serious medical events in paediatric care has also been found . in paediatric oncology , parents are expected to be increasingly involved in the care of their child but the experience of foreign - born parents is not well known in this context . the purpose of this study was to gain knowledge about foreign - born parents ' main concern and their experiences of the situation , including their child 's illness and treatment , and social interactions in the context of paediatric oncology care and to explain how this was dealt with . in this qualitative study , a classical grounded theory approach [ 1416 ] was chosen to discover how participants deal with their social situation and interactions ; resolving their main concern . in accordance with the method [ 1416 ] , sampling was guided by the purpose of the study ; aiming for easily accessible people with personal experience and knowledge about the subject . the first author and a care coordination nurse identified potential participants , at one paediatric oncology centre , through a list of newly diagnosed patients . the child was still undergoing treatment at the childhood cancer care unit . a care coordination nurse , who was not involved either in the research project or in the patients ' direct care , invited potential participants to participate in the study and three declined . the care coordinator nurse also gave them written information in swedish or arabic about the aim and the procedure of the study . after that the first author contacted parents , who consented verbally to participate , to decide on the date , time , and place for the interview . this resulted in 11 parents ( 10 mothers , 1 father ) ; demographic data of the participants are presented in table 1 . the interviews took the form of semistructured conversations where the first author invited participants to tell about their experiences of their child 's illness and treatment . an independent authorized translator translated the questionnaire and the interview guide into arabic , after that an interpreter translated them back to swedish and the two versions were compared to identify differences . relating to participants ' preferences , all but four of the interviews were conducted without interpreters , with the rest facilitated by the same female arabic - speaking authorized healthcare interpreter . field notes and transcribed interviews were coded using the qualitative data analysis software nvivo 2.0 as a tool . during coding , codes were grouped together into concepts , which in classical grounded theory is the naming of a pattern of behaviour . consistent with grounded theory , when the core concept emerged , selective coding was conducted . this was combined with theoretical sampling , thus data was collected , coded , and analysed jointly to enable the process of deciding what data to collect , based on the selected codes , and this modified probing questions used in subsequent interviews [ 1416 ] . the second author validated the codes and concepts by listening to the recordings independently , as well as by reading transcripts and field notes . also literature was used as data in what is termed constant comparison in grounded theory . further , theoretical coding included the exploration of potential theoretical codes that could integrate concepts , in this study approaches is an example of a theoretical code . memos were written throughout the analysis ; these are notes about codes , concepts , and their relationships to each other . most of the analyses were performed using swedish but concepts were also named in english . ethical approval was obtained from the regional ethical review board . to protect confidentiality of participants , in the context of childhood cancer care , the main concern of foreign - born parents is to make it through an uncertain situation of extreme emotional burden and stress by struggling on . foreign - born parents often feel in a position of powerless dependence in relation to the child 's illness and also in relation to healthcare staff . protecting family interests is one aspect of struggling on , which encompasses the engagement in information monitoring . protecting family interests is how parents interact with healthcare staff , which includes cooperation , contesting , and reluctant resigning . as parents enter the healthcare system , often with a feeling of being exposed , in need of help and powerless in relation to the child 's illness , the interaction with healthcare staff can accentuate or lessen these feelings . discrimination and suspicion of discrimination accentuate the feeling of powerless - dependence as well as frustration . furthermore , powerless dependence is influenced by the opportunity for parents to present their point of view and share opinions with healthcare staff and by being listened to . this is influenced by the social status of the parent : i feel that in sweden [ people have the attitude toward me that ] -you do not speak plain swedish , you speak with an accent , what do you know ? ' ( mother from europe of a child with leukaemia . ) additionally , linguistic obstacles negatively influence attempts to present opinions and thereby accentuate the feeling of powerless dependence . the greater the language barrier , the more likely is the feeling of powerless dependence : i did n't have such a great role [ in the treatment decision for my sick child ] because i ca n't speak the language so i could n't exercise my role ( mother from greater middle east of a child with brain tumour . ) however , this can be overcome if parents perceive that they are listened to and that their views matter in decisions about the child 's care and treatment . thus , the more the vulnerable person experience that their views matter and that they are listened to , the less the level of felt powerless - dependence . information monitoring assists parents in gaining some control over the situation and increasing the possibility that family interests will be protected . the type of information sought includes health - related information about the child 's diagnosis , prognosis , the child 's present condition such as blood values , and the available treatment options . the latter include treatment alternatives , side - effects , self - help , and seeking a second opinion . information is sought from different sources , including physicians , nursing staff , other parents , the sick child , the literature , and the internet . for some parents , the level is low because they are already satisfied with , or overwhelmed by , the information at hand , whereas others have a very high level of information seeking and monitoring , as exemplified by we have received so much information ; we have as much as we need and more to . ( mother from europe of a child with a solid tumour . ) ( mother from europe of a child with a solid tumour . ) conversely , disaffection with the information makes it more likely that parents will engage in high level information seeking behaviour to protect family interests . the protection of family interests includes protecting parents ' own interests and principally those of the sick child . parents interact with healthcare staff in such a way as to protect family interests and to achieve the best possible care for the child through cooperation , contesting , and reluctant resigning . families enter the healthcare system with different levels of trust and with different expectations . where there is disparity between these expectations and the possibilities of the delivery of care , parents are more likely to actively protect family interests . the approaches used vary with personality , individual preferences , and perceived levels of powerless - dependence . they will also vary for the same parent , who will fluctuate between different approaches , depending on the situation and conditions . conditions that influence the choice of approaches include the way the family is considered by the staff , attitudes among staff , treatment alternatives , prognosis of the disease , and the family 's previous experiences . sometimes these approaches arise as a consequence of interactions with healthcare staff , for example , the establishment of rapport might lead to a trustful cooperation . at other times approaches are deliberate efforts to influence decisions and manoeuvre the care situation and can be considered strategic . parents try to stay on good terms with the staff and create good relationships to obtain the best possible care for their child while protecting family interests . one parent said the moment when i have given my child into the doctor 's hands ) cooperation is negotiated , particularly in relation to the balancing of responsibilities between parents and staff in relation to the care of the child . this negotiation can be open or closed ; the latter is achieved with subtle , implicit messages , or even assumptions of role divisions . when subtle messages are misunderstood or not perceived at all , this could lead to conflict in the negotiation . at times , parents entrust responsibilities and decisions to the staff , as exemplified by the following quote : then it will be fine , if you think that it is the best , then we are on we say : okay , then we are consenting or what it is called . cooperation is used by all parents , but the level of trust varies . cooperation is motivated by the relationship with healthcare staff and by the wish to protect family interests but can also be partly motivated by , or be a consequence of , powerless dependence in an attempt to decrease vulnerability . when trust is lacking or care expectations are not met , cooperation changes to contesting to protect family interests . contesting is an approach to protect family interests and often includes persuasive communicating , that is , providing convincing information in a persistent way , sometimes with emotional display and threats . convincing information often concerns needs of the child that parents are trying to convince healthcare staff about , for example , the need to be cared for at a specialist unit or the need for extra recourses or support . contesting is often linked to a strong feeling of powerless dependence and sometimes also a mistrust of the healthcare staff or healthcare system . occasionally , contesting is a consequence of a perceived wrongdoing by healthcare staff ; at other times , it is a strategic approach to influence and manoeuvre healthcare staff in care decisions . when parents suspect that their interests are being wrongfully ignored , for instance , because of racism and prejudice , they will try to contest for their rights : i have said outright to the nurses what i think . for many , contesting starts as they try to gain access to care if this is denied , for example , before a serious disease is confirmed , or if they believe that their rights are not being acknowledged : we were very badly treated actually sometimes you can see as a foreigner that it is a little bit unfair then we came there for the third time in a week and her father said that you have to shout at them , you have to tell them that they should examine your daughter properly . contesting is situation dependent and where there is a lack of mutual trust parents are more likely to respond to healthcare staff by contesting . when trusting relations have not been developed , because , for example , of personalities , experience of wrong treatment or obstacles to transcultural caring relationships , parents will try to remain in control by contesting in every possible way to protect family interests : ( mother from europe of a child with leukaemia . ) even in trusting relationships , if family interests are threatened in any way , then parents are likely to engage in contesting to achieve their goals . once goals have been realized , parents attempt to resume trustful cooperation but this can be very difficult to achieve when staff perceive contesting as negative . if this reaction persists , then parents are even more likely to engage in contesting as a response to healthcare staff , thereby creating a causal loop , where parents and staff react to each other in ways that make negative reactions more likely and the reestablishment of trustful cooperation more doubtful . if parents fail to protect their family interests through a combination of cooperation and contesting , then they are likely to resign themselves to the situation through reluctant resigning . reluctant resigning is an approach to protect family interests and is more likely when parents feel powerless dependence . contesting is used when there is some hope of influencing decisions whereas reluctant resigning is often triggered by a critical juncture , when parents feel unable to influence what is happening or decisions being made without consultation . simply , they feel disempowered and unable to do anything about it except to accept the situation as it is , or risk permanently alienating staff and becoming exhausted in the process . both i and my husband were very irritated by them [ physicians ] they tried to persuade us and tell us how we should do this . that was the only thing that i could say i felt like a butchered bird . in this classical grounded theory study with foreign - born parents in paediatric cancer care , struggling on includes information - monitoring such as health information seeking , avoiding and controlling behaviours . it is of great importance to keep parents sufficiently informed , especially because , within paediatric oncology , parents are not currently entirely satisfied with the amount and timing of information [ 20 , 21 ] . furthermore , lack of information to parents in paediatric cancer care leads to a feeling of being unwelcome and abandoned . parents and patients often receive an insufficient level and amount of information , leading to conflict in care . even though information giving has been a constant subject of nursing research for many years , insufficient information giving continues to be an issue as evidenced in this study . information monitoring is influenced by psychosocial , cultural , and sociodemographic factors , such as ethnicity ( johnson , 1997 in ) and it might be expected that there would be a low level of information seeking in this group of foreign - born parents . however , education and social roles also influence information seeking and the findings in the present study that information monitoring is important to protecting family interests might be explained by the fact that participants had a rather high level of education and also by taking into account that they are parents advocating on behalf of their sick children and not themselves . in a previous study , parents did not view their interactions with nurses as collaborative which is considered the ideal relationship between parents and staff for working together in the care of the child . in the context of the present study , cooperation is an approach for sharing and negotiating the care of the child between parents and staff and thus could form the basis for collaboration . this study suggests that contextual demands and feelings of powerless dependence of the interviewed parents are the primary reasons for contesting . also , differences in expectations result in conflicts between parents and healthcare staff in the context of paediatric oncology . thus , careful inquiry needs to be made into what parents ' expectations are and how they can benefit from assistance in assessing the possibilities and realities of the healthcare system , to better support and protect them from disappointment and the risk of ending up in reluctant resigning . contesting might not be an efficient way to protect family interests in care because of the potential risk of alienating staff and the subsequent negative impact on relations and also because healthcare staff prefer trustful cooperation and the development of caring relationships [ 11 , 26 ] . used by nurses as they try to communicate their visualization of subtle changes in patients to physicians in a way that is credible [ 27 , 28 ] . parents ' expressions of anger are often interpreted as a reaction to multiple factors , such as frustration , powerlessness , and suspicions of racism . if contesting includes the expression of negative emotions , it can also have negative consequences for the development of relationships with healthcare staff and has been associated with higher levels of distress in parents . argue that the expression of negative emotions can have negative consequences for communication with healthcare staff . this experience is also evident in several of the quotes of participants in the present study , where lack of proficiency in the swedish language becomes an obstacle in protecting family interests in health care . however , one could argue in accordance with johnstone and kanitsaki that the problem is the inability of the healthcare system to meet the needs of people lacking proficiency in the local language , categorized as institutional prejudice , leading to discrimination . foreign - born parents are often excluded from research because of methodological difficulties related to language differences . not talking the same language leads to a need to use an interpreter which could be considered a limitation , as findings might be influenced by the interpreter 's assumptions and by losing some information in interpretation . however , the voice of foreign - born parents needs to be heard and this is one step from the context of paediatric cancer care in sweden . suggest five criteria for rigor in cross - cultural nursing research : cultural relevance , contextuality , appropriateness , mutual respect , and flexibility . even though this is not a cross - cultural study in the sense of comparing different cultures , these criteria are still relevant . the inductive approach of this study ensures several of these criteria are met including cultural relevance as it aims to study what is of importance to the parents ' interviewed ; flexibility to research question ; respect in allowing participants to tell of their own experience . with regard to contextuality , the researchers were well aware of the situation in paediatric cancer care and had a broad understanding of the situation of foreign - born people in sweden . in the current study , the appropriateness with regard to communication and translation process could be considered fairly good because of the availability of interpreters and the back translation of the written information into arabic , however , the written information was not made available in any other languages than arabic and swedish . the approaches identified in this study can provide a better understanding and anticipation of parents ' approaches to protecting family interests and healthcare staff can adopt strategies to facilitate cooperation . for example , a more frequent use of interpreters could facilitate trustful cooperation as well as persuasive communicating . however , there are potential difficulties with using interpreters , including the risk of triadic relationships which can hinder the caring relationship , loss of information control and information compacting , as well as errors in interpretation . further research is needed on institutional prejudice and the inability of the healthcare system to meet the needs of people lacking proficiency in the local language . implementation research would also be desirable as to enable healthcare staff to use available tools and services to bridge obstacles to communication and at the same time gain knowledge of the social processes . furthermore , healthcare staff need to listen to foreign - born parents and deal with their concerns seriously to prevent powerless - dependence . healthcare staff also need to properly assess foreign - born parents ' expectations of the healthcare system and to further empower them ; ensuring that the best interests of their child and family are upheld . furthermore , it is of great importance to find out what amount and type of information parents want and to give that information in a congruent way . finally , trust is key to preventing reluctant resigning and for a trustful cooperation in the common fight against childhood cancer .

the german decision maker federal joint committee ( fjc ) commissioned the institute for quality and efficiency in health care ( iqwig ) to perform a scientific evaluation on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . on may 20 , 2010 the fjc decided , on the basis of the present systematic review , that routine care can not be made dependent upon whether the opposing arch is provided with fixed or removable partial dentures and has therefore revoked its previous directive on fixed allowances for health care services . in the fourth german dental oral health study ( dms - iv ) it was estimated that , without taking wisdom teeth into account , in persons aged between 35 and 44 years , on average about 2.7 teeth were missing ; 48.5% of missing teeth had been replaced . in persons aged between 65 and 74 years , 14.2 teeth on average were missing and 31.3% had an edentulous upper arch ; 88.7% of missing teeth had been replaced . with the exception of a shortened dental arch , every tooth gap requires the earliest possible prosthetic restoration in order to avoid secondary damage . when deciding upon the type of denture most suitable for the respective patient , besides the nature of the opposing dentition or the type of prosthetic restoration in the opposing arch , the following factors play a crucial role : ( 1 ) size of gap ( number of missing teeth ) , type of gap ( free - end or interdental ) , and gap localization ; ( 2 ) age of patient ; ( 3 ) specific factors in the patient 's lifestyle , including in particular the quality of the patient 's oral hygiene as well as his or her tobacco consumption . the subsequent specifications define the aim of the investigation , which is as follows : assessment of the functionality of fixed and removable partial dentures as test interventions in relation to variations in the opposing dentition and their prosthetic restoration . following patient - relevant outcomes were evaluated : ( 1 ) denture longevity , ( 2 ) change in dietary habits , ( 3 ) oral health - related quality of life to include patient satisfaction and phonetic rehabilitation , which are parameters relevant to quality of life , and ( 4 ) denture cleansability and aftercare required . the test intervention assessed was the treatment of residual dentition by means of fixed partial dentures depending on the nature of the opposing dentition . the comparator intervention assessed was the treatment of residual dentition by means of removable partial dentures or combined fixed / removable partial dentures , again depending on the nature of the opposing dentition . implant - supported dentures ( fixed or a combination of fixed / removable partial dentures ) were also considered in the assessment of the test and comparator interventions . adult patients with residual dentition who had an indication for restoration with partial dentures were included in the investigation . residual dentition was classified as interdental ( kennedy class iii+iv ) as well as free - end gaps ( kennedy class i+ii ) comprising at least the first two molars . all patients with the following conditions were excluded from the investigation : posttraumatic status , status after carcinoma resection , craniofacial deformities and syndromes , and oligodontia . systematic literature searches were carried out in the following databases : central , medline , embase , biosis , scisearch , ccmed , dare , and hta ( search period from 1982 to 2009 , inclusive ) . in addition , a manual search was carried out in german dental journals and the search was extended topic specifically to the following databases : cdsr , cdmr , cdms , nhs eed , cinahl , amed , cab abstracts , global health , istpb + istp / isshp , medikat , and the publisher databases of karger ( secondary search ) , kluwer , springer , thieme , and hogrefe ( secondary search ) . finally , the opportunity to cite additional topic - relevant studies for the timespan until middle of 2008 was provided in july 2008 when interested parties were invited to submit written comments on a preliminary version of the report . randomized controlled trials ( rcts ) together with prospective and retrospective studies without control groups were included for the patient - relevant outcomes , provided the patients were included in the study consecutively and the confounding variables were adequately monitored . also included in the assessment were case reports and case series with a sample size of at least ten and of adequate biometric quality to avoid any bias in selection . the minimum observation time for all studies was a follow - up period of six months . the literature screening was carried out by two reviewers independently of each other . after assessing the biometric quality of the studies , the results from the individual studies were collated according to therapy goals and outcomes , compared , and described . iqwig 's preliminary benefit assessment , the preliminary report , was published on the internet in german language and interested parties invited to submit written comments ( https://www.iqwig.de/download/n05-02_vorbericht_v_1_0_relevanz_der_beschaffenheit_der_gegenbezahnung.pdf ) . in addition to data consistency within the publication , particular attention was paid to the homogeneity of the follow - up period . in prospective studies , this is equivalent to homogeneity in the age of the prostheses fitted . this factor was also examined in the case of retrospective analyses , that is , whether the age of the prostheses deviated considerably from each other or at least whether their age was adequately documented in the studies . in studies using questionnaires , particular focus was placed on the methodology of patient recruitment , paying special attention to the application of a consecutive procedure . the complexity of the topic lies in the great variability of very different dental patterns resulting from the lack of one or more of the total of 32 teeth in the human dentition . to enable subgroup analyses of patients with the numerous teeth patterns , documentation of gap classification according to kennedy was a major factor in the usability of the individual results for the present systematic review . finally , taking the above - mentioned aspects into account , the study and publication quality was assessed by means of a biometric quality tool comprising four grades ( no detectable flaws , minor flaws , major flaws , and unclear ) . exist if it is assumed that their correction would not substantially influence the results and therefore the overall conclusions of the study . in the case of major flaws the overall conclusions of the study would be called in question , as correction of the flaws might lead to different conclusions . initially , a total of twenty five papers were identified that met the inclusion criteria . after more detailed screening , eight studies had to be excluded from the assessment , as the data provided could not be broken up on the basis of variations in the opposing dentition ( figure 1 ) . in five out of 17 papers definitely included , there were prepublications with no additional relevant information . in eight of the 17 studies , there was information about the denture longevity outcome , in five studies information on the change in dietary habits outcome , in four studies information on the oral health - related quality of life and patient satisfaction outcome , and in nine studies information on the denture cleansability and aftercare required outcome , whereby 11 studies reported on one outcome , three studies on two , and three studies on three outcomes . five publications reported only on fixed partial dentures , three publications only on removable partial dentures , one publication on fixed and removable partial dentures , one publication on fixed partial dentures and complete dentures , three publications on removable partial dentures and complete dentures , one publication on removable partial dentures and fully dentulous patients , and three publications on removable partial dentures and complete dentures as well as fully dentulous patients . there were control interventions in nine papers ; however , in eight cases they represented interventions that did not meet the inclusion criteria ( i.e. , complete dentures or fully dentulous patients ) ( see tables 1 and 2 ) . this ultimately resulted in indirect comparisons being carried out on the test interventions of fixed versus removable partial dentures in relation to variations in the opposing dentition . the overall study and publication quality of the relevant studies was for the most part inadequate ( table 3 ) . there was only one prospective trial on the topic under investigation that could be described as randomized controlled , yet it provided no information on the randomization technique used . the six prospective studies [ 1318 ] identified revealed unequal periods of observation and flaws in how study discontinuations were dealt with . the three retrospective studies [ 1921 ] also revealed considerable flaws in the quality of studies and publication . the findings were similar for the seven prevalence studies identified [ 2228 ] , although here it was mainly the selection methods of the patient population that were inadequately described . outcome , one had no detectable flaws , two had minor , and five had major flaws in the biometric quality of the studies and publication . a comparison between the longevity of fixed and removable partial dentures was only possible for one opposing dentition variant ( complete dentures in the opposing arch ) ( tables 4 and 5 ) . only one trial , described as randomized controlled , contains data on the longevity of fixed and removable partial dentures in combination with a complete denture in the opposing arch . the validity of this trial is reduced through the following biometric flaws : ( 1 ) no data on the kennedy classes of the intervention arches ; ( 2 ) incomplete data on prognostic factors and comorbidity ; ( 3 ) inhomogeneous size of tooth gaps in the group with fixed partial denture : 44.4% teeth gaps of two to three teeth , 25.9% gaps of four to five teeth , and 29.7% gaps of nine to 11 teeth ; ( 4 ) no data on teeth gaps for the group with removable partial denture ; ( 5 ) drop - out rate of 18.9% over a follow - up period of five years ; ( 6 ) detailed description of randomization procedure is missing ( only mentioned as a term ) , so it should rather be classed as a nonrandomized controlled trial ; ( 7 ) no significance level given ( p value ) in the subgroup analysis . consequently , a significant difference can not be considered as detected in the 5-year survival rate of fixed partial dentures ( 95.2% ) and removable partial dentures ( 100% ) with a complete denture in the opposing arch . data on the longevity of fixed tooth - borne partial dentures with differing variations in the opposing dentition were found in two studies : in one publication , the 4-year survival rate with natural opposing dentition is given as 86,7% ; in the above - mentioned rct , the 5-year survival rate with complete denture in the opposing arch is given as 95.2% . apart from other differences in the study design and setting , a direct comparison between these two trials is limited by the different follow - up periods , since one of the publications gives no survival rates for shorter periods . data on the longevity of fixed implant - supported partial dentures with differing variations in the opposing dentition were found in two additional trials : the 3-year survival rate in case of natural opposing dentition is given as 97.8% in one publication ; the survival rate for fixed implant - supported partial dentures in the opposing arch after an average followup of 44.5 months is given as 100% in the other publication . apart from other differences in the study design and setting , a direct comparison of these data does not appear to have much value , since the follow - up period in one of the studies does not indicate survival rates for shorter periods . no conclusions can be drawn on the basis of the existing data regarding whether variations in the opposing dentition influence the longevity of fixed or removable partial dentures ( table 6 ) . ( a ) the first trend suggests increased longevity of removable partial dentures , compared to fixed partial dentures , with a fully edentulous opposing arch fitted with a removable prosthesis . ( b ) the second weaker trend suggests increased longevity of implant - supported partial dentures , compared to conventionally fixed partial dentures , with natural opposing dentition or with a removable partial denture in the opposing arch . there were major flaws in the quality of the biometric studies and publication in all 5 studies that contained data on the change in dietary habits in fixed and removable partial dentures in relation to variations in the opposing dentition . as no evaluable data were found on dietary habits in patients fitted with a fixed partial denture , it was not possible to compare with dietary habits in patients fitted with a removable partial denture ( tables 7 and 8) . data on the relevance of opposing dentition for removable partial dentures could only be drawn from one trial . the validity of this trial is reduced due to the following biometric flaws : ( 1 ) inhomogeneous residual dentition in the intervention / opposing arch : on average 17.4 teeth in the opposing natural dentition group , 11.8 teeth in the opposing removable partial denture group , and 5 teeth in the opposing complete denture group ; ( 2 ) no data on the kennedy classes in the intervention arches ; ( 3 ) no data on prognostic factors or on comorbidity ; ( 4 ) only male patients between 67 and 68 years of age ; ( 5 ) inhomogeneous age of prostheses : 35% less than 2 years old , 48% between 2 and 9 years old , and 17% over 10 years old ; ( 6 ) data collection instrument based on 6 hard and 6 soft meals not validated ; ( 7 ) inappropriate percentage analysis of restriction in dietary habits . in another trial , all patients interviewed featured natural dentition in the opposing arch , so that different dentition patterns could not be compared . the existing data does not allow any conclusion to be drawn on whether variations in the opposing dentition influence dietary habits when a fixed or removable partial denture is fitted . the data in another trial show that , in the case of removable partial dentures only , no or little difference can be determined in relation to the opposing dentition when eating soft or hard food ( table 9 ) . out of the four identified trials , one displayed minor flaws and three [ 20 , 22 , 24 ] major flaws in the biometric quality of the studies . it was only possible to compare the satisfaction between a fixed and removable partial denture with one opposing dentition variant ( complete denture in opposing arch ) . one trial described as randomized controlled contained data on patient satisfaction for fixed and removable partial dentures in combination with a complete denture in the opposing arch . due to the biometric flaws already described in the results for the denture longevity a significant difference ( p < 0.05 ) is indicated in patient satisfaction regarding stability in general and during chewing with fixed ( 77.8% and 85.2% of patients , resp . , were satisfied ) and with removable partial denture ( 61.5% and 53.9% , resp . ) . however , the fact that fixed prostheses have greater stability than removable ones appears reasonable . data on general patient satisfaction with removable partial dentures and differing variations in the opposing dentition were found in one trial : the percentage of satisfied patients with removable partial denture in the opposing arch was 37% ( n = 102 ) , and 65% for those with complete denture in the opposing arch ( n = 147 ) . however , due to the variable sampling size and the unequal age of prosthesis ( one to 15 years ) , a comparison of these data would not appear to be worthwhile . based on the existing data , it is not possible to draw any conclusions on whether variations in the opposing dentition influence patient satisfaction when fitting fixed or removable partial dentures . data on denture cleansability and aftercare required when fitting fixed and removable partial dentures were included in nine publications . of these trials , two showed minor [ 12 , 13 ] and seven major flaws [ 2024 , 27 , 28 ] in the biometric quality of studies and publication . it was only possible to compare the maintenance requirements of fixed and removable partial dentures for one opposing dentition variant ( complete denture in the opposing arch ) . as no analysable data were found on prosthesis cleansability or aftercare required in fixed partial dentures , it was not possible to make a comparison with the prosthesis cleansability or aftercare required in removable partial dentures . only one rct contained data on the maintenance requirements of fixed and removable partial dentures when the opposing arch was fitted with a complete denture . however , as already mentioned above , it contained biometric flaws that reduced its validity . consequently , a significant difference could not be proven in the level of repair required for fixed partial dentures ( 22.2% of prostheses ) and removable partial dentures ( 23% ) in combination with a complete denture in the opposing arch . in another trial , data were found on the level of repair required for removable partial dentures with differing variations in the opposing dentition : the number of repairs required within a period of 16 months was 72 for natural opposing dentition , eight for removable partial dentures in the opposing arch , and 18 for removable complete dentures in the opposing arch . however , due to the nondocumented sample size of the individual subgroups and the inhomogeneous age of prosthesis ( one to six years ) , a comparison of these data would not appear to be worthwhile . no conclusions could be drawn on the basis of the existing data as to whether variations in the opposing dentition influence denture cleansability and aftercare in case of a fixed or removable partial denture . after a comprehensive literature search and an assessment of the evidence base on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures , no robust conclusions could be drawn . this is due both to the low quantity and the methodological weaknesses of the few studies identified . only one of the 17 studies compared the two denture types investigated within the context of a controlled prospective study , even though this study can not be regarded as an rct either , due to its inadequately documented randomization procedure . all the remaining data extracted originated from prospectively or retrospectively planned studies without a control group ( one - arm studies ) or from one - off data collections by way of questionnaires . it should be noted , however , that all the studies included were not per se concerned with the question of the relevance of opposing dentition but addressed this question mostly in the form of subgroup analyses . for this reason the literature search for this report was particularly time intensive , as it could mostly be decided only on the basis of full - text screening whether a subgroup analysis considering opposing dentition had been performed . within the framework of the quality assessment for the present systematic review , the serious biometric flaws in study and publication quality identified in nearly all studies refer to the research question investigated here , that is , the relevance of opposing dentition . this quality assessment is not an evaluation of the informative value of the individual studies with regard to their original research questions . the assessment of denture longevity , that is , the survival or success rate of dentures , should follow a clear definition . it must be quite evident what medical , functional , or even aesthetic requirements the dentures must fulfill in order to be classed as functioning or successful . in the present systematic review all irreparable damage was viewed as a failure ( end of denture longevity ) and all repairable damage was allocated to the outcome denture aftercare required . functional and medical reasons ( medical failure = tooth loss ) account for 69.5% and 28.5% of fixed partial dentures failures , respectively ; removable dentures fail nearly exclusively for medical reasons . implant - supported fixed dentures can fail due to prosthesis failure but also through failure of the implants themselves . in most of the studies included , no definition was offered as to what criteria were applied for success of the dentures , nor was the status of the opposing dentition over the course of the entire study period clearly reported . first , some differentiations should be made between the objective chewing performance ( i.e. , the instrumentally measured ability to break down a specific test meal into pieces ) and subjective chewing efficiency ( i.e. , chewing capacity as experienced by the patient ) , depending on the status of the opposing dentition . some objective tests indicate that by means of fixed and removable dentures the chewing function can be equally restored , whereas others claim a better chewing capacity for hard food with fixed prostheses . however , it is still controversial whether the objective effect measured correlates with the patient 's subjective perception . objectively measured improvements in chewing capacity through adaptation of the prosthesis have not been perceived by patients in previous studies . a study in which patients with only one partial prosthesis that estimated their chewing ability to be the same as that of patients with two complete prostheses shows that the parameter subjectively reported chewing efficiency is potentially flawed . independent of the choice of measurement methods , changes in dietary habits should always be compared intraindividually . a further patient - relevant aspect is the change in the perception of taste through dentures . studies were able to show that dissatisfaction due to a changed perception in taste was evident only for prostheses covering the entire palate , that is , for complete prostheses . this aspect plays no role for the removable partial prostheses assessed in this systematic review . oral health - related quality of life was not analyzed in the studies identified ; we therefore extended the outcomes investigated and also included patient satisfaction so as to be able , at least , to draw conclusions on this quality - of - life relevant parameter . every patient should first be provided with removable dentures and then , after assessment of satisfaction , with fixed ones . this approach is more costly than an interindividual assessment , but reduces uncertainty and contradictions in patients ' statements . as an inverse approach is not feasible on practical grounds , an intraindividual approach would , however , have to dispense with randomization with regard to the sequence of the interventions , as the study would not have a cross - over design in the proper sense of the term . particularly in view of the variety of patterns regarding the status of teeth and opposing dentition , this study design represents the most reliable comparison between fixed and removable dentures for the evaluation of patient satisfaction . independently of the nature of the opposing dentition , patient satisfaction has been found to be 96% for fixed dentures and 90% for removable ones . it has also been shown that patient satisfaction diminishes significantly with the number of teeth remaining . patients with at least 25 intact teeth are significantly more satisfied with any type of prosthesis than patients with 1 to 24 intact teeth . above all , however , patient - related factors are of importance : satisfaction with dentures is not only significantly dependent on personality type but also on acceptance of tooth loss . . in the studies included , patient satisfaction was exclusively assessed by means of nonvalidated questionnaires that enquired about patients ' subjective satisfaction . the oral health - related quality of life ( ohqol ) instrument is a validated tool . ohqol also diminishes considerably with the number of intact teeth ( p < 0.001 ) and the condition of those remaining . in patients with less than 20 intact teeth above all , the lack of the front teeth even if these are replaced by a prosthesis substantially lowers the ohqol ( or = 21.5 ) . however , demographic factors such as ethnicity or immigrant status have a stronger influence on the ohqol than the status of the teeth . current studies even come to the conclusion that the ohqol is not meaningful as an indicator for patient satisfaction or the disease burden of patients with tooth gaps . the outcome denture cleansability and aftercare required covers four different patient - related aspects : ( 1 ) effort involved in the cleaning of the remaining teeth , as well as ( 2 ) effort involved in the cleaning of the denture ( the third and fourth aspects are described further below ) . the first and second aspects cover all measures demanded of the patient with regard to oral or prosthesis hygiene . fixed and removable dentures differ fundamentally here , as the cleansability of the remaining teeth is considerably facilitated after the removable dentures are taken out , while extra effort is required for prosthesis hygiene . fixed dentures are to be cleansed within the context required for routine denture care ; not only the demands in time but also the dexterity required by the patient is greater , however , in comparison to the care of natural teeth . in patients no longer possessing sufficient motor capacities due to age or illness , fixed dentures are for this reason often contraindicated . the question as to whether and what influence the nature of the opposing dentition has on oral and prosthesis hygiene could not be answered in the present analysis . in principle it can , however , be assumed that for every denture , independently of the opposing arch , certain efforts are necessary for hygiene , efforts which are doubled , for example , if the opposing arch is equipped with a similar denture . without taking opposing teeth into account , numerous studies have shown that , assuming adequate care , patients with fixed as well as those with removable dentures bear no increased risk of caries [ 5052 ] or periodontitis [ 29 , 53 , 54 ] . for both fixed and removable dentures , adherence to specific regulations on design is essential in the production of the devices in order to keep plaque formation as low as possible and so ensure the hygienic qualities of the dentures . in addition , it is the dentist 's responsibility to inform the patient not only of the necessity and possibilities of oral and prosthesis hygiene , but also to motivate him or her on a regular basis . the reality , however , is that 83% of all denture wearers are not properly informed and 12% of all wearers show no adequate behaviour regarding denture care ; in hospitalized patients the percentage is even 45% . further aspects of the outcome denture cleansability and aftercare required are ( 3 ) the level of repair required for dentures and ( 4 ) the effort involved in denture aftercare . for denture repairs , not only the potential costs incurred but also the often necessary deprivation of the denture during repair in a dental laboratory are relevant issues for patients . the following points were noted in the evaluation of the studies included.to be able to draw robust conclusions , a randomized controlled design of the study is essential . stratified randomization should be used , at least with regard to the different opposing dentition groups . the problem of randomized allocation of patients to the fixed or removable denture group arises from the fact that fixed dentures are more expensive to produce . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the case of non - rcts , the intervention arches should be comparable . as an intact jaw has at least 14 teeth , not less than 2 = 16,384 variations in tooth gaps are possible . to limit the number of subgroups , classification according to type , localization , and size of the gap , as practiced in this report , would be sensible , that is , specification of kennedy class ( iiv ) of the jaw , as well as gap width ( number of missing teeth ) . by this classification these data should be supplemented by the number of intact teeth remaining , which should be similar between groups . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in many publications , these data are only reported within the framework of the demographic description of the patient population , but the results are not presented separately for groups according to opposing dentition . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . as a relevant comorbidity , the periodontal status of the remaining teeth should primarily be considered . every type of denture with the exception of implants results in additional strain on the remaining teeth . if their stability is compromised by periodontal disease , this has an effect on the expected longevity of the denture . the presence of parafunctions such as bruxism is a second important factor , as dentures in people with such a condition are overstressed . prognostic factors include smoking habits and oral hygiene . as these are changeable risk factors , smokers and patients with inadequate oral hygiene need not necessarily be excluded from the study , but could receive counseling for smoking cessation or be given instructions in oral hygiene . as the compliance of study participants is important in every long - term study , this would also represent a preselection with regard to less cooperative patients . many of the studies identified compared newly manufactured prostheses and others that had in part been used for over ten years . in prospective study designs , the age of the prostheses is equivalent to the follow - up period , which should not be subject to deviations . furthermore , attention should be paid to a standardized manufacturing process of the prostheses . for fixed dentures this means the same preparation technique , the same molding material , the same alloy and ceramic material , and if possible the same dental laboratory . for removable dentures it would in principle be important to focus on a uniform , systematic design of the prosthesis , on the same materials , and on a standardized manufacturing process . comparison of the level of repair required for differently fabricated dentures must be regarded as a methodological error in the study design . in retrospectively planned studies it can be assumed that the prostheses were not manufactured under standardized conditions . as a matter of principle , only dental indications should be compared in which both fixed and removable dentures are considered as treatment options . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . regarding the longevity of implants it should be noted that their survival rate depends , among other factors , on their position , length , diameter , and surface . if the jawbone material available is insufficient to fit an implant , then bone augmentation , that is , an increase in the local bone material , must first be performed . the survival rate of implants in augmented areas is again lower than that of those set in original , local bone material . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . overcoming these restrictions ultimately , the conduct of informative studies is urgently recommended in order to successfully clarify the present research questions . to be able to draw robust conclusions , stratified randomization should be used , at least with regard to the different opposing dentition groups . the problem of randomized allocation of patients to the fixed or removable denture group arises from the fact that fixed dentures are more expensive to produce . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the case of non - rcts , the intervention arches should be comparable . as an intact jaw has at least 14 teeth , not less than 2 = 16,384 variations in tooth gaps are possible . to limit the number of subgroups , classification according to type , localization , and size of the gap , as practiced in this report , would be sensible , that is , specification of kennedy class ( iiv ) of the jaw , as well as gap width ( number of missing teeth ) . by this classification the number of subgroups is reduced . these data should be supplemented by the number of intact teeth remaining , which should be similar between groups . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in many publications , these data are only reported within the framework of the demographic description of the patient population , but the results are not presented separately for groups according to opposing dentition . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . as a relevant comorbidity , the periodontal status of the remaining teeth should primarily be considered . every type of denture with the exception of implants results in additional strain on the remaining teeth . if their stability is compromised by periodontal disease , this has an effect on the expected longevity of the denture . the presence of parafunctions such as bruxism is a second important factor , as dentures in people with such a condition are overstressed . prognostic factors include smoking habits and oral hygiene . as these are changeable risk factors , smokers and patients with inadequate oral hygiene need not necessarily be excluded from the study , but could receive counseling for smoking cessation or be given instructions in oral hygiene . as the compliance of study participants is important in every long - term study , this would also represent a preselection with regard to less cooperative patients . suitable variables as indicators of patients ' oral hygiene are plaque and bleeding indices . many of the studies identified compared newly manufactured prostheses and others that had in part been used for over ten years . in prospective study designs , the age of the prostheses is equivalent to the follow - up period , which should not be subject to deviations . furthermore , attention should be paid to a standardized manufacturing process of the prostheses . for fixed dentures this means the same preparation technique , the same molding material , the same alloy and ceramic material , and if possible the same dental laboratory . for removable dentures it would in principle be important to focus on a uniform , systematic design of the prosthesis , on the same materials , and on a standardized manufacturing process . comparison of the level of repair required for differently fabricated dentures must be regarded as a methodological error in the study design . in retrospectively planned studies it can be assumed that the prostheses were not manufactured under standardized conditions . as a matter of principle , only dental indications should be compared in which both fixed and removable dentures are considered as treatment options . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . regarding the longevity of implants it should be noted that their survival rate depends , among other factors , on their position , length , diameter , and surface . if the jawbone material available is insufficient to fit an implant , then bone augmentation , that is , an increase in the local bone material , must first be performed . the survival rate of implants in augmented areas is again lower than that of those set in original , local bone material . the assessment of outcomes must follow a standardized procedure . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . overcoming these restrictions represents a challenge that evidence - based dental medicine should successfully meet . ultimately , the conduct of informative studies is urgently recommended in order to successfully clarify the present research questions . this systematic review assesses the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . there is currently no proof of sufficient certainty regarding the relevance of opposing dentition in removable and fixed partial dentures for any of the following patient - relevant outcomes : denture longevity , change in dietary habits , oral health - related qol , condensed into patient satisfaction , and denture cleansability and aftercare required . no evidence - based statements could be generated as to whether , and if so how , variations in the opposing dentition have a bearing on the decision to fit a partially edentulous arch with a fixed or removable partial denture . there were only few indications of increased patient satisfaction in favor of the fixed partial denture in combination with the opposing dentition variant of complete denture in the opposing arch . however , these indications are based on a small number of methodologically weak studies , and this is characteristic of the field of prosthetic dentistry , as the systematic review shows .

the aim of this systematic review was to evaluate the functionality of fixed and removable partial dentures as test interventions in relation to variations in the opposing dentition and their prosthetic restoration . the abstracts identified in the respective databases were screened independently by two investigators . rcts and uncontrolled studies were considered , provided the patients were included consecutively and the confounding variables were adequately monitored . seventeen papers were included . the study and publication quality was assessed using a biometric quality tool showing an overall poor quality . the reported outcomes , such as survival rates , were in each case obtained from a single study . two possible trends could be deduced for the endpoint longevity : ( a ) the first trend in favor of removable partial dentures , compared to fixed partial dentures , with a fully edentulous opposing arch fitted with a removable prosthesis ; ( b ) the second trend in favor of implant - supported partial dentures , compared to conventionally fixed partial dentures , with natural opposing dentition or with a removable partial denture in the opposing arch . no evidence could be generated as to whether , and if so how , variations in the opposing dentition have a bearing on the decision to fit a partially edentulous arch with a fixed or removable partial denture .

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

the german decision maker federal joint committee ( fjc ) commissioned the institute for quality and efficiency in health care ( iqwig ) to perform a scientific evaluation on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . on may 20 , 2010 the fjc decided , on the basis of the present systematic review , that routine care can not be made dependent upon whether the opposing arch is provided with fixed or removable partial dentures and has therefore revoked its previous directive on fixed allowances for health care services . when deciding upon the type of denture most suitable for the respective patient , besides the nature of the opposing dentition or the type of prosthetic restoration in the opposing arch , the following factors play a crucial role : ( 1 ) size of gap ( number of missing teeth ) , type of gap ( free - end or interdental ) , and gap localization ; ( 2 ) age of patient ; ( 3 ) specific factors in the patient 's lifestyle , including in particular the quality of the patient 's oral hygiene as well as his or her tobacco consumption . the subsequent specifications define the aim of the investigation , which is as follows : assessment of the functionality of fixed and removable partial dentures as test interventions in relation to variations in the opposing dentition and their prosthetic restoration . the test intervention assessed was the treatment of residual dentition by means of fixed partial dentures depending on the nature of the opposing dentition . the comparator intervention assessed was the treatment of residual dentition by means of removable partial dentures or combined fixed / removable partial dentures , again depending on the nature of the opposing dentition . implant - supported dentures ( fixed or a combination of fixed / removable partial dentures ) were also considered in the assessment of the test and comparator interventions . adult patients with residual dentition who had an indication for restoration with partial dentures were included in the investigation . in addition , a manual search was carried out in german dental journals and the search was extended topic specifically to the following databases : cdsr , cdmr , cdms , nhs eed , cinahl , amed , cab abstracts , global health , istpb + istp / isshp , medikat , and the publisher databases of karger ( secondary search ) , kluwer , springer , thieme , and hogrefe ( secondary search ) . randomized controlled trials ( rcts ) together with prospective and retrospective studies without control groups were included for the patient - relevant outcomes , provided the patients were included in the study consecutively and the confounding variables were adequately monitored . also included in the assessment were case reports and case series with a sample size of at least ten and of adequate biometric quality to avoid any bias in selection . after assessing the biometric quality of the studies , the results from the individual studies were collated according to therapy goals and outcomes , compared , and described . to enable subgroup analyses of patients with the numerous teeth patterns , documentation of gap classification according to kennedy was a major factor in the usability of the individual results for the present systematic review . finally , taking the above - mentioned aspects into account , the study and publication quality was assessed by means of a biometric quality tool comprising four grades ( no detectable flaws , minor flaws , major flaws , and unclear ) . after more detailed screening , eight studies had to be excluded from the assessment , as the data provided could not be broken up on the basis of variations in the opposing dentition ( figure 1 ) . five publications reported only on fixed partial dentures , three publications only on removable partial dentures , one publication on fixed and removable partial dentures , one publication on fixed partial dentures and complete dentures , three publications on removable partial dentures and complete dentures , one publication on removable partial dentures and fully dentulous patients , and three publications on removable partial dentures and complete dentures as well as fully dentulous patients . this ultimately resulted in indirect comparisons being carried out on the test interventions of fixed versus removable partial dentures in relation to variations in the opposing dentition . the overall study and publication quality of the relevant studies was for the most part inadequate ( table 3 ) . outcome , one had no detectable flaws , two had minor , and five had major flaws in the biometric quality of the studies and publication . a comparison between the longevity of fixed and removable partial dentures was only possible for one opposing dentition variant ( complete dentures in the opposing arch ) ( tables 4 and 5 ) . only one trial , described as randomized controlled , contains data on the longevity of fixed and removable partial dentures in combination with a complete denture in the opposing arch . the validity of this trial is reduced through the following biometric flaws : ( 1 ) no data on the kennedy classes of the intervention arches ; ( 2 ) incomplete data on prognostic factors and comorbidity ; ( 3 ) inhomogeneous size of tooth gaps in the group with fixed partial denture : 44.4% teeth gaps of two to three teeth , 25.9% gaps of four to five teeth , and 29.7% gaps of nine to 11 teeth ; ( 4 ) no data on teeth gaps for the group with removable partial denture ; ( 5 ) drop - out rate of 18.9% over a follow - up period of five years ; ( 6 ) detailed description of randomization procedure is missing ( only mentioned as a term ) , so it should rather be classed as a nonrandomized controlled trial ; ( 7 ) no significance level given ( p value ) in the subgroup analysis . consequently , a significant difference can not be considered as detected in the 5-year survival rate of fixed partial dentures ( 95.2% ) and removable partial dentures ( 100% ) with a complete denture in the opposing arch . data on the longevity of fixed tooth - borne partial dentures with differing variations in the opposing dentition were found in two studies : in one publication , the 4-year survival rate with natural opposing dentition is given as 86,7% ; in the above - mentioned rct , the 5-year survival rate with complete denture in the opposing arch is given as 95.2% . apart from other differences in the study design and setting , a direct comparison between these two trials is limited by the different follow - up periods , since one of the publications gives no survival rates for shorter periods . data on the longevity of fixed implant - supported partial dentures with differing variations in the opposing dentition were found in two additional trials : the 3-year survival rate in case of natural opposing dentition is given as 97.8% in one publication ; the survival rate for fixed implant - supported partial dentures in the opposing arch after an average followup of 44.5 months is given as 100% in the other publication . apart from other differences in the study design and setting , a direct comparison of these data does not appear to have much value , since the follow - up period in one of the studies does not indicate survival rates for shorter periods . no conclusions can be drawn on the basis of the existing data regarding whether variations in the opposing dentition influence the longevity of fixed or removable partial dentures ( table 6 ) . ( a ) the first trend suggests increased longevity of removable partial dentures , compared to fixed partial dentures , with a fully edentulous opposing arch fitted with a removable prosthesis . ( b ) the second weaker trend suggests increased longevity of implant - supported partial dentures , compared to conventionally fixed partial dentures , with natural opposing dentition or with a removable partial denture in the opposing arch . there were major flaws in the quality of the biometric studies and publication in all 5 studies that contained data on the change in dietary habits in fixed and removable partial dentures in relation to variations in the opposing dentition . as no evaluable data were found on dietary habits in patients fitted with a fixed partial denture , it was not possible to compare with dietary habits in patients fitted with a removable partial denture ( tables 7 and 8) . data on the relevance of opposing dentition for removable partial dentures could only be drawn from one trial . the validity of this trial is reduced due to the following biometric flaws : ( 1 ) inhomogeneous residual dentition in the intervention / opposing arch : on average 17.4 teeth in the opposing natural dentition group , 11.8 teeth in the opposing removable partial denture group , and 5 teeth in the opposing complete denture group ; ( 2 ) no data on the kennedy classes in the intervention arches ; ( 3 ) no data on prognostic factors or on comorbidity ; ( 4 ) only male patients between 67 and 68 years of age ; ( 5 ) inhomogeneous age of prostheses : 35% less than 2 years old , 48% between 2 and 9 years old , and 17% over 10 years old ; ( 6 ) data collection instrument based on 6 hard and 6 soft meals not validated ; ( 7 ) inappropriate percentage analysis of restriction in dietary habits . in another trial , all patients interviewed featured natural dentition in the opposing arch , so that different dentition patterns could not be compared . the existing data does not allow any conclusion to be drawn on whether variations in the opposing dentition influence dietary habits when a fixed or removable partial denture is fitted . the data in another trial show that , in the case of removable partial dentures only , no or little difference can be determined in relation to the opposing dentition when eating soft or hard food ( table 9 ) . it was only possible to compare the satisfaction between a fixed and removable partial denture with one opposing dentition variant ( complete denture in opposing arch ) . one trial described as randomized controlled contained data on patient satisfaction for fixed and removable partial dentures in combination with a complete denture in the opposing arch . , were satisfied ) and with removable partial denture ( 61.5% and 53.9% , resp . ) data on general patient satisfaction with removable partial dentures and differing variations in the opposing dentition were found in one trial : the percentage of satisfied patients with removable partial denture in the opposing arch was 37% ( n = 102 ) , and 65% for those with complete denture in the opposing arch ( n = 147 ) . based on the existing data , it is not possible to draw any conclusions on whether variations in the opposing dentition influence patient satisfaction when fitting fixed or removable partial dentures . data on denture cleansability and aftercare required when fitting fixed and removable partial dentures were included in nine publications . of these trials , two showed minor [ 12 , 13 ] and seven major flaws [ 2024 , 27 , 28 ] in the biometric quality of studies and publication . it was only possible to compare the maintenance requirements of fixed and removable partial dentures for one opposing dentition variant ( complete denture in the opposing arch ) . as no analysable data were found on prosthesis cleansability or aftercare required in fixed partial dentures , it was not possible to make a comparison with the prosthesis cleansability or aftercare required in removable partial dentures . only one rct contained data on the maintenance requirements of fixed and removable partial dentures when the opposing arch was fitted with a complete denture . consequently , a significant difference could not be proven in the level of repair required for fixed partial dentures ( 22.2% of prostheses ) and removable partial dentures ( 23% ) in combination with a complete denture in the opposing arch . in another trial , data were found on the level of repair required for removable partial dentures with differing variations in the opposing dentition : the number of repairs required within a period of 16 months was 72 for natural opposing dentition , eight for removable partial dentures in the opposing arch , and 18 for removable complete dentures in the opposing arch . no conclusions could be drawn on the basis of the existing data as to whether variations in the opposing dentition influence denture cleansability and aftercare in case of a fixed or removable partial denture . after a comprehensive literature search and an assessment of the evidence base on the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures , no robust conclusions could be drawn . for this reason the literature search for this report was particularly time intensive , as it could mostly be decided only on the basis of full - text screening whether a subgroup analysis considering opposing dentition had been performed . within the framework of the quality assessment for the present systematic review , the serious biometric flaws in study and publication quality identified in nearly all studies refer to the research question investigated here , that is , the relevance of opposing dentition . functional and medical reasons ( medical failure = tooth loss ) account for 69.5% and 28.5% of fixed partial dentures failures , respectively ; removable dentures fail nearly exclusively for medical reasons . in most of the studies included , no definition was offered as to what criteria were applied for success of the dentures , nor was the status of the opposing dentition over the course of the entire study period clearly reported . , chewing capacity as experienced by the patient ) , depending on the status of the opposing dentition . some objective tests indicate that by means of fixed and removable dentures the chewing function can be equally restored , whereas others claim a better chewing capacity for hard food with fixed prostheses . this aspect plays no role for the removable partial prostheses assessed in this systematic review . as an inverse approach is not feasible on practical grounds , an intraindividual approach would , however , have to dispense with randomization with regard to the sequence of the interventions , as the study would not have a cross - over design in the proper sense of the term . particularly in view of the variety of patterns regarding the status of teeth and opposing dentition , this study design represents the most reliable comparison between fixed and removable dentures for the evaluation of patient satisfaction . however , demographic factors such as ethnicity or immigrant status have a stronger influence on the ohqol than the status of the teeth . the question as to whether and what influence the nature of the opposing dentition has on oral and prosthesis hygiene could not be answered in the present analysis . in principle it can , however , be assumed that for every denture , independently of the opposing arch , certain efforts are necessary for hygiene , efforts which are doubled , for example , if the opposing arch is equipped with a similar denture . for both fixed and removable dentures , adherence to specific regulations on design is essential in the production of the devices in order to keep plaque formation as low as possible and so ensure the hygienic qualities of the dentures . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . study design used in numerous denture studies , that is , the intraindividual comparison between the left- and right - hand halves of the jaw of the same patient , is only suitable for the assessment of fixed dentures , not removable dentures , which extend over the whole jaw for one - sided tooth gaps . in the description of the opposing dentition , at least the five groups distinguished in this systematic review ( nd , rpd , cd , fpd , and ifp ) should also be assessed separately . in part , for example , the natural opposing dentition is treated as equal to a fixed denture in the opposing arch . in the case of a follow - up period of ten years or longer , it should be noted that , over time , the condition of the opposing dentition may change . due to the loss of teeth in the opposing arch , natural opposing dentition may turn into residual dentition and can eventually lead to a fully edentulous arch . it should further be considered whether only patients with a constant status of their opposing dentition should be included in the assessment in terms of a per - protocol ( pp ) analysis or whether , parallel to that , patients with changes in this status should be evaluated during the follow - up period by way of an intention - to - treat ( itt ) analysis . for fixed dentures this means the same preparation technique , the same molding material , the same alloy and ceramic material , and if possible the same dental laboratory . for removable dentures it would in principle be important to focus on a uniform , systematic design of the prosthesis , on the same materials , and on a standardized manufacturing process . as a matter of principle , only dental indications should be compared in which both fixed and removable dentures are considered as treatment options . for one- or two - sided free - end gaps ( kennedy class i+ii ) with a gap width of one tooth , a fixed denture in the form of an extension bridge is possible , but due to the greater strain arising through the leverage effect , the longevity or level of repair of such bridges is , for example , not comparable to that of conventional bridges in patients with kennedy class iii gaps . although restrictions such as cost aspects , the impossibility of blinding , and the lack of randomization due to patient preferences play an important role in the conduct of dental care studies , some approximation to the state - of - the - art standards implemented in other areas of medical care would be desirable . this systematic review assesses the relevance of variations in the opposing dentition for the functionality of fixed and removable partial dentures . there is currently no proof of sufficient certainty regarding the relevance of opposing dentition in removable and fixed partial dentures for any of the following patient - relevant outcomes : denture longevity , change in dietary habits , oral health - related qol , condensed into patient satisfaction , and denture cleansability and aftercare required . no evidence - based statements could be generated as to whether , and if so how , variations in the opposing dentition have a bearing on the decision to fit a partially edentulous arch with a fixed or removable partial denture . there were only few indications of increased patient satisfaction in favor of the fixed partial denture in combination with the opposing dentition variant of complete denture in the opposing arch .

tuberculosis ( tb ) remains one of the world 's deadliest infectious diseases , causing 1.5 million deaths and 810 million new infections annually . bacille calmette gurin ( bcg ) is currently the only vaccine used for immunoprophylaxis of tb . bcg was included in the who expanded program on immunization in 1974 and more than 4 billion people have been immunized with bcg . over 90% of children worldwide are vaccinated with bcg and > 120 million doses of bcg are administered annually , making it the world 's most widely used vaccine . clinical studies have confirmed that bcg protects against disseminated tb including meningitis and miliary tb in children . however , in nonendemic countries , bcg vaccination is not performed due to the variable effectiveness ( ranging from 0 to 80% ) in preventing pulmonary tb in adults and the relatively low incidence of disease in these regions . although bcg is generally considered very safe , there is a substantially higher risk of disseminated bcg disease in children with primary immune deficiencies or hiv infection , which apparently outweighs the potential benefit of tb prevention . as a result , the current who policy recommends that children who are known to be hiv infected , even if asymptomatic , should not be immunized with bcg . bcg was derived from a virulent strain of mycobacterium bovis through in vitro attenuation ( 230 times passaging ) from 1908 to 1921 . beginning in 1924 , bcg was distributed to multiple countries worldwide , leading to its diversification into a number of genetically distinct substrains . comparative genome analyses using a variety of techniques ( including subtractive hybridization , spotted oligonucleotide arrays , microarray based resequencing , and whole genome sequencing ) have uncovered numerous large sequence polymorphisms including deletions and duplications , as well as single nucleotide polymorphisms among bcg strains . for example , a tandem duplication-2 ( du2 ) occurs in all bcg strains examined so far , but du2 exists in four different forms ( table 1 ) . consequently , bcg strains are divided into four major groups based on the du2 forms : group i ( bcg - russia , bcg - japan , and bcg - moreau ) , ii ( bcg - sweden and bcg - birkhaug ) , iii ( bcg - danish , bcg - prague , bcg - glaxo , and bcg - china ) , and iv ( bcg - phipps , bcg - tice , bcg - frappier , and bcg - pasteur ) ( table 1 ) . the genetic clustering of bcg strains is generally consistent with the historical records of bcg dissemination . for example , bcg strains of du2 groups i and ii were disseminated before 1927 ( the early strains ) and the strains of groups iii and iv were distributed after 1927 ( the late strains ) . groups iii and iv strains also exhibit a deletion in the region of difference-2 ( rd2 ) ( table 1 ) . it is unequivocal that bcg has evolved over time , but whether this matters in terms of bcg effectiveness against tb is being debated . reviews of clinical trials led to a conclusion that the variation in bcg strains is not a significant determinant of overall efficacy . however , these analyses were based on very limited data available from human studies . considering the vast number of publications on bcg , clinical and animal studies directly comparing the effectiveness of different bcg strains have been remarkably scarce ( reviewed in ref . given the recent delineation of genetic differences between bcg strains , a head - to - head comparison of bcg strains of genetic diversity is of great interest . in this study , we compared the virulence and efficacy of 13 bcg strains , representing all genetic lineages , in scid and balb / c mice , respectively . scid mice , which lack t- and b lymphocytes and are highly immunocompromised , are the reference model for the safety test of live vaccines including recombinant bcg and attenuated m. tb strains in preclinical studies , consented among tb vaccine researchers and regulatory bodies . the safety of a live vaccine is inferred from its virulence in scid mice , which is reflected in the ability of the vaccine to replicate in the animal and to cause mortality . to directly compare the virulence of bcg strains , we performed scid mice infection of 13 bcg strains and monitored the survival of animals over 18 weeks . strikingly , there were significant differences in the survival curves of scid mice infected with different groups of bcg strains ( p < 0.0001 , log - rank test ; figure 1a ) . the majority of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were dead by week 10 , and comparison of their survival curves revealed no significant differences among them ( log - rank test ) . the median survival time of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were 48 , 53.5 , 55 , and 58 days , respectively . in contrast , all scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague survived during the 18-week experiment . the survival rate of scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague were 93.75 , 100 , 100 , 100 , and 100% , respectively , which were the same as the phosphate - buffered saline ( pbs ) control group ( 87.5% ) . the median survival time of scid mice infected with bcg - china , bcg - moreau , bcg - russia , and bcg - danish were 83 , 94.5 , 99.5 , and 114 days , respectively ( figure 1a ) . accordingly , the 13 bcg strains fall into five groups based on their virulence levels : bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice > bcg - china > bcg - russia , bcg - moreau > bcg - danish > bcg - glaxo , bcg - prague , bcg - japan , bcg - sweden , and bcg - birkhaug . the differences were statistically significant between groups , but not between members of the same group ( log - rank analysis ) . there was a general parallel between the scid mice survival curves and the ability of individual bcg strains to replicate in the animals . at week 4 postinfection , bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice exhibited the highest counts in the lungs of individual scid mice , reaching 7.35 log10 , 7.34 log10 , 6.76 log10 , and 7.11 log10 colony - forming units ( cfus ) , respectively , which were on average 2.5 log10 higher than their counts at the week 1 timepoint ( 4.11 log10 , 4.92 log10 , 4.25 log10 , and 5.04 log10 cfus for bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice , respectively ) ( figure 1b ) . bcg - japan , bcg - birkhaug , bcg - prague , and bcg - glaxo showed little growth in the lungs of scid mice during the same period , with an average of 4.3 log10 cfus at week 4 postinfection , which was essentially the same as the average counts ( 4.1 log10 cfus ) at the week 1 time point . bcg - russia , bcg - moreau , bcg - china , bcg - danish , and bcg - sweden showed intermediate levels of replication , ranging from 1.2 to 2.1 log10 cfus higher in the lungs of scid mice at week 4 than at week 1 postinfection ( figure 1b ) . a similar trend was observed for bcg counts in the spleen of scid mice ( figure 1c ) . in a separate experiment , three bcg strains ( bcg - pasteur , bcg - russia , bcg - japan ) representing different virulence levels were selected to repeat the scid mice infection experiment . consistent with the results above , all scid mice infected with bcg - japan survived the 18-week experiment , while mice infected with bcg - pasteur and bcg - russia had a median survival time of 42 and 84 days , respectively . immunocompetent inbred mice ( balb / c and c57bl/6 ) are widely used for tb vaccine studies because of the low cost and the availability of immunological reagents . to compare the protective efficacy of bcg strains , we used a low - dose ( 100 cfus of m. tb h37rv ) , aerosol infection mouse ( balb / c ) model to mimic the natural m. tb infection in humans . as classically demonstrated , the m. tb infection of balb / c and c57bl/6 mice by aerosol challenge is followed by two phases . the first is the progressive phase in which m. tb grows essentially uninhibitedly for the first 34 weeks , resulting in 10~10 cfus in lungs . the progressive phase ends with the inhibition of further m. tb growth by adaptive immunity . this is followed by a stationary phase , in which the bacterial burden remains largely unchanged for 912 months before the mice succumb to tb . at week 4 post - m . tb challenge , the nonvaccinated group of balb / c mice had a mean m. tb burden of 7.27 log10 cfus in the lungs , and there was a significant difference between the mean m. tb burden in the lungs of animal groups vaccinated with various bcg strains and the pbs control group ( p < 0.0001 , one - way analysis of variance ( anova ) ) ( figure 2a ; table 2 ) . for multiple pair - wise comparisons , one - way anova and it was found that mice vaccinated with bcg - phipps , bcg - frappier , bcg - pasteur , bcg - tice , bcg - danish , bcg - prague , bcg - russia , and bcg - moreau had significantly lower m. tb burdens than the nonvaccinated pbs group ( figure 2a ) , ranging from 0.58 log10 ( bcg - moreau group ) to 1.03 log10 cfus ( bcg - phipps group ) lower ( table 2 ) . mice vaccinated with bcg - china , bcg - glaxo , bcg - japan , bcg - birkhaug , and bcg - sweden had 0.250.52 log10 lower m. tb counts than the pbs group but the differences were not statistically significant ( table 2 ) . in a comparison between the bcg - vaccinated groups , mice vaccinated with bcg - phipps had significantly lower m. tb counts in the lungs than animals vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.0001 ) , bcg - sweden ( cfu = 0.71 log10 , p < 0.001 ) , bcg - japan ( cfu = 0.54 log10 , p < 0.05 ) , and bcg - glaxo ( cfu = 0.56 log10 , p < 0.05 ) . vaccinated mice also had significantly lower m. tb burdens than those vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.05 ) . the infection of mice via the respiratory route begins to disseminate to the spleen and liver ~2 weeks after aerosol challenge , and due to the short period of m. tb growth , the bacterial burden in these organs are much lower than in the lungs . consistently , our data showed that the amount of m. tb disseminated to the spleen at week 4 postinfection was lower than that in the lungs by ~1.02.0 log10 ( figure 2b ; table 2 ) . the levels of m. tb burden in the spleen generally correlated with that in the lungs of individual animal groups . mice vaccinated with bcg - pasteur , bcg - tice , and bcg - danish had the lowest m. tb burden in the spleen , on average 0.78 log10 cfus lower than the pbs control group ( p < 0.05 , one - way anova and tukey 's test ; figure 2b ) . mice vaccinated with bcg - phipps , bcg - frappier , bcg - prague , and bcg - moreau also had , on average , 0.67 log10 lower m. tb counts than the pbs group , although the differences were not statistically significant . the m. tb burden in animals vaccinated with the remaining bcg strains , except bcg - china , were lower than the pbs control group , ranging from 0.34 log10 ( bcg - japan ) to 0.58 log10 cfus ( bcg - russia ) , but these differences were not statistically significant ( one - way anova and tukey 's post hoc test ) . at week 9 postchallenge , the infection has entered into the stationary phase and the m. tb burden in the pbs group was stabilized at 6.77 log10 and 6.07 log10 cfus in the lungs and spleen , respectively ( table 2 ) . the differences in m. tb burden between the bcg - vaccinated groups and the pbs group diminished . bcg - vaccinated animals had on average 6.65 log10 and 5.89 log10 cfus in the lungs and spleen , respectively , and none of the bcg - vaccinated animal groups were significantly different than the pbs control group ( table 2 ) . to determine if the results above were reproducible , two bcg strains of each genetic lineage ( du2 groups i iv ) were selected and the experiment was performed at a different laboratory under similar conditions ( i.e. , s.c . vaccination with bcg strains followed by aerosol infection of m. tb ) . the m. tb burden in the lungs of animals at week bcg - tice , bcg - danish , bcg - russia , and bcg - moreau had significantly lower m. tb burden in the lungs , ranging from 0.54 to 0.83 log10 cfus lower than the pbs group ( p < 0.05 , one - way anova and tukey 's post hoc test ; figure 2c ) . no significant differences were found between the bacterial burden in mice vaccinated with bcg - sweden , bcg - birkhaug , and bcg - glaxo compared to the pbs group . the protective efficacy of bcg strains appeared to correlate with their genetic clustering based on tandem duplications . we replotted the lung m. tb burdens of figure 2a by combining data of bcg strains of the same du2 group ( figure 3a ) . the mean m. tb burden of animals vaccinated with groups i iv bcg strains were 6.70 log10 , 6.98 log10 , 6.63 log10 , and 6.47 log10 cfus in the lungs , respectively . one - way anova and bonferroni 's multiple - comparison test showed that strains of group iv ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg -tice ) and group iii ( bcg - china , bcg - danish , bcg - prague , and bcg - glaxo ) afforded significantly better protection than strains of group ii ( bcg - sweden and bcg - birkhaug ) in vaccinated mice . the most protective bcg group , du2 group iv , also had the highest level of virulence in scid mice , and there appeared to be a correlation between virulence and efficacy . in figure 3b , we replotted the data of figure 2a by dividing the bcg strains into three groups based on the virulence level ( figure 1a ) , the most virulent group ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice ) , the intermediate virulent group ( bcg - china , bcg - russia , bcg - moreau , and bcg - danish ) , and the least virulent group ( bcg - japan , bcg - glaxo , bcg - prague , bcg - sweden , and bcg - birkhaug ) . the mean m. tb burden for these three groups were 6.47 log10 , 6.64 log10 , and 6.81 log10 cfus in the lungs , respectively , and the difference between the most virulent group and the least virulent group was statistically significant ( p < 0.0001 , one - way anova and bonferroni 's test ) . scid mice , which lack t- and b lymphocytes and are highly immunocompromised , are the reference model for the safety test of live vaccines including recombinant bcg and attenuated m. tb strains in preclinical studies , consented among tb vaccine researchers and regulatory bodies . the safety of a live vaccine is inferred from its virulence in scid mice , which is reflected in the ability of the vaccine to replicate in the animal and to cause mortality . to directly compare the virulence of bcg strains , we performed scid mice infection of 13 bcg strains and monitored the survival of animals over 18 weeks . strikingly , there were significant differences in the survival curves of scid mice infected with different groups of bcg strains ( p < 0.0001 , log - rank test ; figure 1a ) . the majority of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were dead by week 10 , and comparison of their survival curves revealed no significant differences among them ( log - rank test ) . the median survival time of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were 48 , 53.5 , 55 , and 58 days , respectively . in contrast , all scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague survived during the 18-week experiment . the survival rate of scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague were 93.75 , 100 , 100 , 100 , and 100% , respectively , which were the same as the phosphate - buffered saline ( pbs ) control group ( 87.5% ) . the median survival time of scid mice infected with bcg - china , bcg - moreau , bcg - russia , and bcg - danish were 83 , 94.5 , 99.5 , and 114 days , respectively ( figure 1a ) . accordingly , the 13 bcg strains fall into five groups based on their virulence levels : bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice > bcg - china > bcg - russia , bcg - moreau > bcg - danish > bcg - glaxo , bcg - prague , bcg - japan , bcg - sweden , and bcg - birkhaug . the differences were statistically significant between groups , but not between members of the same group ( log - rank analysis ) . there was a general parallel between the scid mice survival curves and the ability of individual bcg strains to replicate in the animals . at week 4 postinfection , bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice exhibited the highest counts in the lungs of individual scid mice , reaching 7.35 log10 , 7.34 log10 , 6.76 log10 , and 7.11 log10 colony - forming units ( cfus ) , respectively , which were on average 2.5 log10 higher than their counts at the week 1 timepoint ( 4.11 log10 , 4.92 log10 , 4.25 log10 , and 5.04 log10 cfus for bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice , respectively ) ( figure 1b ) . bcg - japan , bcg - birkhaug , bcg - prague , and bcg - glaxo showed little growth in the lungs of scid mice during the same period , with an average of 4.3 log10 cfus at week 4 postinfection , which was essentially the same as the average counts ( 4.1 log10 cfus ) at the week 1 time point . bcg - russia , bcg - moreau , bcg - china , bcg - danish , and bcg - sweden showed intermediate levels of replication , ranging from 1.2 to 2.1 log10 cfus higher in the lungs of scid mice at week 4 than at week 1 postinfection ( figure 1b ) . a similar trend was observed for bcg counts in the spleen of scid mice ( figure 1c ) . in a separate experiment , three bcg strains ( bcg - pasteur , bcg - russia , bcg - japan ) representing different virulence levels were selected to repeat the scid mice infection experiment . consistent with the results above , all scid mice infected with bcg - japan survived the 18-week experiment , while mice infected with bcg - pasteur and bcg - russia had a median survival time of 42 and 84 days , respectively . immunocompetent inbred mice ( balb / c and c57bl/6 ) are widely used for tb vaccine studies because of the low cost and the availability of immunological reagents . to compare the protective efficacy of bcg strains , we used a low - dose ( 100 cfus of m. tb h37rv ) , aerosol infection mouse ( balb / c ) model to mimic the natural m. tb infection in humans . as classically demonstrated , the m. tb infection of balb / c and c57bl/6 mice by aerosol challenge is followed by two phases . the first is the progressive phase in which m. tb grows essentially uninhibitedly for the first 34 weeks , resulting in 10~10 cfus in lungs . the progressive phase ends with the inhibition of further m. tb growth by adaptive immunity . this is followed by a stationary phase , in which the bacterial burden remains largely unchanged for 912 months before the mice succumb to tb . at week 4 post - m . tb challenge , the nonvaccinated group of balb / c mice had a mean m. tb burden of 7.27 log10 cfus in the lungs , and there was a significant difference between the mean m. tb burden in the lungs of animal groups vaccinated with various bcg strains and the pbs control group ( p < 0.0001 , one - way analysis of variance ( anova ) ) ( figure 2a ; table 2 ) . for multiple pair - wise comparisons , one - way anova and tukey 's post hoc tests were performed . it was found that mice vaccinated with bcg - phipps , bcg - frappier , bcg - pasteur , bcg - tice , bcg - danish , bcg - prague , bcg - russia , and bcg - moreau had significantly lower m. tb burdens than the nonvaccinated pbs group ( figure 2a ) , ranging from 0.58 log10 ( bcg - moreau group ) to 1.03 log10 cfus ( bcg - phipps group ) lower ( table 2 ) . mice vaccinated with bcg - china , bcg - glaxo , bcg - japan , bcg - birkhaug , and bcg - sweden had 0.250.52 log10 lower m. tb counts than the pbs group but the differences were not statistically significant ( table 2 ) . in a comparison between the bcg - vaccinated groups , mice vaccinated with bcg - phipps had significantly lower m. tb counts in the lungs than animals vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.0001 ) , bcg - sweden ( cfu = 0.71 log10 , p < 0.001 ) , bcg - japan ( cfu = 0.54 log10 , p < 0.05 ) , and bcg - glaxo ( cfu = 0.56 log10 , p < 0.05 ) . bcg - frappier vaccinated mice also had significantly lower m. tb burdens than those vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.05 ) . the infection of mice via the respiratory route begins to disseminate to the spleen and liver ~2 weeks after aerosol challenge , and due to the short period of m. tb growth , the bacterial burden in these organs are much lower than in the lungs . consistently , our data showed that the amount of m. tb disseminated to the spleen at week 4 postinfection was lower than that in the lungs by ~1.02.0 log10 ( figure 2b ; table 2 ) . the levels of m. tb burden in the spleen generally correlated with that in the lungs of individual animal groups . mice vaccinated with bcg - pasteur , bcg - tice , and bcg - danish had the lowest m. tb burden in the spleen , on average 0.78 log10 cfus lower than the pbs control group ( p < 0.05 , one - way anova and tukey 's test ; figure 2b ) . mice vaccinated with bcg - phipps , bcg - frappier , bcg - prague , and bcg - moreau also had , on average , 0.67 log10 lower m. tb counts than the pbs group , although the differences were not statistically significant . the m. tb burden in animals vaccinated with the remaining bcg strains , except bcg - china , were lower than the pbs control group , ranging from 0.34 log10 ( bcg - japan ) to 0.58 log10 cfus ( bcg - russia ) , but these differences were not statistically significant ( one - way anova and tukey 's post hoc test ) . at week 9 postchallenge , the infection has entered into the stationary phase and the m. tb burden in the pbs group was stabilized at 6.77 log10 and 6.07 log10 cfus in the lungs and spleen , respectively ( table 2 ) . the differences in m. tb burden between the bcg - vaccinated groups and the pbs group diminished . bcg - vaccinated animals had on average 6.65 log10 and 5.89 log10 cfus in the lungs and spleen , respectively , and none of the bcg - vaccinated animal groups were significantly different than the pbs control group ( table 2 ) . to determine if the results above were reproducible , two bcg strains of each genetic lineage ( du2 groups i iv ) were selected and the experiment was performed at a different laboratory under similar conditions ( i.e. , s.c . vaccination with bcg strains followed by aerosol infection of m. tb ) . the m. tb burden in the lungs of animals at week 4 postinfection were determined and analyzed . consistently , mice vaccinated with bcg - phipps , bcg - tice , bcg - danish , bcg - russia , and bcg - moreau had significantly lower m. tb burden in the lungs , ranging from 0.54 to 0.83 log10 cfus lower than the pbs group ( p < 0.05 , one - way anova and tukey 's post hoc test ; figure 2c ) . no significant differences were found between the bacterial burden in mice vaccinated with bcg - sweden , bcg - birkhaug , and bcg - glaxo compared to the pbs group . the protective efficacy of bcg strains appeared to correlate with their genetic clustering based on tandem duplications . we replotted the lung m. tb burdens of figure 2a by combining data of bcg strains of the same du2 group ( figure 3a ) . the mean m. tb burden of animals vaccinated with groups i iv bcg strains were 6.70 log10 , 6.98 log10 , 6.63 log10 , and 6.47 log10 cfus in the lungs , respectively . one - way anova and bonferroni 's multiple - comparison test showed that strains of group iv ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg -tice ) and group iii ( bcg - china , bcg - danish , bcg - prague , and bcg - glaxo ) afforded significantly better protection than strains of group ii ( bcg - sweden and bcg - birkhaug ) in vaccinated mice . the most protective bcg group , du2 group iv , also had the highest level of virulence in scid mice , and there appeared to be a correlation between virulence and efficacy . in figure 3b , we replotted the data of figure 2a by dividing the bcg strains into three groups based on the virulence level ( figure 1a ) , the most virulent group ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice ) , the intermediate virulent group ( bcg - china , bcg - russia , bcg - moreau , and bcg - danish ) , and the least virulent group ( bcg - japan , bcg - glaxo , bcg - prague , bcg - sweden , and bcg - birkhaug ) . the mean m. tb burden for these three groups were 6.47 log10 , 6.64 log10 , and 6.81 log10 cfus in the lungs , respectively , and the difference between the most virulent group and the least virulent group was statistically significant ( p < 0.0001 , one - way anova and bonferroni 's test ) . in this study , we performed a head - to - head comparison of the virulence and efficacy of 13 bcg strains representing different genetic lineages in murine models . previously , there had been over a dozen studies comparing the effectiveness of different bcg strains using several animal models including mice , guinea pigs , hamsters , and bank vole ( reviewed in ref . the number of bcg strains included for comparative analyses were limited , and only three studies compared 10 strains of bcg . differences in the animal model and study designs make it difficult to compare results from different studies . as such early studies in the 1970s by ladefoged and coworkers compared 11 bcg strains in bank voles and 12 bcg strains in guinea pigs . however , in the bank vole study , the 11 bcg strains were compared in six separate experiments , each involving 5 strains , and bcg - danish was the only strain common in all experiments , which limits the comparison of the results . in the guinea pig study , the m. tb strain used in the challenge was inadvertently attenuated and the data on protective efficacy was unavailable . a more recent study by castillo - rodal et al . compared the effectiveness of 10 bcg strains in balb / c mice . however , 6 of the 10 bcg strains ( bcg - phipps , bcg - tice , bcg - pasteur , bcg - frappier , bcg - connaught , and bcg - mexico ) included in this study are from the du2 group iv , while only 1 strain each from group i ( bcg - moreau ) and group iii ( bcg - danish ) and 2 strains from group ii ( bcg - sweden and bcg - birkhaug ) were included . therefore , the bcg strains chosen for this study are not equally represented . recognizing the limitations of the previous studies , we performed comparative studies of 13 bcg strains in the same experiment , which has the advantage of removing variability between experiments . the bcg strains chosen for our experiments are also well characterized genetically ( e.g. , the availability of genome sequences ) , and these bcg strains represent all genetic lineages identified so far , including three , two , four , and four strains from du2 groups i iv , respectively . in addition , we also compared the virulence of these 13 bcg strains in scid mice , which , to the best of our knowledge , has never been performed previously . one limitation of the present study is that laboratory strains instead of commercial bcg vaccines were used for experiments . however , analyses of commercial bcg vaccines are complicated by the observation that commercial preparations could have mixed bcg strains . the segregation of the virulence of bcg strains generally coincides with their genetic clustering based on genome tandem duplications ( figure 1 ) . the most virulent strains ( bcg - phipps , bcg - pasteur , bcg - frappier , and bcg - tice ) all belong to the du2 group iv ( figure 1 ) , and the two strains of the du2 group ii ( bcg - sweden and bcg - birkhaug ) are among the least virulent group . tandem duplications are a major mechanism of bcg adaptation to in vitro growth conditions , thus it is not surprising that gene amplification via tandem duplication has a major influence on bcg properties . comparison of the du2 groups reveals that groups iii and iv contain a 15765-bp ( 3,590,9023,606,667 ) duplication that does not occur in groups i and ii ( table 1 ) . sigh plays a critical role in the oxidative stress responses in m. tb and whib1 is a nitric oxide - responsive transcription factor . elevated expression of sigh and whib1 in bcg strains of groups iii and iv may enhance their replication , thereby exhibiting higher virulence in scid mice . bcg - glaxo is less virulent than bcg - china and bcg - danish of the same group ( group iii ) likely because bcg - glaxo is naturally deficient in the production of phthiocerol dimycocerosates and phenolic glycolipids . phthiocerol dimycocerosates and phenolic glycolipids are multiple methyl - branched fatty acid - containing lipids in the mycobacterial cell wall and their critical roles in virulence have been demonstrated in multiple pathogenic mycobacteria including m. tb , m. bovis , and m. marinum . the low virulence of bcg - prague may be related to the phop mutation in this strain . phopr controls the synthesis and export of multiple virulence factors in m. tb including esxa , an effector of the type vii secretion system esx-1 , and lipids of polyacyltrehalose and sulfolipid families , and therefore is critical for m. tb virulence . however , since bcg has lost the rd1 region encoding the esx-1 , in addition to the impaired phopr regulation system in m. bovis and bcg due to single nucleotide polymorphisms in this locus , the extent to which the phop mutation contributes to the attenuation of bcg - prague remains unknown . bcg - japan has a lower level of virulence than bcg - russia and bcg - moreau in group i strains presumably because it is deficient in the production of phthiocerol dimycocerosates / phenolic glycolipids and triacylglycerols . for bcg - sweden and bcg - birkhaug of group ii , the deletion of whib3 , a reductive stress regulator , and trcr , a response regulator of the trcr - trcs two - component system , may account for their low virulence . strain - specific single nucleotide polymorphisms revealed by whole genome sequencing may also account for the differential virulence of individual bcg strains . for example , bcg - russia and bcg - moreau of group i are more virulent than bcg - danish but less virulent than bcg - china of the same group ( group iii ) . there appears to be a general trend that more virulent bcg strains are also more protective . bcg strains of the most virulent group ( bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice ) demonstrated better protection than bcg - sweden and bcg - birkhaug of group ii . among the group i strains , bcg - japan is the least virulent and also less protective than bcg - russia and bcg - moreau . consistent with this notion , a previous study found that recombinant bcg strains complemented with the rd1 region exhibited increased virulence in scid mice but also better protection in c57bl/6 mice and guinea pigs . the correlation is less straightforward when comparing strains in group iii ( bcg - danish , bcg - china , bcg - prague , and bcg - glaxo ) . for example , bcg - prague is quite attenuated but well protecting , suggesting that multiple factors are involved . current strategies to develop the next generation of tb vaccines include the construction of recombinant bcg . candidates that have entered clinical trials include rbcg30 and vpm1002 ( rbcg::urec hly ) , which in the preclinical animal studies ( mice or guinea pigs ) , consistently reduced the m. tb burden by 0.51.0 log10 cfus compared to the corresponding parental bcg strains . notably , we found that mice vaccinated with bcg - phipps and bcg - frappier had 0.50.8 log10 fewer m. tb than those vaccinated with bcg - birkhaug , bcg - sweden , bcg - japan , or bcg - glaxo ( figure 2a ) , highlighting the importance of selecting specific bcg strain(s ) for the construction of recombinant bcg . the most widely used bcg vaccines include both early ( bcg - japan , bcg - moreau , bcg - russia ) and late bcg ( bcg - pasteur , bcg - danish , bcg - connaught , distributed after 1927 ) strains . reviews of clinical trial data found no evidence that efficacy was associated with the bcg strain . however , it should be noted that these analyses were limited by the paucity of randomized trials directly comparing different bcg strains . the conclusion drawn was based on comparisons between different clinical trials , which is compounded by multiple factors including differences in trial method and the population of the study . a randomized trial study comparing two bcg strains in 300,000 infants in hong kong found that a more virulent strain , bcg - pasteur , administered at a lower dosage , provided a significantly greater ( 40% ) protection against childhood forms of tb than a less virulent strain , bcg - glaxo . however , in a retrospective analysis of cohorts in kazakhstan , vaccination of neonates by one of the least virulent strains , bcg - japan , reduced the risk of tb by 69% , by 43% after bcg - serbia vaccination , and only by 22% after bcg - russia . while it is difficult to compare results between clinical trial studies , and between animal and human studies , these results suggest there are significant differences in effectiveness against tb between bcg strains . multiple studies have also demonstrated that bcg exhibits strain - dependent variations in immune responses in humans . taken together , these pieces of evidence calls for a clinical trial study directly comparing the effectiveness of different bcg strains . based on the findings of our study , we suggest that the trial should include bcg strains from a diverse genetic background that is , representatives of each of the four major groups ( du2 group i iv ) . the outcome of such a clinical trial may not only identify the most effective bcg strain(s ) for current clinical use , but also uncover genetic factors that influence the vaccine effectiveness , which will be useful for the development of the next generation of tb vaccines . bcg strains and m. tb h37rv were grown at 37 c in middlebrook 7h9 broth ( difco ) supplemented with 0.2% glycerol , 10% albumin dextrose catalase ( adc ; bd bbl , shanghai , china ) , and 0.05% tween-80 or on middlebrook 7h11 agar ( difco ) supplemented with 0.5% glycerol and 10% oleic acid albumin dextrose catalase ( oadc ; bd bbl ) . female scid mice were purchased from beijing hfk bioscience and the mice were age matched ( 78 weeks ) within each experiment . mice ( 27 per group ) were infected i.v . via the tail vein with 10 cfu of the different bcg strains in 0.1 ml postinfection , two mice from each group were sacrificed and the lungs and spleens were harvested , homogenized in pbs , and plated on 7h11 agar to enumerate bacterial burden . this was performed to confirm the actual infection dosage . to analyze the replication of bcg strains in scid mice , four mice from each group at week 1 and week 4 postinfection were sacrificed and the cfus of bcg in lungs and spleen were determined . groups of 12 female balb / c mice were vaccinated s.c . on the scruff of the neck with 10 cfu of the bcg strains in 0.1 ml pbs/0.01% tween-80 or pbs/0.01% tween-80 alone as a control . at 8 weeks postvaccination , mice were aerogenically challenged with 100 cfu of m. tb h37rv using a glascol nebulizer . mice were euthanized at 4 and 9 weeks postchallenge ( five mice per group per time point ) to harvest the lungs and spleen , which were then homogenized and plated on 7h11 agar to enumerate the burden of m. tb . actual infection dose was confirmed by homogenizing whole lungs of two mice at day 1 postinfection and plating on 7h11 to enumerate m. tb . statistical analysis . scid mice survival was plotted using the kaplan meier method and differences between curves were analyzed using the log - rank test . one - way anova with tukey 's multiple comparisons were performed for m. tb burdens ( log10-transformed cfu data ) when there are more than six groups . one - way anova and bonferroni 's multiple comparisons were performed for six or fewer groups .

bacille calmette gurin ( bcg ) , an attenuated strain of mycobacterium bovis , is the only vaccine available for tuberculosis ( tb ) control . however , bcg is not an ideal vaccine and has two major limitations : bcg exhibits highly variable effectiveness against the development of tb both in pediatric and adult populations and can cause disseminated bcg disease in immunocompromised individuals . bcg comprises a number of substrains that are genetically distinct . whether and how these genetic differences affect bcg efficacy remains largely unknown . in this study , we performed comparative analyses of the virulence and efficacy of 13 bcg strains , representing different genetic lineages , in scid and balb / c mice . our results show that bcg strains of the du2 group iv ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice ) exhibit the highest levels of virulence , and bcg strains of the du2 group ii ( bcg - sweden , bcg - birkhaug ) are among the least virulent group . these distinct levels of virulence may be explained by strain - specific duplications and deletions of genomic dna . there appears to be a general trend that more virulent bcg strains are also more effective in protection against mycobacterium tuberculosis challenge . our findings have important implications for current bcg vaccine programs and for future tb vaccine development .

Introduction Results Evaluation of virulence of BCG strains in SCID mice Evaluation of efficacy of BCG strains in BALB/c mice Discussion Materials and Methods

bacille calmette gurin ( bcg ) is currently the only vaccine used for immunoprophylaxis of tb . however , in nonendemic countries , bcg vaccination is not performed due to the variable effectiveness ( ranging from 0 to 80% ) in preventing pulmonary tb in adults and the relatively low incidence of disease in these regions . consequently , bcg strains are divided into four major groups based on the du2 forms : group i ( bcg - russia , bcg - japan , and bcg - moreau ) , ii ( bcg - sweden and bcg - birkhaug ) , iii ( bcg - danish , bcg - prague , bcg - glaxo , and bcg - china ) , and iv ( bcg - phipps , bcg - tice , bcg - frappier , and bcg - pasteur ) ( table 1 ) . in this study , we compared the virulence and efficacy of 13 bcg strains , representing all genetic lineages , in scid and balb / c mice , respectively . to directly compare the virulence of bcg strains , we performed scid mice infection of 13 bcg strains and monitored the survival of animals over 18 weeks . the majority of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were dead by week 10 , and comparison of their survival curves revealed no significant differences among them ( log - rank test ) . the median survival time of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were 48 , 53.5 , 55 , and 58 days , respectively . in contrast , all scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague survived during the 18-week experiment . the survival rate of scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague were 93.75 , 100 , 100 , 100 , and 100% , respectively , which were the same as the phosphate - buffered saline ( pbs ) control group ( 87.5% ) . accordingly , the 13 bcg strains fall into five groups based on their virulence levels : bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice > bcg - china > bcg - russia , bcg - moreau > bcg - danish > bcg - glaxo , bcg - prague , bcg - japan , bcg - sweden , and bcg - birkhaug . at week 4 postinfection , bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice exhibited the highest counts in the lungs of individual scid mice , reaching 7.35 log10 , 7.34 log10 , 6.76 log10 , and 7.11 log10 colony - forming units ( cfus ) , respectively , which were on average 2.5 log10 higher than their counts at the week 1 timepoint ( 4.11 log10 , 4.92 log10 , 4.25 log10 , and 5.04 log10 cfus for bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice , respectively ) ( figure 1b ) . bcg - russia , bcg - moreau , bcg - china , bcg - danish , and bcg - sweden showed intermediate levels of replication , ranging from 1.2 to 2.1 log10 cfus higher in the lungs of scid mice at week 4 than at week 1 postinfection ( figure 1b ) . in a separate experiment , three bcg strains ( bcg - pasteur , bcg - russia , bcg - japan ) representing different virulence levels were selected to repeat the scid mice infection experiment . to compare the protective efficacy of bcg strains , we used a low - dose ( 100 cfus of m. tb h37rv ) , aerosol infection mouse ( balb / c ) model to mimic the natural m. tb infection in humans . for multiple pair - wise comparisons , one - way anova and it was found that mice vaccinated with bcg - phipps , bcg - frappier , bcg - pasteur , bcg - tice , bcg - danish , bcg - prague , bcg - russia , and bcg - moreau had significantly lower m. tb burdens than the nonvaccinated pbs group ( figure 2a ) , ranging from 0.58 log10 ( bcg - moreau group ) to 1.03 log10 cfus ( bcg - phipps group ) lower ( table 2 ) . mice vaccinated with bcg - china , bcg - glaxo , bcg - japan , bcg - birkhaug , and bcg - sweden had 0.250.52 log10 lower m. tb counts than the pbs group but the differences were not statistically significant ( table 2 ) . in a comparison between the bcg - vaccinated groups , mice vaccinated with bcg - phipps had significantly lower m. tb counts in the lungs than animals vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.0001 ) , bcg - sweden ( cfu = 0.71 log10 , p < 0.001 ) , bcg - japan ( cfu = 0.54 log10 , p < 0.05 ) , and bcg - glaxo ( cfu = 0.56 log10 , p < 0.05 ) . mice vaccinated with bcg - pasteur , bcg - tice , and bcg - danish had the lowest m. tb burden in the spleen , on average 0.78 log10 cfus lower than the pbs control group ( p < 0.05 , one - way anova and tukey 's test ; figure 2b ) . mice vaccinated with bcg - phipps , bcg - frappier , bcg - prague , and bcg - moreau also had , on average , 0.67 log10 lower m. tb counts than the pbs group , although the differences were not statistically significant . no significant differences were found between the bacterial burden in mice vaccinated with bcg - sweden , bcg - birkhaug , and bcg - glaxo compared to the pbs group . one - way anova and bonferroni 's multiple - comparison test showed that strains of group iv ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg -tice ) and group iii ( bcg - china , bcg - danish , bcg - prague , and bcg - glaxo ) afforded significantly better protection than strains of group ii ( bcg - sweden and bcg - birkhaug ) in vaccinated mice . the most protective bcg group , du2 group iv , also had the highest level of virulence in scid mice , and there appeared to be a correlation between virulence and efficacy . in figure 3b , we replotted the data of figure 2a by dividing the bcg strains into three groups based on the virulence level ( figure 1a ) , the most virulent group ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice ) , the intermediate virulent group ( bcg - china , bcg - russia , bcg - moreau , and bcg - danish ) , and the least virulent group ( bcg - japan , bcg - glaxo , bcg - prague , bcg - sweden , and bcg - birkhaug ) . to directly compare the virulence of bcg strains , we performed scid mice infection of 13 bcg strains and monitored the survival of animals over 18 weeks . the majority of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were dead by week 10 , and comparison of their survival curves revealed no significant differences among them ( log - rank test ) . the median survival time of scid mice infected with bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice were 48 , 53.5 , 55 , and 58 days , respectively . in contrast , all scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague survived during the 18-week experiment . the survival rate of scid mice infected with bcg - japan , bcg - birkhaug , bcg - sweden , bcg - glaxo , and bcg - prague were 93.75 , 100 , 100 , 100 , and 100% , respectively , which were the same as the phosphate - buffered saline ( pbs ) control group ( 87.5% ) . accordingly , the 13 bcg strains fall into five groups based on their virulence levels : bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice > bcg - china > bcg - russia , bcg - moreau > bcg - danish > bcg - glaxo , bcg - prague , bcg - japan , bcg - sweden , and bcg - birkhaug . at week 4 postinfection , bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice exhibited the highest counts in the lungs of individual scid mice , reaching 7.35 log10 , 7.34 log10 , 6.76 log10 , and 7.11 log10 colony - forming units ( cfus ) , respectively , which were on average 2.5 log10 higher than their counts at the week 1 timepoint ( 4.11 log10 , 4.92 log10 , 4.25 log10 , and 5.04 log10 cfus for bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice , respectively ) ( figure 1b ) . bcg - russia , bcg - moreau , bcg - china , bcg - danish , and bcg - sweden showed intermediate levels of replication , ranging from 1.2 to 2.1 log10 cfus higher in the lungs of scid mice at week 4 than at week 1 postinfection ( figure 1b ) . in a separate experiment , three bcg strains ( bcg - pasteur , bcg - russia , bcg - japan ) representing different virulence levels were selected to repeat the scid mice infection experiment . to compare the protective efficacy of bcg strains , we used a low - dose ( 100 cfus of m. tb h37rv ) , aerosol infection mouse ( balb / c ) model to mimic the natural m. tb infection in humans . it was found that mice vaccinated with bcg - phipps , bcg - frappier , bcg - pasteur , bcg - tice , bcg - danish , bcg - prague , bcg - russia , and bcg - moreau had significantly lower m. tb burdens than the nonvaccinated pbs group ( figure 2a ) , ranging from 0.58 log10 ( bcg - moreau group ) to 1.03 log10 cfus ( bcg - phipps group ) lower ( table 2 ) . mice vaccinated with bcg - china , bcg - glaxo , bcg - japan , bcg - birkhaug , and bcg - sweden had 0.250.52 log10 lower m. tb counts than the pbs group but the differences were not statistically significant ( table 2 ) . in a comparison between the bcg - vaccinated groups , mice vaccinated with bcg - phipps had significantly lower m. tb counts in the lungs than animals vaccinated with bcg - birkhaug ( cfu = 0.78 log10 , p < 0.0001 ) , bcg - sweden ( cfu = 0.71 log10 , p < 0.001 ) , bcg - japan ( cfu = 0.54 log10 , p < 0.05 ) , and bcg - glaxo ( cfu = 0.56 log10 , p < 0.05 ) . mice vaccinated with bcg - pasteur , bcg - tice , and bcg - danish had the lowest m. tb burden in the spleen , on average 0.78 log10 cfus lower than the pbs control group ( p < 0.05 , one - way anova and tukey 's test ; figure 2b ) . mice vaccinated with bcg - phipps , bcg - frappier , bcg - prague , and bcg - moreau also had , on average , 0.67 log10 lower m. tb counts than the pbs group , although the differences were not statistically significant . consistently , mice vaccinated with bcg - phipps , bcg - tice , bcg - danish , bcg - russia , and bcg - moreau had significantly lower m. tb burden in the lungs , ranging from 0.54 to 0.83 log10 cfus lower than the pbs group ( p < 0.05 , one - way anova and tukey 's post hoc test ; figure 2c ) . no significant differences were found between the bacterial burden in mice vaccinated with bcg - sweden , bcg - birkhaug , and bcg - glaxo compared to the pbs group . one - way anova and bonferroni 's multiple - comparison test showed that strains of group iv ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg -tice ) and group iii ( bcg - china , bcg - danish , bcg - prague , and bcg - glaxo ) afforded significantly better protection than strains of group ii ( bcg - sweden and bcg - birkhaug ) in vaccinated mice . the most protective bcg group , du2 group iv , also had the highest level of virulence in scid mice , and there appeared to be a correlation between virulence and efficacy . in figure 3b , we replotted the data of figure 2a by dividing the bcg strains into three groups based on the virulence level ( figure 1a ) , the most virulent group ( bcg - phipps , bcg - frappier , bcg - pasteur , and bcg - tice ) , the intermediate virulent group ( bcg - china , bcg - russia , bcg - moreau , and bcg - danish ) , and the least virulent group ( bcg - japan , bcg - glaxo , bcg - prague , bcg - sweden , and bcg - birkhaug ) . in this study , we performed a head - to - head comparison of the virulence and efficacy of 13 bcg strains representing different genetic lineages in murine models . however , in the bank vole study , the 11 bcg strains were compared in six separate experiments , each involving 5 strains , and bcg - danish was the only strain common in all experiments , which limits the comparison of the results . however , 6 of the 10 bcg strains ( bcg - phipps , bcg - tice , bcg - pasteur , bcg - frappier , bcg - connaught , and bcg - mexico ) included in this study are from the du2 group iv , while only 1 strain each from group i ( bcg - moreau ) and group iii ( bcg - danish ) and 2 strains from group ii ( bcg - sweden and bcg - birkhaug ) were included . recognizing the limitations of the previous studies , we performed comparative studies of 13 bcg strains in the same experiment , which has the advantage of removing variability between experiments . in addition , we also compared the virulence of these 13 bcg strains in scid mice , which , to the best of our knowledge , has never been performed previously . the most virulent strains ( bcg - phipps , bcg - pasteur , bcg - frappier , and bcg - tice ) all belong to the du2 group iv ( figure 1 ) , and the two strains of the du2 group ii ( bcg - sweden and bcg - birkhaug ) are among the least virulent group . for bcg - sweden and bcg - birkhaug of group ii , the deletion of whib3 , a reductive stress regulator , and trcr , a response regulator of the trcr - trcs two - component system , may account for their low virulence . for example , bcg - russia and bcg - moreau of group i are more virulent than bcg - danish but less virulent than bcg - china of the same group ( group iii ) . there appears to be a general trend that more virulent bcg strains are also more protective . bcg strains of the most virulent group ( bcg - phipps , bcg - pasteur , bcg - frappier , bcg - tice ) demonstrated better protection than bcg - sweden and bcg - birkhaug of group ii . among the group i strains , bcg - japan is the least virulent and also less protective than bcg - russia and bcg - moreau . notably , we found that mice vaccinated with bcg - phipps and bcg - frappier had 0.50.8 log10 fewer m. tb than those vaccinated with bcg - birkhaug , bcg - sweden , bcg - japan , or bcg - glaxo ( figure 2a ) , highlighting the importance of selecting specific bcg strain(s ) for the construction of recombinant bcg . a randomized trial study comparing two bcg strains in 300,000 infants in hong kong found that a more virulent strain , bcg - pasteur , administered at a lower dosage , provided a significantly greater ( 40% ) protection against childhood forms of tb than a less virulent strain , bcg - glaxo . however , in a retrospective analysis of cohorts in kazakhstan , vaccination of neonates by one of the least virulent strains , bcg - japan , reduced the risk of tb by 69% , by 43% after bcg - serbia vaccination , and only by 22% after bcg - russia .

complex molecule networks are involved in the cell 's response to external signals and cross - communication among different physiological systems of the organism . transduction of cell signalling , which is mainly mediated by second messenger cascades , allows faster responses to a wide variety of stimuli [ 36 ] , which control cellular functions such as proliferation , growth , and differentiation . among extracellular stimuli are steroid and protein hormones which are able to induce phosphorylation of membrane receptors with serine - threonine - tyrosine kinase activity which constitutes the initial process that will subsequently orchestrate the whole transduction pathway [ 710 ] . the mitogen - activated protein kinase family ( mapk ) that contain various signaling molecules have received increasing attention due to their implications on cell growth and proliferation . furthermore , mapks are highly conserved along the evolutionary scale [ 11 , 12 ] from invertebrates to mammals . mapks are divided into three main groups : ( 1 ) jun nh2 terminal kinases ( jnk 1/2/3 ) which are mainly activated by cytokines and induce dna damage . ( 2 ) p38 ( p38 /// ) that switches on in response to stress stimuli and has been related to osmoregulation and cell cycle entry . ( 3 ) extracellular signal - regulated kinases ( erk 1/2 ) that can be activated by hormones associated to g - protein coupled receptors ( gpcrs ) , growth factors , stress stimuli , cytokines , and raf kinases , among others . two main isoforms have been described for erk , erk1 , and erk2 , which have around 80% of homology in protein sequence [ 2 , 13 ] . during cell proliferation , erk 1/2 can activate , through specific phosphorylation of serine - proline / threonine - proline residues , several nuclear transcription factors , such as c - fos and c - jun , elk1 , c - myc , and stat3 , among others . the role of erk 1/2 during the molecular crosstalk between host and parasite is not clear yet , although it is present when the host synthesizes hormones and growth factors which directly benefit the parasite . this bidirectional communication between the two organisms can occur due to the presence of parasitic structures that simulate the hosts ' hormone receptors and ligand - binding proteins . concomitantly , little is known about the presence and function of erk 1/2 in parasites . for instance , erk 1/2 has been implied in the response of echinococcus multilocularis to human epidermal growth factor ( erk - like map kinase ) . on the other hand , an erk - like protein has been reported for trypanosoma brucei and is involved in the parasite 's growth rate . although some information is available regarding the presence of erk homologs in parasites , not much is known on their function and possible role in mediating the parasite 's response to the host 's hormonal microenvironment , which occurs during an infection process , for example , in murine cysticercosis caused by taenia crassiceps . experimentally induced murine cysticercosis represents an alternative model to study the parasite - host interaction similar to that occurring during human / porcine infection caused by taenia solium . murine cysticercosis may be induced in both male and female mice by injecting the metacestode stage of the helminth parasite t. crassiceps into the mouse peritoneal cavity . interestingly , female mice of any strain are more susceptible than male mice and this effect is strongly related to the sex - steroid microenvironment . it has been shown that 17-estradiol ( e2 ) induces an increase in the parasite load by two mechanisms : tilting the hosts ' immune response towards a parasite - permissive th2 response and directly promoting parasite growth and reproduction . in this context , we reported that e2 increases in vitro reproduction of t. crassiceps while androgens inhibit it . the proliferative effect of e2 on the parasite seems to be mediated by the estrogen - induced ap-1 transcription factor expression in the parasite . estrogen receptor ( er ) expression by cysticerci and activation of the ap-1 complex , in addition to the inhibition of proliferation by tamoxifen , support the idea that one of the proliferative mechanisms mediated by e2 is a classical nuclear receptor - dependent pathway [ 22 , 24 ] . however , the proliferative effect of e2 on t. crassiceps reproduction is not fully inhibited by the antiestrogen tamoxifen suggesting that the parasite may respond to estrogens by alternative pathways , such as those mediated by the interaction between steroid hormones and gpcrs . for instance , in excitable cells from mouse reproductive tissue , estrogens can promote gpcr activity , triggering to the epidermal growth factor receptor activation as well as phosphorylation of the plc - pkc pathway . on the other hand , e2 effects are not only mediated by unspecific receptors localized in the cellular membrane but also by means of the specific nuclear er [ 28 , 29 ] . in fact , the binding between e2 and its membrane er activates group i and ii of the metabotropic glutamate receptor . it should here be mentioned that er is able to bind to src kinases through their highly conserved sh2 domains , which could considerably modify the effect of erk 1/2 on the phosphorylation pattern of this transcription factor . nevertheless , information on this type of mechanisms in parasitic cells is scarce [ 16 , 17 , 32 ] . therefore further studies on these mechanisms are required particularly for drug design since the knowledge obtained on the metabolic pathways that regulate parasite growth and establishment could provide with specific potential targets for therapeutic treatment . these may involve enzymes , genes , and transduction molecules which are exclusively present in the parasite . additionally , this would also avoid host damage and nonspecific cross responses . the aim of this work was to find experimental evidence on the functional participation of alternative molecules that can respond to the estrogenic stimulus , as may be the case of a parasite erk - like protein , which could mediate the proliferative effects of exogenous 17-estradiol on the helminth parasite taenia crassiceps . the study of signal transduction in this parasite may potentially benefit not only the understanding of the host - parasite molecular crosstalk but also the design of drugs that specifically arrest the activity of important parasite molecules such as transduction proteins and transcription factors . a new stock of t. crassiceps cysticerci ( orf - kuhn2 strain ) was donated to our laboratory by r. kuhn in 2000 and was kept by serial intraperitoneal passage in balb / c ann female mice approximately every four months [ 19 , 33 ] . cysticerci for each experimental session were obtained from intraperitoneally infected female mice and placed in tubes containing sterile pbs ( 1x ) supplemented with 100 u / ml of antibiotics fungizone ( gibco , grand island ) . the tubes were centrifuged for 10 minutes at 1,500 rpm and 4c , and the supernatant was discarded . packed cysticerci were incubated in aim - v serum - free medium ( sigma , st . these were then centrifuged 3 times at 1,500 rpm for 10 minutes for washing . after the final wash , the numbers of viable cysticerci ( complete , translucent , and motile cystic structures ) were counted under a binocular microscope . ten viable nonbudding cysticerci of approximately 2 mm in diameter were then selected and dispensed into each well of 24-well culture plates ( falcon , becton dickinson labware , franklin lakes , new jersey ) in 1 ml aim - v medium ( gibco brl ) and incubated at 37c and 5% co2 . a sufficient number of culture wells were prepared to accommodate the complete experimental design to evaluate the effects of in vitro treatment of estradiol on cysticerci . culture grade erk inhibitor ii was obtained from calbiochem . for in vitro tests , water - soluble e2 was dissolved in dmem serum - free culture medium , and erk inhibitor ii was dissolved in 3% dmso . each one was prepared to 100 m stock concentration and then sterilized by passage through a 0.2 mm millipore filter . each of the following experimental conditions was applied to 24 parasite - loaded wells for concentration - response curves : ( a ) supplemented with the vehicle where hormones were dissolved ( control groups ) , ( b ) separately supplemented with 0.1 , 0.5 , 1 , and 10 m of e2 and ( c ) separately supplemented with 0.1 , 0.5 , 1 , and 10 m of erk inhibitor ii . optimal concentrations of e2 and erk inhibitor ii were selected from the concentration - response curves and used further on in time - response curves . the used concentrations were as follows : 0.1 m of e2 and 0.5 m of erk inhibitor ii . the number of buds per cysticercus as a function of days in culture was assessed as the response variable . in parasites treated at the same time with estrogen and inhibitor , parasite reproduction was measured by counting the total number of buds in the ten cysticerci in each well . bud count , as well as viability , was checked daily under an inverted light microscope ( olympus , mo21 , tokyo , japan ) at 4 and 10 magnification . injury to cysticerci was recognized microscopically by progressive internal disorganization , development of whitish opaque areas on the parasite 's tegument , and loss of motility . total rna was isolated from hormone and erk inhibitor ii - treated t. crassiceps cysticerci and balb / c ann female mouse spleen ( used as controls for specific erk gene amplification , data not shown ) by the single - step method based on guanidine isothiocyanate / phenol / chloroform extraction using trizol reagent ( invitrogen , carlsbad , ca ) . briefly , cysticerci were disrupted in trizol reagent ( 1 ml/0.1 g tissue ) , and 0.2 ml of chloroform was added per 1 ml of trizol . rna was precipitated with isopropyl alcohol , washed with 75% ethanol , and dissolved in rnase - free water . rna concentration was determined by absorbance at 260 nm and its purity was determined by electrophoresis in 1.0% denaturing agarose gel in the presence of 2.2 m formaldehyde . once properly quantified , total rna was reverse transcribed , followed by specific pcr amplification of erk - like gene from parasite tissue , at the same time as -actin ( control gene ) , as previously described . briefly , 10 g of total rna were incubated at 37c for h with 40 units of m - mlv reverse transcriptase ( applied biosystems , usa ) in 20 l of reaction volume containing 50 m of each dntp and 0.05 g oligo ( dt ) primer ( gibco , ny ) . ten l of the cdna reaction were subjected to pcr in order to amplify the sequences of the specified genes . primer design was based on the most conserved regions of sequenced genes of all species reported in the database . sequences of primers are as follows : erk sense 5-acaaagttcgagttgctatca-3 and antisense 5-attgatgccaatgatgttctc-3 and -actin sense 5-gggtcagaaggattcctatg and antisense 5-ggtctcaaacatgatctggg . the 50 l pcr reaction included 10 l of previously synthesized cdna , 5 l of 10 pcr buffer ( perkin - elmer , usa ) , 1 mm mgcl , 0.2 mm of each dntp , 0.05 m of each primer , and 2.5 units of taq dna ( biotecnologias universitarias , mexico ) . after an initial denaturation step at 95c for 5 minutes , temperature cycling was as follows : 95c for 30 seconds , 57c for 45 seconds , and 72c for 45 seconds during 35 cycles . the 50 l of the pcr reaction were electrophoresed on 2% agarose gel in the presence of a 100 bp ladder as molecular weight marker ( gibco , brl , ny ) . the pcr products obtained were visualized by staining with ethidium bromide . in both cases , different pcr conditions were assessed until a single band corresponding to the expected molecular weight of the gene was found . the -actin gene is a constitutively expressed gene , and it was used as internal control for differences in the amplification procedure between experiments and to stain different gels . briefly , untreated , e2 and erk inhibitor ii - treated cysticerci were disrupted in tris - hcl ( 1 ml/0.1 g tissue ) , proteinase k ( 100 units / ml ) , and proteases inhibitor cocktail ( calbiochem ) . bw cells were used as internal control of protein extraction and integrity ( data not shown ) . the supernatant was recovered after 15 minutes centrifugation at 1,500 rpm and the pellet was discarded . protein concentration was obtained by absorbance at 595 nm using the bradford - lowry method . total protein extracts of t. crassiceps cysticerci and bw cells were boiled in reducing laemmli sample buffer , separated by sds - page ( 10% acrylamide ) , and electroblotted onto nitrocellulose membranes . the membranes were blocked overnight in tbst buffer ( 10 mm tris - hcl , ph 7.4 , 100 mm nacl , 0.5% tween 20 ) containing 1% bsa with 3% dry milk . then , different membranes were washed five times in tbst and separately incubated for 2.5 hours in presence of -erk ( 1 g/l , santa cruz biotechnology ) and -perk ( 1 : 2000 , cell signaling ) . after this first incubation , membranes were washed three times in tbst and subsequently incubated for 1 hour in presence of -mouse igg hrp ( 1 : 2000 , amersham ) for perk and -rabbit igg hrp ( 1 : 1500 , amersham ) for erk . immediately after , the bands were visualized using the ecl system according to the manufacturer 's instructions ( super signal ecl , pierce ) . t. crassiceps and mouse spleen cells were extracted by tissue disruption from cultured treated and untreated parasites . 2 10 cells for each treatment were incubated at 4c for 20 minutes in presence of -cd3 , -cd4 , -cd8 , -cd19 , and -macrophages antibodies ( as the surface antibodies ) and subsequently washed in sterile pbs 1x staining . next , cells were centrifuged at 1,500 rpm for 5 minutes and incubated in golgiplug ( bd , biosciences ) for 3 hours . immediately , cells were washed in perm / wash buffer ( bd , biosciences ) and centrifuged at 1,500 rpm for 5 minutes . after this , cells were separately incubated in presence of -erk 1 g/l ( santa cruz , biotech ) at room temperature for 20 minutes , and subsequently washed in pbs 1x staining . cell pellets were separately resuspended in presence of fitc - conjugated goat anti - rabbit antibody and incubated at 4c for 30 minutes in the dark . after this , cells were washed in pbs 1x - staining and centrifuged at 1,500 rpm for 5 minutes . cell pellets were resuspended in 500 l of pbs 1x staining in absence of light and analyzed by flow cytometry using an facs calibur ( bd , biosciences ) . e 2-treated t. crassiceps cysticerci were washed with pbs 1x , embedded in tissuetek ( triangle biomedical science ) , and frozen at 80c . parasite tissue sections ( 10 m ) were fixed with 4% paraformaldehyde for 30 minutes , washed three times in pbs , and blocked for 30 minutes with rpmi medium containing 0.5% bsa and 5% fbs ( hernndez - bello et al . ) . cross - sections were then incubated with a 1 : 500 dilution of the polyclonal -erk ( santa cruz , biotech ) for 45 minutes at 37c , washed with pbs , and then incubated with fluorescein isothiocyanate ( fitc)-conjugated goat anti - rabbit antibody ( zymed ) at 1 : 500 dilution . control experiments were assessed incubating the thick tissue sections in presence of only the fitc - conjugated goat anti - rabbit antibody at the same dilution . to eliminate background fluorescence , after two single washings , samples were mounted in vectashield mounting medium ( vector laboratories inc . ) and examined with a carl zeiss epifluorescence microscope at 10x , 40x and 100x magnifications ( carl zeiss , germany ) . total protein from cultured t. crassiceps cysticerci was extracted and properly quantified as described before . protein samples were placed in a buffer containing 8 m urea , 2% chaps , 50 mm dtt , ipg ph 47 ( bio - rad ) , and bromophenol blue . immediately after , protein samples were incubated overnight with the first - dimension gel ( amersham ) . once they were properly hydrated , the first - dimension gel was isoelectrofocused with a constant voltage on a lineal electric gradient . after this , the gel was equilibrated in a buffer containing 6 m urea , 2% sds , 375 mm tris ph 8.8 , 2% dtt , and 20% glycerol for 15 minutes . next , the same equilibration process was performed using iodoacetamide 25 mg / ml instead of dtt . once equilibrated , the gel was separated according to the molecular size of each protein in a second - dimension gel ( page - sds al 12.5% ) . finally , the bidimensionally separated gel was electroblotted onto nitrocellulose membranes and a -erk western blot was carried out , as mentioned before . only the immunodetected point , corresponding to the expected molecular weight and to the predicted isoelectrical point , was cut out and sequenced by mass spectrometry . e 2 and erk inhibitor ii concentration - response and time - response curves were estimated in six independent experiments , each performed with ten cysticerci , freshly extracted from different infected donor mice , and replicated in 24 different wells . the response variable used in statistical analysis was the sum of buds in the 24 wells with each treatment along the time of exposure of each experiment . data of the six replications of each experiment were pooled and expressed as their average + / standard deviation . all optical densitometries as well as the mean of the fluorescence in the flow cytometry analysis was calculated for six different experiments and expressed as the average + / standard deviation . data were analyzed using either student 's t - test or one - way anova and a subsequent dunnet 's multiple comparison test , depending on the experimental design . the number of buds ( which directly reflects the reproductive rate in this parasite ) in cultured t. crassiceps cysticerci clearly increased by addition of 17-estradiol in a concentration - dependent manner . compared to control groups , e2 increased the parasite reprodcution rate up to 3-folds , from the lowest concentration on 100 nm up to higher pharmacologic concentrations such as 1 and 10 m , without affecting parasite viability ( figure 1(a ) ) . in contrast , the erk inhibitor showed the opposite effect on the number of cysticercus buds , although this effect was significant only at the highest concentrations ( 1 and 10 m ) , which suggests that the erk inhibitor effect was also dependent on concentration ( figure 1(a ) ) . in addition to the concentration effects , the proliferative action of e2 on parasite reproduction was maintained throughout the culture time , reaching a 3-fold higher number of buds than that in untreated cysticerci at around five days of in vitro culture ( figure 1(b ) ) . interestingly , compared with control cysticerci , erk inhibitor treatment of cysticerci had no significant effects on parasite reproduction ( figure 1(b ) ) . however , it was clear that the erk inhibitor completely blocked the e2-dependent proliferative effect ( figure 1(b ) ) , emphasizing the importance of both e2 and erk - like in the reproduction of the t. crassiceps cysticerci . using specific primers designed considering the most conserved sequences of all reported erks 1/2 , a band of 112 bp was amplified and corresponds to the expected molecular weight of the erk - like gene in t. crassiceps and in the mouse ( internal control of expression , data not shown ) . interestingly , a significant increase in expression of the erk - like gene was observed in response to e2 and erk inhibitor , even if the inhibitor was in presence of the steroid hormone ( figure 2 ) . native erk - like protein ( 55 kda ) was detected in untreated , e2 treated , and erk inhibitor - treated parasites . contrary to the observations made by rt - pcr , no changes in total protein quantity were observed with any of the treatments ( figure 3(a ) ) . interestingly , when the phosphorylated form of erk - like ( perk - like ) protein was analyzed , it was evident that e2 increases erk - like protien phosphorylation 4 folds compared to untreated parasites ( figure 3(b ) ) . interestingly , erk inhibitor treatment partially blocked the estradiol stimulated erk - like protein phosphorylation , without returning it to basal levels ( figure 3(b ) ) . flow cytometry analysis firstly showed that t. crassiceps cells were different in size and complexity from mouse spleen cells ( figure 4 ) . in fact , parasite cells were approximately 3 folds smaller than mouse spleen cells and exhibited poor complexity ( data not shown ) . in addition , parasite cells showed no expression of the membrane markers cd3 , cd4 , cd8 , and cd19 , typically present in different types of mammalian leukocytes ( figure 4(a ) ) . however , paramyosin ( ag - b , an exclusive component of the cytoskeleton of cestodes and insects ) was clearly detected in t. crassiceps cells but not in host splenocytes ( figure 4(a ) ) . thus , the erk - like protein was exclusively recognized on t. crassiceps cells , because the facs analysis was performed on parasite cells ag - b / cd3/cd4/cd8/cd19 . no significant differences were observed in the fluorescence intensity related to the erk - like protein in untreated and e2-treated parasites ( figure 4(b ) ) . up to this point , experimental evidence indicates that the erk - like protein is expressed in t. crassiceps cysticerci and that this is not a contamination product from host cells . in addition , on well - preserved t. crassiceps tissue ( figure 5(a ) ) , immunochemistry assays were perfomed and the results showed that parasite cells express erk - like protein mainly in the subtegument tissue of the cysticercus but not in tegument ( figures 5(c ) and 5(d ) ) . as expected , t. crassiceps tissue incubated only in presence of the secondary antibody did not give any positive signal related to erk ( figure 5(b ) ) , which suggests that the experimental conditions were optimal for detecting exclusively parasite cells presenting erk - like molecules without false positive signals . total proteins from e2-treated parasites were separated in a ph range of 47 , according to their isoelectrical point ( ip ) and molecular weight ( figure 6(a ) ) . moreover , a well - defined dot around ph 6.00 , corresponding to the ip of most of the sequenced erk 1/2 , was recognized with high specificity by means of a single western blot ( figure 6(b ) ) . interestingly , the erk - like sequence showed high homology ( 60% ) to erk 1/2 from yeast , chicken , and mouse . concomitantly , we were able to detect the activation motif tey ( threonine - glutamine - tyrosine ) on the parasite 's erk - like protein sequence , which is typically present in all erk 1/2 sequenced from several vertebrate and invertebrate organisms . finally , the highly conserved amino acid sequence , niltlhnvantvmknpamisknllr , was identified from the parasite 's erk - like protein , which strongly suggests that this protein also conserves serine - threonine kinase activity and could influence phosphorylation of other parasite kinases ( data not shown ) . as previously reported , 17-estradiol exerts a direct proliferative effect on taenia crassiceps cysticercus reproduction , which is not necessarily mediated by the host immune system , but through a classic nuclear receptor in the parasite . however , alternative mechanisms through which e2 can affect the parasite , securing its reproduction and establishment in an immuncompetent host , have not been completely explored yet . the aim of the present study was to investigate the participation of a signaling pathway mediated by second messenger cascades , which may be responsive to sex steroids that are highly conserved throughout the species and involved in cell proliferative processes . thus , we explored the signaling pathway dependent on erk - like protein , a member of the mapk kinases which can be activated by several extracellular signals and generate rapid responses inside the cell , and the effect of the erk inhibitor on erk - like protein participation in parasite reproduction . interestingly , we found that , at progressively higher 17-estradiol concentrations , the number of t. crassiceps cysticercus buds reached a maximum value . as concentration of erk inhibitor increased , parasite reproduction progressively decreased . on the other hand , the e2 effect was enhanced as time passed , reaching its maximum effect on parasite reproduction at day five of in vitro culture , which supports that e2 effects are concentration dependent . nevertheless , no time - dependent response was found when erk inhibitor was tested , although this inhibitor completely blocked the e2-dependent proliferative effect , suggesting that parasite erk - like protein is directly involved in mediating the crosstalk between the hormonal microenvironment ( exogenous e2 ) and parasite reproduction . these findings show a marked concentration and time - dependent pattern in the effects of e2 on cysticercus reproduction . this observation is relevant since it suggests that sex steroids may have similar effects on mammals and parasitic cestodes , a hypothesis that evokes the wide range of steroid hormone actions not only in many different cell types but also along the phylogenetic scale , among distantly related organisms . moreover , determination of the actions of e2 and erk inhibitor on t. crassiceps cysticerci was important . this was approached by analyzing the erk - like gene expression in the parasite as well as its translation to functional proteins that mediate the e2 effects . in view of this , a band corresponding to erk - like gene was amplified from t. crassiceps larval tissue using specific primers designed for the most conserved regions of these genes , previously reported in mammals such as mouse , rat , and human , as well as in yeast , birds , and amphibian . unexpectedly , not only the erk - like gene is expressed in the parasite , but it can also be overregulated by 17-estradiol . however , a similar effect was observed when parasites were cultured in presence of erk inhibitor . this unexpected finding suggests that helminth parasites could have developed a positive feedback mechanism which is able to sense changes in the expression of very essential molecules such as mapk kinases and maintain their activity in order to assure viability and reproduction of the parasite . on the other hand , the effects of sex steroids on parasite mapk kinases gene expression , which are in charge of mediating the functionality of the whole pathway , have been scarcely explored . the results obtained in this study showed that this type of parasite genes is differentially regulated by exogenous sex steroids , which could be occurring in vivo from host to parasite . also , this finding offers an alternative explanation of why t. crassiceps cysticerci grow better in the female mouse than in the male , emphasizing the molecular crosstalk between host and parasite , which is in turn differentially influenced by the hormonal microenvironment of each gender . it is important to underline that proteins involved in signal transduction pathways , lead their functions by being phosphorylated ; it was therefore crucial to determine the corresponding erk - like protein in the parasite and to analyze its phophorylation pattern in response to 17-estradiol and erk inhibitor treatments . unexpectedly , one single band corresponding to erk - like protein was detected in t. crassiceps , while most mammalian species exhibit a characteristic doublet . this finding suggests that , along many years of coevolution between the host and the parasite , several molecules , such as genes or proteins , may have been lost or , passing their functions onto others that conserve similar structure and can therefore trigger the same effects due to a complex process of molecular hypertrophy the fact that the erk - like protein differs from its homolog in mammalian cells supports two very important aspects of this study : ( a ) that the mapk kinase detected in the parasite is not produced by contamination from host immune cells and ( b ) that , although both proteins have different characteristics , they probably conserve a high degree of similarity in their catalytic domains , which makes them recognizable with the same antibody and assures the suitable function for transducing signals in both organisms . in addition , 17-estradiol treatment differentially stimulated the phosphorylation of erk - like protein , which strongly supports that only parasitic erk - like protein is expressed and regulated by exogenous estrogens but also that this mapk kinase can be translated to a functional protein , able to be activated through e2-stimulated phosphorylation processes . this result suggests again that the mechanisms of action of steroid hormones on target cells are poorly diversified among species , maintaining similar and successful strategies from the simplest to the most complex organisms . on the other hand , it was critical to determine that the detected and analyzed erk - like protein was exclusively found in the taenia crassiceps cysticercus and not a consequence of host immune cell contamination , because , as shown elsewhere , there is extremely high interaction between parasites and immune cells , which may eventually lead to leukocyte invasion into several parasitic tissues . for this reason , an alternative use of flow cytometry was used to differentiate proteins from t. crassiceps and the murine host by identifying exclusive molecules of the parasite which are neither synthesized nor expressed by the host . this is the case of paramyosin , a muscle protein found only in invertebrates such as drosophila melanogaster , caenorhabditis elegans , taenia solium , and t. saginata . the flow cytometry studies showed that presence and phosphorylation pattern of the analyzed erk - like protein belonged specifically to the parasite , because paramyosin was only detected in t. crassiceps cells , where this mapk kinase was also found and studied . in contrast , cells extracted from mouse spleen were not recognized by the -paramyosin antibody , but they were positive for cd3 , cd4 , cd8 , cd19 , and macrophage antibodies , contrary to parasite cells . these results demonstrate that the analyzed parasite erk - like protein is in fact from taenia crassiceps origin and not from other sources and simultaneously accentuate the potential use of flow cytometry for differential identification of molecules from organisms with extremely close contact , such as this parasite and its host . worth of mention is that the erk - like protein was not only detected at messenger rna and protein level but also localized inside the parasite cell . interestingly , parasitic cells expressing erk - like protein were exclusively located at the subtegument tissue , where most of the muscle and nephridium cells are found . this result suggests that the erk - like protein is importantly involved in parasite motility and excretion , as well as in reproductive functions , which together maintain parasite viability and proliferation . finally , the protein sequence of the erk - like protein showed a remarkable degree of conservation of certain segments , such as the activation domain , and functions , such as the serine - threonine kinase activity , both of them characteristic of these mapk kinases . in fact , protein sequences from mammalian and high invertebrate erk 1/2 are different to the parasite erk - like protein , but closely related regarding the conserved domains , which suggests that parasites have developed similar structures as their host homologs , probably keeping only the catalytic domains in order to secure the basic functions of important proteins such as the erk family . this hypothesis supports that development of proteins which can recognize the host 's growth factors represents advantages in parasite metabolism economy , because the pathogen does not need to synthesize all molecules involved in a pathway but can take them directly from its host . this benefits the processes of reproduction , establishment , and immune evasion , among other important aspects of parasite life . in conclusion this mapk kinase showed great capacity to transduce signals evoked by 17-estradiol in the parasite . it is important to mention that much information about the effects of the host hormonal microenvironment on parasite physiology has been generated in recent years [ 15 , 3739 ] . our results then contribute to understand the mechanisms by which the host microenvironment affects the parasite . furthermore , the evolutionary origin of the molecules described herein , which facilitate exploitation of the host 's hormones , is worth studying . in particular , whether those genes were acquired by the parasite through horizontal gene transfer or evolved by mimicry , or simply from common ancestral genes , remains to be defined . finally , our findings may help to understand , at molecular and evolutionary levels , several aspects of the crosstalk between host and parasite , of the sexual dimorphism of the immune response , and may provide information on new parasite targets for designing specific antihelminth drugs .

map kinases ( mapk ) are involved in the regulation of cellular processes such as reproduction and growth . in parasites , the role of mapk has been scarcely studied . here , we describe the participation of an erk - like protein in estrogen - dependent reproduction of the helminth parasite taenia crassiceps . our results show that 17-estradiol induces a concentration - dependent increase in the bud number of in vitro cultured cysticerci . if parasites are also incubated in presence of an erk - inhibitor , the stimulatory effect of estrogen is blocked . the expression of erk - like mrna and its corresponding protein was detected in the parasite . the erk - like protein was over - expressed by all treatments . nevertheless , a strong induction of phosphorylation of this protein was observed only in response to 17-estradiol . cross - contamination by host cells was discarded by flow cytometry analysis . parasite cells expressing the erk - like protein were exclusively located at the subtegument tissue by confocal microscopy . finally , the erk - like protein was separated by bidimensional electrophoresis and then sequenced , showing the conserved tey activation motif , typical of all known erk 1/2 proteins . our results show that an erk - like protein is involved in the molecular signalling during the interaction between the host and t. crassiceps , and may be considered as target for anti - helminth drugs design .

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

complex molecule networks are involved in the cell 's response to external signals and cross - communication among different physiological systems of the organism . ( 2 ) p38 ( p38 /// ) that switches on in response to stress stimuli and has been related to osmoregulation and cell cycle entry . ( 3 ) extracellular signal - regulated kinases ( erk 1/2 ) that can be activated by hormones associated to g - protein coupled receptors ( gpcrs ) , growth factors , stress stimuli , cytokines , and raf kinases , among others . during cell proliferation , erk 1/2 can activate , through specific phosphorylation of serine - proline / threonine - proline residues , several nuclear transcription factors , such as c - fos and c - jun , elk1 , c - myc , and stat3 , among others . the role of erk 1/2 during the molecular crosstalk between host and parasite is not clear yet , although it is present when the host synthesizes hormones and growth factors which directly benefit the parasite . concomitantly , little is known about the presence and function of erk 1/2 in parasites . for instance , erk 1/2 has been implied in the response of echinococcus multilocularis to human epidermal growth factor ( erk - like map kinase ) . on the other hand , an erk - like protein has been reported for trypanosoma brucei and is involved in the parasite 's growth rate . although some information is available regarding the presence of erk homologs in parasites , not much is known on their function and possible role in mediating the parasite 's response to the host 's hormonal microenvironment , which occurs during an infection process , for example , in murine cysticercosis caused by taenia crassiceps . murine cysticercosis may be induced in both male and female mice by injecting the metacestode stage of the helminth parasite t. crassiceps into the mouse peritoneal cavity . it has been shown that 17-estradiol ( e2 ) induces an increase in the parasite load by two mechanisms : tilting the hosts ' immune response towards a parasite - permissive th2 response and directly promoting parasite growth and reproduction . in this context , we reported that e2 increases in vitro reproduction of t. crassiceps while androgens inhibit it . the proliferative effect of e2 on the parasite seems to be mediated by the estrogen - induced ap-1 transcription factor expression in the parasite . however , the proliferative effect of e2 on t. crassiceps reproduction is not fully inhibited by the antiestrogen tamoxifen suggesting that the parasite may respond to estrogens by alternative pathways , such as those mediated by the interaction between steroid hormones and gpcrs . in fact , the binding between e2 and its membrane er activates group i and ii of the metabotropic glutamate receptor . it should here be mentioned that er is able to bind to src kinases through their highly conserved sh2 domains , which could considerably modify the effect of erk 1/2 on the phosphorylation pattern of this transcription factor . these may involve enzymes , genes , and transduction molecules which are exclusively present in the parasite . the aim of this work was to find experimental evidence on the functional participation of alternative molecules that can respond to the estrogenic stimulus , as may be the case of a parasite erk - like protein , which could mediate the proliferative effects of exogenous 17-estradiol on the helminth parasite taenia crassiceps . the study of signal transduction in this parasite may potentially benefit not only the understanding of the host - parasite molecular crosstalk but also the design of drugs that specifically arrest the activity of important parasite molecules such as transduction proteins and transcription factors . a sufficient number of culture wells were prepared to accommodate the complete experimental design to evaluate the effects of in vitro treatment of estradiol on cysticerci . each of the following experimental conditions was applied to 24 parasite - loaded wells for concentration - response curves : ( a ) supplemented with the vehicle where hormones were dissolved ( control groups ) , ( b ) separately supplemented with 0.1 , 0.5 , 1 , and 10 m of e2 and ( c ) separately supplemented with 0.1 , 0.5 , 1 , and 10 m of erk inhibitor ii . in parasites treated at the same time with estrogen and inhibitor , parasite reproduction was measured by counting the total number of buds in the ten cysticerci in each well . rna concentration was determined by absorbance at 260 nm and its purity was determined by electrophoresis in 1.0% denaturing agarose gel in the presence of 2.2 m formaldehyde . once properly quantified , total rna was reverse transcribed , followed by specific pcr amplification of erk - like gene from parasite tissue , at the same time as -actin ( control gene ) , as previously described . the 50 l of the pcr reaction were electrophoresed on 2% agarose gel in the presence of a 100 bp ladder as molecular weight marker ( gibco , brl , ny ) . total protein extracts of t. crassiceps cysticerci and bw cells were boiled in reducing laemmli sample buffer , separated by sds - page ( 10% acrylamide ) , and electroblotted onto nitrocellulose membranes . 2 10 cells for each treatment were incubated at 4c for 20 minutes in presence of -cd3 , -cd4 , -cd8 , -cd19 , and -macrophages antibodies ( as the surface antibodies ) and subsequently washed in sterile pbs 1x staining . after this , cells were separately incubated in presence of -erk 1 g/l ( santa cruz , biotech ) at room temperature for 20 minutes , and subsequently washed in pbs 1x staining . cell pellets were separately resuspended in presence of fitc - conjugated goat anti - rabbit antibody and incubated at 4c for 30 minutes in the dark . cross - sections were then incubated with a 1 : 500 dilution of the polyclonal -erk ( santa cruz , biotech ) for 45 minutes at 37c , washed with pbs , and then incubated with fluorescein isothiocyanate ( fitc)-conjugated goat anti - rabbit antibody ( zymed ) at 1 : 500 dilution . control experiments were assessed incubating the thick tissue sections in presence of only the fitc - conjugated goat anti - rabbit antibody at the same dilution . once equilibrated , the gel was separated according to the molecular size of each protein in a second - dimension gel ( page - sds al 12.5% ) . all optical densitometries as well as the mean of the fluorescence in the flow cytometry analysis was calculated for six different experiments and expressed as the average + / standard deviation . the number of buds ( which directly reflects the reproductive rate in this parasite ) in cultured t. crassiceps cysticerci clearly increased by addition of 17-estradiol in a concentration - dependent manner . in contrast , the erk inhibitor showed the opposite effect on the number of cysticercus buds , although this effect was significant only at the highest concentrations ( 1 and 10 m ) , which suggests that the erk inhibitor effect was also dependent on concentration ( figure 1(a ) ) . in addition to the concentration effects , the proliferative action of e2 on parasite reproduction was maintained throughout the culture time , reaching a 3-fold higher number of buds than that in untreated cysticerci at around five days of in vitro culture ( figure 1(b ) ) . however , it was clear that the erk inhibitor completely blocked the e2-dependent proliferative effect ( figure 1(b ) ) , emphasizing the importance of both e2 and erk - like in the reproduction of the t. crassiceps cysticerci . using specific primers designed considering the most conserved sequences of all reported erks 1/2 , a band of 112 bp was amplified and corresponds to the expected molecular weight of the erk - like gene in t. crassiceps and in the mouse ( internal control of expression , data not shown ) . interestingly , a significant increase in expression of the erk - like gene was observed in response to e2 and erk inhibitor , even if the inhibitor was in presence of the steroid hormone ( figure 2 ) . native erk - like protein ( 55 kda ) was detected in untreated , e2 treated , and erk inhibitor - treated parasites . interestingly , when the phosphorylated form of erk - like ( perk - like ) protein was analyzed , it was evident that e2 increases erk - like protien phosphorylation 4 folds compared to untreated parasites ( figure 3(b ) ) . interestingly , erk inhibitor treatment partially blocked the estradiol stimulated erk - like protein phosphorylation , without returning it to basal levels ( figure 3(b ) ) . flow cytometry analysis firstly showed that t. crassiceps cells were different in size and complexity from mouse spleen cells ( figure 4 ) . in addition , parasite cells showed no expression of the membrane markers cd3 , cd4 , cd8 , and cd19 , typically present in different types of mammalian leukocytes ( figure 4(a ) ) . however , paramyosin ( ag - b , an exclusive component of the cytoskeleton of cestodes and insects ) was clearly detected in t. crassiceps cells but not in host splenocytes ( figure 4(a ) ) . thus , the erk - like protein was exclusively recognized on t. crassiceps cells , because the facs analysis was performed on parasite cells ag - b / cd3/cd4/cd8/cd19 . no significant differences were observed in the fluorescence intensity related to the erk - like protein in untreated and e2-treated parasites ( figure 4(b ) ) . up to this point , experimental evidence indicates that the erk - like protein is expressed in t. crassiceps cysticerci and that this is not a contamination product from host cells . in addition , on well - preserved t. crassiceps tissue ( figure 5(a ) ) , immunochemistry assays were perfomed and the results showed that parasite cells express erk - like protein mainly in the subtegument tissue of the cysticercus but not in tegument ( figures 5(c ) and 5(d ) ) . as expected , t. crassiceps tissue incubated only in presence of the secondary antibody did not give any positive signal related to erk ( figure 5(b ) ) , which suggests that the experimental conditions were optimal for detecting exclusively parasite cells presenting erk - like molecules without false positive signals . moreover , a well - defined dot around ph 6.00 , corresponding to the ip of most of the sequenced erk 1/2 , was recognized with high specificity by means of a single western blot ( figure 6(b ) ) . interestingly , the erk - like sequence showed high homology ( 60% ) to erk 1/2 from yeast , chicken , and mouse . concomitantly , we were able to detect the activation motif tey ( threonine - glutamine - tyrosine ) on the parasite 's erk - like protein sequence , which is typically present in all erk 1/2 sequenced from several vertebrate and invertebrate organisms . finally , the highly conserved amino acid sequence , niltlhnvantvmknpamisknllr , was identified from the parasite 's erk - like protein , which strongly suggests that this protein also conserves serine - threonine kinase activity and could influence phosphorylation of other parasite kinases ( data not shown ) . as previously reported , 17-estradiol exerts a direct proliferative effect on taenia crassiceps cysticercus reproduction , which is not necessarily mediated by the host immune system , but through a classic nuclear receptor in the parasite . the aim of the present study was to investigate the participation of a signaling pathway mediated by second messenger cascades , which may be responsive to sex steroids that are highly conserved throughout the species and involved in cell proliferative processes . thus , we explored the signaling pathway dependent on erk - like protein , a member of the mapk kinases which can be activated by several extracellular signals and generate rapid responses inside the cell , and the effect of the erk inhibitor on erk - like protein participation in parasite reproduction . interestingly , we found that , at progressively higher 17-estradiol concentrations , the number of t. crassiceps cysticercus buds reached a maximum value . on the other hand , the e2 effect was enhanced as time passed , reaching its maximum effect on parasite reproduction at day five of in vitro culture , which supports that e2 effects are concentration dependent . nevertheless , no time - dependent response was found when erk inhibitor was tested , although this inhibitor completely blocked the e2-dependent proliferative effect , suggesting that parasite erk - like protein is directly involved in mediating the crosstalk between the hormonal microenvironment ( exogenous e2 ) and parasite reproduction . this was approached by analyzing the erk - like gene expression in the parasite as well as its translation to functional proteins that mediate the e2 effects . in view of this , a band corresponding to erk - like gene was amplified from t. crassiceps larval tissue using specific primers designed for the most conserved regions of these genes , previously reported in mammals such as mouse , rat , and human , as well as in yeast , birds , and amphibian . unexpectedly , not only the erk - like gene is expressed in the parasite , but it can also be overregulated by 17-estradiol . however , a similar effect was observed when parasites were cultured in presence of erk inhibitor . this unexpected finding suggests that helminth parasites could have developed a positive feedback mechanism which is able to sense changes in the expression of very essential molecules such as mapk kinases and maintain their activity in order to assure viability and reproduction of the parasite . on the other hand , the effects of sex steroids on parasite mapk kinases gene expression , which are in charge of mediating the functionality of the whole pathway , have been scarcely explored . also , this finding offers an alternative explanation of why t. crassiceps cysticerci grow better in the female mouse than in the male , emphasizing the molecular crosstalk between host and parasite , which is in turn differentially influenced by the hormonal microenvironment of each gender . it is important to underline that proteins involved in signal transduction pathways , lead their functions by being phosphorylated ; it was therefore crucial to determine the corresponding erk - like protein in the parasite and to analyze its phophorylation pattern in response to 17-estradiol and erk inhibitor treatments . unexpectedly , one single band corresponding to erk - like protein was detected in t. crassiceps , while most mammalian species exhibit a characteristic doublet . this finding suggests that , along many years of coevolution between the host and the parasite , several molecules , such as genes or proteins , may have been lost or , passing their functions onto others that conserve similar structure and can therefore trigger the same effects due to a complex process of molecular hypertrophy the fact that the erk - like protein differs from its homolog in mammalian cells supports two very important aspects of this study : ( a ) that the mapk kinase detected in the parasite is not produced by contamination from host immune cells and ( b ) that , although both proteins have different characteristics , they probably conserve a high degree of similarity in their catalytic domains , which makes them recognizable with the same antibody and assures the suitable function for transducing signals in both organisms . in addition , 17-estradiol treatment differentially stimulated the phosphorylation of erk - like protein , which strongly supports that only parasitic erk - like protein is expressed and regulated by exogenous estrogens but also that this mapk kinase can be translated to a functional protein , able to be activated through e2-stimulated phosphorylation processes . on the other hand , it was critical to determine that the detected and analyzed erk - like protein was exclusively found in the taenia crassiceps cysticercus and not a consequence of host immune cell contamination , because , as shown elsewhere , there is extremely high interaction between parasites and immune cells , which may eventually lead to leukocyte invasion into several parasitic tissues . for this reason , an alternative use of flow cytometry was used to differentiate proteins from t. crassiceps and the murine host by identifying exclusive molecules of the parasite which are neither synthesized nor expressed by the host . this is the case of paramyosin , a muscle protein found only in invertebrates such as drosophila melanogaster , caenorhabditis elegans , taenia solium , and t. saginata . the flow cytometry studies showed that presence and phosphorylation pattern of the analyzed erk - like protein belonged specifically to the parasite , because paramyosin was only detected in t. crassiceps cells , where this mapk kinase was also found and studied . these results demonstrate that the analyzed parasite erk - like protein is in fact from taenia crassiceps origin and not from other sources and simultaneously accentuate the potential use of flow cytometry for differential identification of molecules from organisms with extremely close contact , such as this parasite and its host . worth of mention is that the erk - like protein was not only detected at messenger rna and protein level but also localized inside the parasite cell . interestingly , parasitic cells expressing erk - like protein were exclusively located at the subtegument tissue , where most of the muscle and nephridium cells are found . this result suggests that the erk - like protein is importantly involved in parasite motility and excretion , as well as in reproductive functions , which together maintain parasite viability and proliferation . finally , the protein sequence of the erk - like protein showed a remarkable degree of conservation of certain segments , such as the activation domain , and functions , such as the serine - threonine kinase activity , both of them characteristic of these mapk kinases . in fact , protein sequences from mammalian and high invertebrate erk 1/2 are different to the parasite erk - like protein , but closely related regarding the conserved domains , which suggests that parasites have developed similar structures as their host homologs , probably keeping only the catalytic domains in order to secure the basic functions of important proteins such as the erk family . it is important to mention that much information about the effects of the host hormonal microenvironment on parasite physiology has been generated in recent years [ 15 , 3739 ] . our results then contribute to understand the mechanisms by which the host microenvironment affects the parasite . furthermore , the evolutionary origin of the molecules described herein , which facilitate exploitation of the host 's hormones , is worth studying . finally , our findings may help to understand , at molecular and evolutionary levels , several aspects of the crosstalk between host and parasite , of the sexual dimorphism of the immune response , and may provide information on new parasite targets for designing specific antihelminth drugs .

the accurate diagnosis and proper treatment of dental trauma is a challenging area in dentistry . traumatic injuries to the pulp and periodontal structures produce damages that may impair the esthetics and function of the stomatognathic system , in addition to causing serious emotional and psychological problems to the patient7,14 . the main pulp changes secondary to dental trauma are pulp necrosis , root canal obliteration and resorption5,8 . since pulp necrosis is the most frequent8,16 , early diagnosis is necessary before microbial invasion and the onset of external resorptions , for achievement of a favorable prognosis . however , this diagnosis is limited and may present failures , since the diagnostic methods available to determine the pulp status after trauma are not precise or standardized5,13 . some criteria have been suggested to diagnose pulp necrosis in traumatized teeth , such as the negative response to pulp vitality tests , progressive discoloration of the crown , periapical radiolucency , interruption of root formation or positive response to the vertical percussion test6 . however , the criteria are not standardized and may have different meanings along the period after trauma19 , giving rise to insecurity as to the adequate moment for endodontic intervention . the thermal pulp vitality tests to heat and cold and electric tests are not completely reliable for detection of pulp necrosis25 . discoloration of the tooth crown is also not pathognomonic of pulp necrosis , because pulp hemorrhage after trauma might lead to the overflow of erythrocytes and byproducts of their decomposition to the surrounding tissue , making the tooth crown pinkish or grayish . after reestablishment of normal circulation at the region , these byproducts could be eliminated and the normal color of the tooth crown could be recovered4 . the presence of radiolucent areas at the periapical region of luxated teeth may represent intermediate stages of pulp repair , not necessarily indicating pulp necrosis2 . a histobacteriological assessment of removed pulps with clinical diagnosis of pulp necrosis after dental trauma revealed that 9% to 14% of these pulps presented potential for repair , and thus they should not have been removed3 . the presence of microorganisms in the root canal system should also be evaluated , which may definitely impair the processes of revascularization and regeneration of the ischemic pulp tissue after luxation14 . previous studies demonstrated that , in traumatized teeth with intact pulp chambers , the necrotic pulp may be either infected or free of microorganisms for a long time , which raises questions on the actual need of endodontic intervention9,11,21,27 . the purpose of the present study was to evaluate the presence of microorganisms and analyze microscopically the pulp of traumatized human permanent teeth with intact crowns and clinical diagnosis of pulp necrosis . the study sample was composed of 20 patients from the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois ( brazil ) and approved by the respective institutional review board . the inclusion criteria comprised teeth with history of trauma to the periodontal tissues ( subluxation 8 teeth , extrusive luxation 3 teeth , lateral luxation 2 teeth , and avulsion 7 teeth ) , presenting intact crowns and clinical diagnosis of pulp necrosis . teeth presenting root fracture , periapical lesion , periodontal pocket , resorptions or root canal obliteration were excluded . the following clinical data were recorded : a ) type of trauma ; b ) date of trauma and time elapsed between dental trauma and the onset of endodontic intervention ; c ) crown shade ; d ) response to thermal and electric pulp vitality tests ( pvt ) ( positive or negative ) - ( heat : a piece of heated guttapercha was applied to the center of the buccal aspect of the affected tooth , previously lubricated with petroleum jelly , with cotton roll isolation ; cold : a cold spray at -50c ( endo frost;roeko , langenau , germany ) was applied to the center of the buccal aspect of the affected tooth , with cotton roll isolation ; electric test : this was performed with the endoanalyser device ( kerr corporation , orange , ca , usa ) , placing the electrode tip on the center of the buccal aspect lubricated with fluoride gel , with cotton roll isolation , and recording the response value ) ; e ) response to vertical ( vpt ) and horizontal ( hpt ) percussion tests ( positive or negative ) ; f ) pain upon palpation ( absent or present ) ; g ) symptomatology ( absent or present ) ; h ) mobility ( normal or increased ) . all these clinical data were also recorded for the contralateral teeth , in order to check the response pattern of each patient . radiographic examination was performed with a film holder for adult patients ( indusbelo , londrina , brazil ) and insight radiographic film ( eastman kodak company , rochester ny , usa ) , with the same x - ray machine , to assure constant power and current intensity . the radiographs were analyzed in an indirect light environment , at a different moment than clinical examination . black cardboard masks were fabricated to improve the observation of radiographs on the film viewer . after clinical diagnosis of pulp necrosis based on the association of at least three clinical criteria , the teeth were submitted to endodontic treatment . for each selected tooth , rubber dam was placed and antisepsis was performed with 1% sodium hypochlorite solution ( probem , catanduva , sp , brazil ) . following , microbiological collection of the root canal was performed , based on previous studies17,26 , with the aid of autoclaved absorbent paper points n. 20 ( tanari , manacapuru , am , brazil ) . each point was introduced in the root canal up to the provisional working length with sterile pliers and kept for 30 s , removed and placed in a test tube containing srensen phosphate buffer solution ( pbs ) . the samples were sent to the department of microbiology , immunology , parasitology and pathology of the institute of tropical pathology and community health of the federal university of gois for microbiological processing . when present , the pulp tissue was removed at the provisional working length with hedstrm files ( maillefer , ballaigues , switzerland ) of compatible size with the root canal , which was placed on an autoclaved cardboard and then in a flask containing 10% buffered formalin labeled with the patient number for later laboratory processing , which was performed at the oral pathology laboratory of the dental school of the federal university of gois . each tooth was then prepared according to the protocol of endodontic treatment for traumatized teeth of the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois . the patients were followed at every 3 months by clinical and radiographic examination , until root canal obturation was indicated . for establishment of the number of colony forming units ( cfu ) of bacteria , the collected material was plated on chocolate agar ( total bacterial counting ) , mitis salivarius agar ( oral streptococci counting ) and sb20 agar ( mutans streptococci counting ) . the plated petri dishes were incubated in anaerobiosis jars ( microaerophilia ) at 37c for up to 96 s. after the incubation period , the developed colonies were counted according to their macroscopic characteristics , with the aid of a stereomicroscope . the removed pulp samples were processed in an automated histoprocessor ( oma - dm20 ) , in which they were dehydrated with a series of increasing ethanol concentrations , cleared with xylol and immersed in liquid paraffin to obtain blocks . serial 5.0-mm - thick sections were cut with a rotary microtome ( model rm2155 ; leica instruments , germany ) , placed on microscopic glass slides and routinely stained with hematoxylin and eosin ( h.e ) . the tissue sections were analyzed on a trinocular microscope ( axiostar plus , carl zeiss , jena , germany ) , which allowed the investigation of possible morphological changes and related inflammatory processes in the pulp of traumatized teeth . linear regression was used to evaluate the time elapsed from trauma until onset of endodontic intervention , as to the number of microorganisms in the root canal of traumatized teeth , at a significance level of 5% . the diagnostic analysis was adopted to evaluate the sensitivity , specificity and accuracy of the vertical percussion test as to the presence of microorganisms in the root canals of these teeth . the sections stained with h.e . were analyzed subjectively , based on histological study of pulps extirpated after luxation injuries3 , to evaluate the occurrence of collagen hyalinization , presence of blood vessels , cell nuclei and nerve bundles . the study sample was composed of 20 patients from the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois ( brazil ) and approved by the respective institutional review board . the inclusion criteria comprised teeth with history of trauma to the periodontal tissues ( subluxation 8 teeth , extrusive luxation 3 teeth , lateral luxation 2 teeth , and avulsion 7 teeth ) , presenting intact crowns and clinical diagnosis of pulp necrosis . teeth presenting root fracture , periapical lesion , periodontal pocket , resorptions or root canal obliteration were excluded . the following clinical data were recorded : a ) type of trauma ; b ) date of trauma and time elapsed between dental trauma and the onset of endodontic intervention ; c ) crown shade ; d ) response to thermal and electric pulp vitality tests ( pvt ) ( positive or negative ) - ( heat : a piece of heated guttapercha was applied to the center of the buccal aspect of the affected tooth , previously lubricated with petroleum jelly , with cotton roll isolation ; cold : a cold spray at -50c ( endo frost;roeko , langenau , germany ) was applied to the center of the buccal aspect of the affected tooth , with cotton roll isolation ; electric test : this was performed with the endoanalyser device ( kerr corporation , orange , ca , usa ) , placing the electrode tip on the center of the buccal aspect lubricated with fluoride gel , with cotton roll isolation , and recording the response value ) ; e ) response to vertical ( vpt ) and horizontal ( hpt ) percussion tests ( positive or negative ) ; f ) pain upon palpation ( absent or present ) ; g ) symptomatology ( absent or present ) ; h ) mobility ( normal or increased ) . all these clinical data were also recorded for the contralateral teeth , in order to check the response pattern of each patient . radiographic examination was performed with a film holder for adult patients ( indusbelo , londrina , brazil ) and insight radiographic film ( eastman kodak company , rochester ny , usa ) , with the same x - ray machine , to assure constant power and current intensity . the radiographs were analyzed in an indirect light environment , at a different moment than clinical examination . black cardboard masks were fabricated to improve the observation of radiographs on the film viewer . after clinical diagnosis of pulp necrosis based on the association of at least three clinical criteria , the teeth were submitted to endodontic treatment . for each selected tooth , rubber dam was placed and antisepsis was performed with 1% sodium hypochlorite solution ( probem , catanduva , sp , brazil ) . following , microbiological collection of the root canal was performed , based on previous studies17,26 , with the aid of autoclaved absorbent paper points n. 20 ( tanari , manacapuru , am , brazil ) . each point was introduced in the root canal up to the provisional working length with sterile pliers and kept for 30 s , removed and placed in a test tube containing srensen phosphate buffer solution ( pbs ) . the samples were sent to the department of microbiology , immunology , parasitology and pathology of the institute of tropical pathology and community health of the federal university of gois for microbiological processing . when present , the pulp tissue was removed at the provisional working length with hedstrm files ( maillefer , ballaigues , switzerland ) of compatible size with the root canal , which was placed on an autoclaved cardboard and then in a flask containing 10% buffered formalin labeled with the patient number for later laboratory processing , which was performed at the oral pathology laboratory of the dental school of the federal university of gois . each tooth was then prepared according to the protocol of endodontic treatment for traumatized teeth of the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois . the patients were followed at every 3 months by clinical and radiographic examination , until root canal obturation was indicated . for establishment of the number of colony forming units ( cfu ) of bacteria , the collected material was plated on chocolate agar ( total bacterial counting ) , mitis salivarius agar ( oral streptococci counting ) and sb20 agar ( mutans streptococci counting ) . the plated petri dishes were incubated in anaerobiosis jars ( microaerophilia ) at 37c for up to 96 s. after the incubation period , the developed colonies were counted according to their macroscopic characteristics , with the aid of a stereomicroscope . the removed pulp samples were processed in an automated histoprocessor ( oma - dm20 ) , in which they were dehydrated with a series of increasing ethanol concentrations , cleared with xylol and immersed in liquid paraffin to obtain blocks . serial 5.0-mm - thick sections were cut with a rotary microtome ( model rm2155 ; leica instruments , germany ) , placed on microscopic glass slides and routinely stained with hematoxylin and eosin ( h.e ) . the tissue sections were analyzed on a trinocular microscope ( axiostar plus , carl zeiss , jena , germany ) , which allowed the investigation of possible morphological changes and related inflammatory processes in the pulp of traumatized teeth . linear regression was used to evaluate the time elapsed from trauma until onset of endodontic intervention , as to the number of microorganisms in the root canal of traumatized teeth , at a significance level of 5% . the diagnostic analysis was adopted to evaluate the sensitivity , specificity and accuracy of the vertical percussion test as to the presence of microorganisms in the root canals of these teeth . the sections stained with h.e . were analyzed subjectively , based on histological study of pulps extirpated after luxation injuries3 , to evaluate the occurrence of collagen hyalinization , presence of blood vessels , cell nuclei and nerve bundles . among the 20 root canal samples obtained from traumatized teeth , only 3 ( 15% ) did not present microbial development . the following microorganisms were isolated : oral streptococci ( 31% ) , facultative anaerobic bacteria gram - positive cocci , alpha - hemolytic gram - positive cocci and alpha - hemolytic gram - positive bacilli ( 52% ) , staphylococcus sp ( 6.7% ) and sporulated gram - positive bacilli ( 10.3% ) . the time elapsed from the occurrence of dental trauma up to onset of endodontic intervention ranged from 15 days to 31 months ( table 1 ) . this period was not statistically significant ( p=0.591 ) in relation to the number of microorganisms present in the root canal , when all types were analyzed in combination . however , the individual analysis of each type of microorganism revealed that the time elapsed from dental trauma up to endodontic intervention was statistically significant ( p=0.047 ) as to the number of oral streptococci isolated from the root canal , with an increase in this number with the increase in the time period ( figure 1 ) . the vertical percussion test was analyzed as to the presence of microorganisms in traumatized teeth , exhibiting high sensitivity ( 80% ) ( table 2 ) . sensitivity = 80% ; specificity = 0% ; accuracy = 60% ; n : number of teeth . among the 20 traumatized teeth selected for this study , pulp samples from the remaining 17 teeth were analyzed , among which 3 ( 15% ) presented partial necrosis ( figure 2 ) and 14 ( 70% ) exhibited complete necrosis ( figure 3 ) . in the pulps with partial necrosis , despite the evident necrotic and degenerative phenomena in some regions , nerve bundles could be observed in some areas , as well as collagen , cell nuclei and blood vessels , but without chance of regeneration . the pulps with complete necrosis demonstrated partial or complete collagen hyalinization , and absence or advanced degeneration of blood vessels , cell nuclei and nerve bundles . based on the microscopic findings , none of the clinical criteria employed in the present study was pathognomonic . however , the pulp sensitivity tests to heat ( 90% ) and cold ( 85% ) and the vertical percussion test ( 75% ) were more reliable than the others ( table 3 ) . ( - ) : negative response ; ( + ) : positive response ; pvt : pulp vitality test ; vpt : vertical percussion test ; hpt : horizontal percussion test . among the 20 root canal samples obtained from traumatized teeth , only 3 ( 15% ) did not present microbial development . the following microorganisms were isolated : oral streptococci ( 31% ) , facultative anaerobic bacteria gram - positive cocci , alpha - hemolytic gram - positive cocci and alpha - hemolytic gram - positive bacilli ( 52% ) , staphylococcus sp ( 6.7% ) and sporulated gram - positive bacilli ( 10.3% ) . the time elapsed from the occurrence of dental trauma up to onset of endodontic intervention ranged from 15 days to 31 months ( table 1 ) . this period was not statistically significant ( p=0.591 ) in relation to the number of microorganisms present in the root canal , when all types were analyzed in combination . however , the individual analysis of each type of microorganism revealed that the time elapsed from dental trauma up to endodontic intervention was statistically significant ( p=0.047 ) as to the number of oral streptococci isolated from the root canal , with an increase in this number with the increase in the time period ( figure 1 ) . the vertical percussion test was analyzed as to the presence of microorganisms in traumatized teeth , exhibiting high sensitivity ( 80% ) ( table 2 ) . sensitivity = 80% ; specificity = 0% ; accuracy = 60% ; n : number of teeth . among the 20 traumatized teeth selected for this study , 3 did not present pulp tissue , being characterized as complete autolysis . pulp samples from the remaining 17 teeth were analyzed , among which 3 ( 15% ) presented partial necrosis ( figure 2 ) and 14 ( 70% ) exhibited complete necrosis ( figure 3 ) . in the pulps with partial necrosis , despite the evident necrotic and degenerative phenomena in some regions , nerve bundles could be observed in some areas , as well as collagen , cell nuclei and blood vessels , but without chance of regeneration . the pulps with complete necrosis demonstrated partial or complete collagen hyalinization , and absence or advanced degeneration of blood vessels , cell nuclei and nerve bundles . based on the microscopic findings , none of the clinical criteria employed in the present study was pathognomonic . however , the pulp sensitivity tests to heat ( 90% ) and cold ( 85% ) and the vertical percussion test ( 75% ) were more reliable than the others ( table 3 ) . ( - ) : negative response ; ( + ) : positive response ; pvt : pulp vitality test ; vpt : vertical percussion test ; hpt : horizontal percussion test . the present study was conducted on patients with permanent teeth with history of trauma to the periodontal tissues ( subluxation , extrusive luxation , lateral luxation and avulsion ) , with intact crowns and clinical diagnosis of pulp necrosis . the groups did not have similar numbers of samples because the study did not aim to correlate the type of dental trauma with the occurrence of pulp necrosis , but rather to verify if the clinical and radiographic criteria available for this diagnosis in traumatized teeth are concordant with their microbiological and microscopic characteristics . the teeth did not present carious lesions , restorations , fractures or periodontal disorders , which might represent possible sources of microbial invasion in the pulp tissue1,24 . teeth with periapical lesion or resorptions were also excluded because the presence of free microorganisms in the root canal , adhered to the root canal walls and/or penetrating the dentinal tubules has been demonstrated in these cases22,23 . collection of material from traumatized teeth to determine the number of colony forming units of bacteria was performed by sequential introduction of three sterile absorbent paper points in the root canal for 30 s. the procedure was based on studies conducted by engstrom17 and serene and mcdonald26 , in which the most reliable indication for collection with positive culture was obtained by utilization of three paper points for this time period . the development of pulp necrosis in traumatized teeth is determined by three factors : presence of microorganisms , which may impair the processes of revascularization and pulp regeneration14 ; type of trauma , which is associated with the damage to the pulp - periodontal complex ; and the stage of root development , which is related to the repair capacity5,6 . the concussion and subluxation , which cause minimal aggression to the periodontium and the pulp , present limited risk of developing pulp necrosis . conversely , this risk is increased in avulsions and extrusive , lateral and intrusive luxation due to the greater damage to the pulp , periodontal ligament and alveolar bone5,6 . teeth with incompletely formed roots also presented reduced risk to develop pulp necrosis due to the greater possibility of pulp revascularization5,6,14 . among the 20 samples obtained from the root canals of traumatized teeth , 17 ( 85% ) exhibited microbial development . this result is higher than those reported by arwill , et al.9 and bergenholtz11 , who observed microbial growth in traumatized teeth in 28.6% and 64% of cases , respectively , and lower than those of taklan27 and le goff , et al.21 , who found microbial development in traumatized teeth with intact pulp chambers in 100% and 90% of cases , respectively . the time elapsed from dental trauma up to endodontic intervention in the selected teeth was also analyzed , because little is known on when pulp necrosis may be accurately diagnosed in these teeth . the time period between dental trauma and onset of endodontic intervention in the present study ranged from 15 days to 31 months . the teeth indicated for treatment after 15 days involved the avulsion of teeth with completely formed roots , since andreasen4 stated that pulp necrosis is the only outcome expected in these cases . moreover , in 14 teeth ( 70% ) , the clinical diagnosis of pulp necrosis was performed in up to three months . this result agrees with the report of andreasen4 and is close to the findings of jacobsen19 , namely four months . conversely , barkin10 stated that the vitality of traumatized teeth can not be predicted in the first three months . according to andreasen and vestergaard - pedersen6 and andreasen4 , different chronological patterns are observed for different types of trauma ; pulp necrosis may occur after three months or even after two years in cases of lateral and intrusive luxation . analysis of this time length also aimed to investigate the correlation between it and the number of microorganisms present in the root canal . even though bergenholtz11 reported that the number of microorganisms present in the root canal was increased with the increase in the time elapsed from dental trauma up to endodontic intervention , in the present study this increase was not statistically significant as to the total number of isolated microorganisms , except for oral streptococci . this fact may be explained , since rupture of the nerve and blood supply to the pulp after dental trauma may lead to ischemia and thus jeopardize the pulp to microbial invasion . since oral streptococci are predominant and are always present in the oral cavity , root canal colonization by them is facilitated . also , these microorganisms are able to survive in environments with few nutrients20 . some hypotheses have been proposed to explain how the microorganisms may reach the intact pulp chamber of traumatized teeth . this origin would be related to the rupture of blood vessels in the periodontium , allowing the penetration of microorganisms through the gingival sulcus , by the collateral or even the systemic blood circulation18,27 , and through the dentinal tubules in cases of coronal fracture associated or not with luxation11 . this invasion might also occur due to penetration of microorganisms through cracks present in the enamel and dentin of avulsed teeth22 . for these teeth , microorganisms coming from the oral cavity or the root surface , contaminated during the extra - alveolar time , might reach the apical region of the replanted tooth through the blood clot present in the alveolus before replantation or formed during the initial healing periods , since the blood clot is an excellent substrate for microbial proliferation15 . the present results revealed that 85% of pulps submitted to light microscopy analysis presented necrosis , being 15% with partial necrosis without possibility of repair and 70% with complete necrosis . the remaining 3 cases ( 15% ) exhibited complete autolysis , adding up to 100% of nonvital teeth . these results disagree with the findings of arwill , et al.9 , who investigated a sample of 25 pulps removed from teeth with clinical diagnosis of pulp necrosis after trauma and observed that only 56% were really necrotic on microscopic examination . cipriano and walton12 , in turn , did not confirm microscopically the clinical diagnosis of pulp necrosis after trauma in 88% of analyzed teeth . also , andreasen2 conducted microscopic evaluation of a sample of 66 teeth with clinical diagnosis of pulp necrosis and observed that 9 to 14% of removed pulps still presented possibility of repair and should not have been removed . this difference between the present results and the reports in the literature2,9,17 could be first assigned to the year of accomplishment of studies , considering the limitation of diagnostic methods available at the time for establishment of pulp necrosis after trauma , as well as the utilization of isolated clinical criteria adopted to diagnose pulp necrosis in the most recent studies . in the present study , the criteria adopted for clinical diagnosis of pulp necrosis comprised discoloration of the crown , negative response to thermal and electric pulp vitality tests , positive response to vertical and horizontal percussion tests , pain upon palpation and increased mobility5,6,19 . the clinical diagnosis of pulp necrosis of traumatized teeth was only achieved if at least three of these clinical criteria were present . none of the criteria was pathognomonic ( 100% ) to diagnose pulp necrosis in traumatized teeth . however , the heat ( 90% ) , cold ( 85% ) and vertical percussion ( 75% ) pulp vitality tests were more reliable than the others , also when compared to the electric test ( 50% ) . peterson , et al.25 also observed the superior outcomes of thermal pulp vitality tests ( 87.5% ) compared to the electric test ( 62.5% ) in the diagnosis of pulp necrosis . the last test has also been associated with false - negative responses in traumatized teeth , because it stimulates the nerve fibers in the pulp to produce response , yet it does not provide information on the blood supply of the pulp . since the blood supply presents faster reestablishment than the nerve supply of the pulp after dental trauma , the tooth may be vital yet insensitive13,19 . however , this is still an important and non - subjective clinical criterion , because it provides a value that may be monitored . discoloration of the crown after dental trauma should not be expected as a definite sign of pulp necrosis , yet it should be associated with the other clinical criteria for diagnosis5,13 . the vertical percussion test has been a clinical criterion with simple accomplishment and good results . previous studies revealed that teeth with infected necrotic pulps exhibited higher sensitivity to percussion when compared to teeth with non - infected necrotic pulps , indicating that a positive response to the vertical percussion test would be associated with pulp necrosis with presence of microorganisms3,5 . this finding was confirmed in the present study , in which this test exhibited high sensitivity ( 80% ) for detection of microorganisms in the root canals of traumatized teeth . in summary , even though the inclusion and exclusion criteria determined a limited sample ( 20 cases ) , the present study demonstrated that complete and careful follow up of the traumatized tooth , by the association of at least three clinical tests well performed and interpreted , may determine a more accurate diagnosis of pulp necrosis , as demonstrated by the microscopic and microbiological findings . notwithstanding , the present results agree with those of other authors who state that full understanding of pulp necrosis occurring after trauma requires further microscopic and microbiological studies on larger samples and using standardized methodologies . under the tested conditions and within the limitations of the employed methodology , the following conclusions may be drawn : 1 . regarding the microbiological results , 85% of teeth presented microorganisms in the root canal , despite the presence of intact crowns ; 2 . regarding the microscopic findings , 100% of traumatized teeth exhibited pulp necrosis ; 3 . the pulp vitality tests to heat , cold and vertical percussion were the most reliable to diagnose pulp necrosis in traumatized teeth .

objective : this study evaluated the presence of microorganisms and analyzed microscopically the pulp of 20 traumatized human teeth with intact crowns and clinical diagnosis of pulp necrosis , based on the association of at least three of the clinical criteria : crown discoloration , negative response to thermal and electric pulp vitality tests , positive response to vertical and horizontal percussion , pain on palpation or mobility.material and methods : microbiological collection was performed from the root canals to evaluate the presence of microorganisms . the pulp samples were stained with hematoxylin and eosin ( h.e . ) for histological evaluation of possible morphological alterations.results:analysis of results was performed by statistical tests ( linear regression test and diagnostic analysis ) and subjective analysis of the sections stained with h.e . and revealed that only 15% of the sample did not exhibit microbial development . the time elapsed between dental trauma and onset of endodontic intervention ranged from 15 days to 31 months ; the percussion test presented high sensitivity ( 80% ) for detection of microorganisms in the root canal of traumatized teeth ; 3 teeth ( 15% ) did not present pulp tissue , being characterized as complete autolysis ; analysis of pulp samples was performed on the other 17 cases , among which 3 ( 15% ) exhibited partial necrosis without possibility of repair and 14 presented complete necrosis ; none of the clinical criteria employed for the diagnosis of pulp necrosis in traumatized teeth was pathognomonic.conclusions:the present results allowed the following conclusions : with regard to microbiological findings , 85% of teeth presented microorganisms in the root canal , despite the presence of an intact crown . concerning the microscopic findings , 100% of traumatized teeth presented pulp necrosis ; the pulp vitality tests based on pulp response to heat , cold and vertical percussion were the most reliable to diagnose pulp necrosis in traumatized teeth .

INTRODUCTION MATERIAL AND METHODS Clinical Procedures Microbiological and Microscopic Processing Analysis of Results RESULTS Microbiological Evaluation Microscopic Analysis DISCUSSION CONCLUSIONS

some criteria have been suggested to diagnose pulp necrosis in traumatized teeth , such as the negative response to pulp vitality tests , progressive discoloration of the crown , periapical radiolucency , interruption of root formation or positive response to the vertical percussion test6 . the thermal pulp vitality tests to heat and cold and electric tests are not completely reliable for detection of pulp necrosis25 . discoloration of the tooth crown is also not pathognomonic of pulp necrosis , because pulp hemorrhage after trauma might lead to the overflow of erythrocytes and byproducts of their decomposition to the surrounding tissue , making the tooth crown pinkish or grayish . a histobacteriological assessment of removed pulps with clinical diagnosis of pulp necrosis after dental trauma revealed that 9% to 14% of these pulps presented potential for repair , and thus they should not have been removed3 . the presence of microorganisms in the root canal system should also be evaluated , which may definitely impair the processes of revascularization and regeneration of the ischemic pulp tissue after luxation14 . previous studies demonstrated that , in traumatized teeth with intact pulp chambers , the necrotic pulp may be either infected or free of microorganisms for a long time , which raises questions on the actual need of endodontic intervention9,11,21,27 . the purpose of the present study was to evaluate the presence of microorganisms and analyze microscopically the pulp of traumatized human permanent teeth with intact crowns and clinical diagnosis of pulp necrosis . the study sample was composed of 20 patients from the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois ( brazil ) and approved by the respective institutional review board . the inclusion criteria comprised teeth with history of trauma to the periodontal tissues ( subluxation 8 teeth , extrusive luxation 3 teeth , lateral luxation 2 teeth , and avulsion 7 teeth ) , presenting intact crowns and clinical diagnosis of pulp necrosis . the following clinical data were recorded : a ) type of trauma ; b ) date of trauma and time elapsed between dental trauma and the onset of endodontic intervention ; c ) crown shade ; d ) response to thermal and electric pulp vitality tests ( pvt ) ( positive or negative ) - ( heat : a piece of heated guttapercha was applied to the center of the buccal aspect of the affected tooth , previously lubricated with petroleum jelly , with cotton roll isolation ; cold : a cold spray at -50c ( endo frost;roeko , langenau , germany ) was applied to the center of the buccal aspect of the affected tooth , with cotton roll isolation ; electric test : this was performed with the endoanalyser device ( kerr corporation , orange , ca , usa ) , placing the electrode tip on the center of the buccal aspect lubricated with fluoride gel , with cotton roll isolation , and recording the response value ) ; e ) response to vertical ( vpt ) and horizontal ( hpt ) percussion tests ( positive or negative ) ; f ) pain upon palpation ( absent or present ) ; g ) symptomatology ( absent or present ) ; h ) mobility ( normal or increased ) . after clinical diagnosis of pulp necrosis based on the association of at least three clinical criteria , the teeth were submitted to endodontic treatment . following , microbiological collection of the root canal was performed , based on previous studies17,26 , with the aid of autoclaved absorbent paper points n. 20 ( tanari , manacapuru , am , brazil ) . when present , the pulp tissue was removed at the provisional working length with hedstrm files ( maillefer , ballaigues , switzerland ) of compatible size with the root canal , which was placed on an autoclaved cardboard and then in a flask containing 10% buffered formalin labeled with the patient number for later laboratory processing , which was performed at the oral pathology laboratory of the dental school of the federal university of gois . serial 5.0-mm - thick sections were cut with a rotary microtome ( model rm2155 ; leica instruments , germany ) , placed on microscopic glass slides and routinely stained with hematoxylin and eosin ( h.e ) . the tissue sections were analyzed on a trinocular microscope ( axiostar plus , carl zeiss , jena , germany ) , which allowed the investigation of possible morphological changes and related inflammatory processes in the pulp of traumatized teeth . linear regression was used to evaluate the time elapsed from trauma until onset of endodontic intervention , as to the number of microorganisms in the root canal of traumatized teeth , at a significance level of 5% . the diagnostic analysis was adopted to evaluate the sensitivity , specificity and accuracy of the vertical percussion test as to the presence of microorganisms in the root canals of these teeth . the sections stained with h.e . were analyzed subjectively , based on histological study of pulps extirpated after luxation injuries3 , to evaluate the occurrence of collagen hyalinization , presence of blood vessels , cell nuclei and nerve bundles . the study sample was composed of 20 patients from the research and extension project " teeth should be in the mouth " , conducted by the dental school of the federal university of gois ( brazil ) and approved by the respective institutional review board . the inclusion criteria comprised teeth with history of trauma to the periodontal tissues ( subluxation 8 teeth , extrusive luxation 3 teeth , lateral luxation 2 teeth , and avulsion 7 teeth ) , presenting intact crowns and clinical diagnosis of pulp necrosis . the following clinical data were recorded : a ) type of trauma ; b ) date of trauma and time elapsed between dental trauma and the onset of endodontic intervention ; c ) crown shade ; d ) response to thermal and electric pulp vitality tests ( pvt ) ( positive or negative ) - ( heat : a piece of heated guttapercha was applied to the center of the buccal aspect of the affected tooth , previously lubricated with petroleum jelly , with cotton roll isolation ; cold : a cold spray at -50c ( endo frost;roeko , langenau , germany ) was applied to the center of the buccal aspect of the affected tooth , with cotton roll isolation ; electric test : this was performed with the endoanalyser device ( kerr corporation , orange , ca , usa ) , placing the electrode tip on the center of the buccal aspect lubricated with fluoride gel , with cotton roll isolation , and recording the response value ) ; e ) response to vertical ( vpt ) and horizontal ( hpt ) percussion tests ( positive or negative ) ; f ) pain upon palpation ( absent or present ) ; g ) symptomatology ( absent or present ) ; h ) mobility ( normal or increased ) . after clinical diagnosis of pulp necrosis based on the association of at least three clinical criteria , the teeth were submitted to endodontic treatment . following , microbiological collection of the root canal was performed , based on previous studies17,26 , with the aid of autoclaved absorbent paper points n. 20 ( tanari , manacapuru , am , brazil ) . when present , the pulp tissue was removed at the provisional working length with hedstrm files ( maillefer , ballaigues , switzerland ) of compatible size with the root canal , which was placed on an autoclaved cardboard and then in a flask containing 10% buffered formalin labeled with the patient number for later laboratory processing , which was performed at the oral pathology laboratory of the dental school of the federal university of gois . serial 5.0-mm - thick sections were cut with a rotary microtome ( model rm2155 ; leica instruments , germany ) , placed on microscopic glass slides and routinely stained with hematoxylin and eosin ( h.e ) . the tissue sections were analyzed on a trinocular microscope ( axiostar plus , carl zeiss , jena , germany ) , which allowed the investigation of possible morphological changes and related inflammatory processes in the pulp of traumatized teeth . linear regression was used to evaluate the time elapsed from trauma until onset of endodontic intervention , as to the number of microorganisms in the root canal of traumatized teeth , at a significance level of 5% . the diagnostic analysis was adopted to evaluate the sensitivity , specificity and accuracy of the vertical percussion test as to the presence of microorganisms in the root canals of these teeth . the sections stained with h.e . were analyzed subjectively , based on histological study of pulps extirpated after luxation injuries3 , to evaluate the occurrence of collagen hyalinization , presence of blood vessels , cell nuclei and nerve bundles . among the 20 root canal samples obtained from traumatized teeth , only 3 ( 15% ) did not present microbial development . the time elapsed from the occurrence of dental trauma up to onset of endodontic intervention ranged from 15 days to 31 months ( table 1 ) . this period was not statistically significant ( p=0.591 ) in relation to the number of microorganisms present in the root canal , when all types were analyzed in combination . however , the individual analysis of each type of microorganism revealed that the time elapsed from dental trauma up to endodontic intervention was statistically significant ( p=0.047 ) as to the number of oral streptococci isolated from the root canal , with an increase in this number with the increase in the time period ( figure 1 ) . the vertical percussion test was analyzed as to the presence of microorganisms in traumatized teeth , exhibiting high sensitivity ( 80% ) ( table 2 ) . among the 20 traumatized teeth selected for this study , pulp samples from the remaining 17 teeth were analyzed , among which 3 ( 15% ) presented partial necrosis ( figure 2 ) and 14 ( 70% ) exhibited complete necrosis ( figure 3 ) . in the pulps with partial necrosis , despite the evident necrotic and degenerative phenomena in some regions , nerve bundles could be observed in some areas , as well as collagen , cell nuclei and blood vessels , but without chance of regeneration . based on the microscopic findings , none of the clinical criteria employed in the present study was pathognomonic . however , the pulp sensitivity tests to heat ( 90% ) and cold ( 85% ) and the vertical percussion test ( 75% ) were more reliable than the others ( table 3 ) . ( - ) : negative response ; ( + ) : positive response ; pvt : pulp vitality test ; vpt : vertical percussion test ; hpt : horizontal percussion test . among the 20 root canal samples obtained from traumatized teeth , only 3 ( 15% ) did not present microbial development . the time elapsed from the occurrence of dental trauma up to onset of endodontic intervention ranged from 15 days to 31 months ( table 1 ) . this period was not statistically significant ( p=0.591 ) in relation to the number of microorganisms present in the root canal , when all types were analyzed in combination . however , the individual analysis of each type of microorganism revealed that the time elapsed from dental trauma up to endodontic intervention was statistically significant ( p=0.047 ) as to the number of oral streptococci isolated from the root canal , with an increase in this number with the increase in the time period ( figure 1 ) . the vertical percussion test was analyzed as to the presence of microorganisms in traumatized teeth , exhibiting high sensitivity ( 80% ) ( table 2 ) . among the 20 traumatized teeth selected for this study , 3 did not present pulp tissue , being characterized as complete autolysis . pulp samples from the remaining 17 teeth were analyzed , among which 3 ( 15% ) presented partial necrosis ( figure 2 ) and 14 ( 70% ) exhibited complete necrosis ( figure 3 ) . in the pulps with partial necrosis , despite the evident necrotic and degenerative phenomena in some regions , nerve bundles could be observed in some areas , as well as collagen , cell nuclei and blood vessels , but without chance of regeneration . based on the microscopic findings , none of the clinical criteria employed in the present study was pathognomonic . however , the pulp sensitivity tests to heat ( 90% ) and cold ( 85% ) and the vertical percussion test ( 75% ) were more reliable than the others ( table 3 ) . ( - ) : negative response ; ( + ) : positive response ; pvt : pulp vitality test ; vpt : vertical percussion test ; hpt : horizontal percussion test . the present study was conducted on patients with permanent teeth with history of trauma to the periodontal tissues ( subluxation , extrusive luxation , lateral luxation and avulsion ) , with intact crowns and clinical diagnosis of pulp necrosis . the groups did not have similar numbers of samples because the study did not aim to correlate the type of dental trauma with the occurrence of pulp necrosis , but rather to verify if the clinical and radiographic criteria available for this diagnosis in traumatized teeth are concordant with their microbiological and microscopic characteristics . the teeth did not present carious lesions , restorations , fractures or periodontal disorders , which might represent possible sources of microbial invasion in the pulp tissue1,24 . teeth with periapical lesion or resorptions were also excluded because the presence of free microorganisms in the root canal , adhered to the root canal walls and/or penetrating the dentinal tubules has been demonstrated in these cases22,23 . collection of material from traumatized teeth to determine the number of colony forming units of bacteria was performed by sequential introduction of three sterile absorbent paper points in the root canal for 30 s. the procedure was based on studies conducted by engstrom17 and serene and mcdonald26 , in which the most reliable indication for collection with positive culture was obtained by utilization of three paper points for this time period . the development of pulp necrosis in traumatized teeth is determined by three factors : presence of microorganisms , which may impair the processes of revascularization and pulp regeneration14 ; type of trauma , which is associated with the damage to the pulp - periodontal complex ; and the stage of root development , which is related to the repair capacity5,6 . among the 20 samples obtained from the root canals of traumatized teeth , 17 ( 85% ) exhibited microbial development . this result is higher than those reported by arwill , et al.9 and bergenholtz11 , who observed microbial growth in traumatized teeth in 28.6% and 64% of cases , respectively , and lower than those of taklan27 and le goff , et al.21 , who found microbial development in traumatized teeth with intact pulp chambers in 100% and 90% of cases , respectively . the time elapsed from dental trauma up to endodontic intervention in the selected teeth was also analyzed , because little is known on when pulp necrosis may be accurately diagnosed in these teeth . the time period between dental trauma and onset of endodontic intervention in the present study ranged from 15 days to 31 months . moreover , in 14 teeth ( 70% ) , the clinical diagnosis of pulp necrosis was performed in up to three months . analysis of this time length also aimed to investigate the correlation between it and the number of microorganisms present in the root canal . even though bergenholtz11 reported that the number of microorganisms present in the root canal was increased with the increase in the time elapsed from dental trauma up to endodontic intervention , in the present study this increase was not statistically significant as to the total number of isolated microorganisms , except for oral streptococci . for these teeth , microorganisms coming from the oral cavity or the root surface , contaminated during the extra - alveolar time , might reach the apical region of the replanted tooth through the blood clot present in the alveolus before replantation or formed during the initial healing periods , since the blood clot is an excellent substrate for microbial proliferation15 . the present results revealed that 85% of pulps submitted to light microscopy analysis presented necrosis , being 15% with partial necrosis without possibility of repair and 70% with complete necrosis . the remaining 3 cases ( 15% ) exhibited complete autolysis , adding up to 100% of nonvital teeth . these results disagree with the findings of arwill , et al.9 , who investigated a sample of 25 pulps removed from teeth with clinical diagnosis of pulp necrosis after trauma and observed that only 56% were really necrotic on microscopic examination . cipriano and walton12 , in turn , did not confirm microscopically the clinical diagnosis of pulp necrosis after trauma in 88% of analyzed teeth . also , andreasen2 conducted microscopic evaluation of a sample of 66 teeth with clinical diagnosis of pulp necrosis and observed that 9 to 14% of removed pulps still presented possibility of repair and should not have been removed . this difference between the present results and the reports in the literature2,9,17 could be first assigned to the year of accomplishment of studies , considering the limitation of diagnostic methods available at the time for establishment of pulp necrosis after trauma , as well as the utilization of isolated clinical criteria adopted to diagnose pulp necrosis in the most recent studies . in the present study , the criteria adopted for clinical diagnosis of pulp necrosis comprised discoloration of the crown , negative response to thermal and electric pulp vitality tests , positive response to vertical and horizontal percussion tests , pain upon palpation and increased mobility5,6,19 . the clinical diagnosis of pulp necrosis of traumatized teeth was only achieved if at least three of these clinical criteria were present . none of the criteria was pathognomonic ( 100% ) to diagnose pulp necrosis in traumatized teeth . however , the heat ( 90% ) , cold ( 85% ) and vertical percussion ( 75% ) pulp vitality tests were more reliable than the others , also when compared to the electric test ( 50% ) . peterson , et al.25 also observed the superior outcomes of thermal pulp vitality tests ( 87.5% ) compared to the electric test ( 62.5% ) in the diagnosis of pulp necrosis . the last test has also been associated with false - negative responses in traumatized teeth , because it stimulates the nerve fibers in the pulp to produce response , yet it does not provide information on the blood supply of the pulp . discoloration of the crown after dental trauma should not be expected as a definite sign of pulp necrosis , yet it should be associated with the other clinical criteria for diagnosis5,13 . previous studies revealed that teeth with infected necrotic pulps exhibited higher sensitivity to percussion when compared to teeth with non - infected necrotic pulps , indicating that a positive response to the vertical percussion test would be associated with pulp necrosis with presence of microorganisms3,5 . this finding was confirmed in the present study , in which this test exhibited high sensitivity ( 80% ) for detection of microorganisms in the root canals of traumatized teeth . in summary , even though the inclusion and exclusion criteria determined a limited sample ( 20 cases ) , the present study demonstrated that complete and careful follow up of the traumatized tooth , by the association of at least three clinical tests well performed and interpreted , may determine a more accurate diagnosis of pulp necrosis , as demonstrated by the microscopic and microbiological findings . regarding the microbiological results , 85% of teeth presented microorganisms in the root canal , despite the presence of intact crowns ; 2 . regarding the microscopic findings , 100% of traumatized teeth exhibited pulp necrosis ; 3 . the pulp vitality tests to heat , cold and vertical percussion were the most reliable to diagnose pulp necrosis in traumatized teeth .

dwi has been primarily applied in acute brain ischaemia , which is characterised in its early phase by cytotoxic oedema . thanks to its sensitivity in detecting hyperacute ischaemic strokes , dwi has gained a primary role in routine brain mr protocols when dealing with acute neurological syndromes . the frequent utilisation of dwi has unexpectedly revealed peculiar signal abnormalities in several other cerebral diseases , such as abscesses , tumour and head trauma , widening even further the spectrum of brain diseases where this sequence helps to address the diagnosis . the reduction of the apparent diffusion coefficient ( adc ) values depends on the disease studied , and it might involve an increase in cell debris , cellularity , intramyelin and axonal oedema . the neuroradiologist is often challenged by neurologists and paediatricians with neuroimaging studies that document white matter lesions characterised by adc restriction . in this pictorial we focused on different conditions ( pertaining to adult or child neurology ) other than territorial ischaemic stroke where dwi signal abnormalities and adc value reduction are confined within the white matter . the dw images throughout the study were performed with a b value of 800 or 1,000 s / mm . x - linked charcot - marie - tooth disease ( cmtx ) is the second most common form of inherited motor and sensory demyelinating neuropathy next to the cmt type 1a ; it accounts for approximately 1020% of all hereditary demyelinating neuropathies . it is associated with different mutations in the protein connexin 32 ( cx32 ) , a gap junction protein widely expressed in schwann cells , oligodendrocytes and neurons [ 1 , 2 ] . even if this disease is considered a disorder of the peripheral nervous system , some cx32-mutated subjects might present with acute onset of central nervous system dysfunctions such as hemiparesis , ataxia or aphasia associated with transient white matter changes in the brain . in particular , neuroradiological mr findings in the acute phase show symmetrical , largely confluent cerebral deep white matter signal abnormalities , predominantly in the posterior centrum semiovale , the posterior limb of the internal capsule , the splenium of the corpus callosum ( fig . 1 ) and sometimes in the middle cerebellar peduncles [ 13 ] . bilateral t2 and dwi signal abnormalities might disappear within 1 week or might persist for several months , unrelated to the clinical picture that often appears clearly asymmetric and usually improves rapidly.fig . 1a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas as adc reduction might reverse after a few months , signal abnormalities can not be interpreted as simple cytotoxic oedema , which usually lasts less than 2 weeks , while the increase in the magnetisation transfer ratios is not consistent with demyelination or oedema . as gap junction dysfunctions may disrupt the diffusion of small molecules and ions across myelin sheaths , the more likely explanation of the reversible white matter lesions remains myelin splitting and intra - myelin oedema with compression of the extracellular spaces . maple syrup urine disease ( msud ) is a rare autosomic recessive disorder caused by a deficiency of the branched - chain -keto acid dehydrogenase mitochondrial multi - enzyme complex . a sudden metabolic decompensation with encephalopathy occurs spontaneously or in concomitance with minor illnesses ; high concentrations of upstream metabolites are thought to exert the neurotoxic effect . clinical presentation and outcome are heterogeneous ; in the classical phenotype the prognosis is very poor , and untreated surviving children are left with psychomotor retardation , spasticity , generalised dystonia and cerebral blindness . in the classical neonatal form , which accounts for about 80% of cases , brain mri may provide a highly suggestive pattern of lesions with a generalised cerebral oedema and a striking increase in dwi and t2 signal intensity in all areas that are already myelinated at birth ( fig . 2 ) . these peculiar histological changes are thought to explain the observed reduction in adc values and its reversibility . early diagnosis and treatment may normalise clinical and neuroradiological abnormalities , although inter - current illnesses throughout life may lead to further metabolic derangements .fig . 2diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . the myelinated areas shown as symmetrical , markedly high intensity signal on a rather homogeneous brain parenchyma depict the cortico - spinal tract ( a , b ) and the cerebello - pontine white matter ( c ) . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . the myelinated areas shown as symmetrical , markedly high intensity signal on a rather homogeneous brain parenchyma depict the cortico - spinal tract ( a , b ) and the cerebello - pontine white matter ( c ) . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) menkes disease is an x - linked disorder caused by defective copper - transporting adenosine triphosphatase , which results in low levels of intracellular copper . male children are normal in the first 24 months of life , and then present with loss of developmental milestones , hypotonia , seizures and failure to thrive ; these patients usually have short , hypopigmented , kinked hair ; death often occurs early in childhood . brain mri might disclose diffuse brain atrophy , subdural effusion , or haematoma and vascular tortuosity . asymmetrical localised white matter lesions involving lobar white matter have been described [ 5 , 6 ] . recently , lee et al . reported that white matter lesions are bright on dwi and present decreased adc values consistent with cytotoxic oedema ( fig . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . adc maps ( b ) of the lesions showed low intensity signal an 8-month - old child affected by menkes disease . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . adc maps ( b ) of the lesions showed low intensity signal as in cmt and msud disease , the pathogenesis of these findings is uncertain . according to hsich et al . , the tortuosity of the vessels might produce erratic and turbulent blood flow , thus predisposing to deep white matter ischaemia . alternatively , brain tissue abnormalities may be related to a deficiency in atp7a mitochondrial copper - containing enzyme with consequent energy failure . glutaric aciduria type 1 ( ga-1 ) is a rare autosomal recessive disorder caused by deficiency of the mitochondrial enzyme glutaryl coa dehydrogenase , necessary for the metabolism of three essential amino acids : lysine , hydroxylysine and tryptophan . the metabolic blocking of these amino acids results in increased levels of glutaric acid and 3-hydroxy glutaric acid . because excessive glutaric acid is probably toxic to the striatal cells , atrophy in these nuclei occurs with age . patients may be asymptomatic or present with acute metabolic decompensation and striatal necrosis , which often leads to severe dyskinesia and cognitive impairment . magnetic resonance imaging findings in ga-1 include wide cerebrospinal fluid spaces anterior to the temporal lobes and within the sylvian fissure , subdural collections and brain atrophy . oedema and increased t2 signal intensity within both caudate heads and lentiform nuclei are seen during metabolic derangement followed by necrosis and shrinking of these structures . in older children as well as in asymptomatic patients , a peculiar and persistent dwi hyperintensity of white matter with a peculiar strip - like involvement of the corpus callosum has been noted . 4axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . ( b ) revealed mildly reduced values in the affected areas axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . adc maps ( b ) revealed mildly reduced values in the affected areas no convincing , definitive explanation has yet been found for the long persistence of these signal abnormalities . phenylketonuria ( pku ) is a severe autosomal recessive disorder due to phenylalanine hydroxylase deficiency [ 4 , 12 , 13 ] that strongly benefits from early diagnosis and dietary treatment . brain mr examination of adolescents and adults discloses white matter abnormalities both in untreated pku patients and those treated early . typically , white matter lesions are occipital - parietal periventricular signal abnormalities on t2 sequences and are related to phenylalanine blood concentrations without significant correlation with the neurological impairment . recently , restricted diffusion of water molecules has been reported in the areas of white matter abnormalities [ 4 , 12 , 14 , 15 ] , appearing hyperintense on dwi in an otherwise rather homogeneously grey brain parenchyma ( fig . these findings have been interpreted as cytotoxic oedema because of a reduction in the na+k+-atpase activity or alternatively as dysmyelination with intra - myelin oedema , but the long persistence of the signal abnormalities does not support these hypotheses .fig . 5magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol / l n.v . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) watershed infarctions account for 0.73.2% of all strokes and may be confined to the deep white matter . in the acute phase , mr examination may reveal peri - ventricular round foci of restricted diffusion , with a string of pearls appearance within the deep white matter ( fig . 6 ) . despite a multi - focal bilateral appearance , all these lesions functionally represent a single vascular territory between superficial branches and deep perforators of cerebral arteries . this territory is particularly vulnerable in conditions of severely impaired cerebral blood flow such as prolonged hypotensive events , cardiac arrest with resuscitation and moyamoya disease.fig . 6a 53-year - old male patient affected by dissection of the left internal carotid artery . axial diffusion ( a ) and t2-weighted images ( c ) revealed hyperintense foci in the subcortical watershed territories in a characteristic parasagittal chain - like distribution ( arrows ) . adc maps disclosed the cytotoxic nature of the lesions ( b ) a 53-year - old male patient affected by dissection of the left internal carotid artery . axial diffusion ( a ) and t2-weighted images ( c ) revealed hyperintense foci in the subcortical watershed territories in a characteristic parasagittal chain - like distribution ( arrows ) . adc maps disclosed the cytotoxic nature of the lesions ( b ) on mri lesions might also be unilateral in the case of an ipsilateral internal carotid artery occlusion or tight stenosis or main intracranial artery stenosis , especially when intracranial collateral flow is not compensatory ( e.g. because of the incompleteness of the circle of willis ) . contrary to patients with territorial infarction , patients with wi often do have a history of transient ischaemic attacks , generally have better outcomes , and usually do not benefit from thrombolysis . grey matter is known to be more vulnerable to diffuse hypoxic - ischaemic injury than neighbouring white matter , but recent reports have highlighted that white matter is more sensitive to ischaemia than previously thought and may be primarily involved , sometimes in an isolated fashion [ 19 , 20 ] . global hypoxic - ischaemic encephalopathy of the adult is generally secondary to prolonged cardiac arrest , cardiac arrhythmias , drug overdose , respiratory failure or carbon monoxide poisoning . even though prominent symmetrical , restricted diffusion abnormalities ( fig . 7 ) have been described in the early stages of diffuse hypoxic - ischaemic injury , white matter abnormalities typically appear in the late - subacute period ( 1420 days ) and may normalise thereafter.fig . 7magnetic resonance images obtained during the subacute stage of co intoxication in a 47-year - old man with a history of heroin abuse found unconscious at home . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . on adc maps ( b ) , as co intoxication and heroin - associated spongiform leukoencephalopathy may produce similar mri findings , both events could have resulted in this peculiar mr picture in our patient magnetic resonance images obtained during the subacute stage of co intoxication in a 47-year - old man with a history of heroin abuse found unconscious at home . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . on adc maps ( b ) , as co intoxication and heroin - associated spongiform leukoencephalopathy may produce similar mri findings , both events could have resulted in this peculiar mr picture in our patient histologically , diffuse white matter abnormalities on mr imaging have correlated with deep white matter vacuolar degeneration and with adjacent axonal injury in normal - appearing white matter . pontine and extrapontine myelinolysis is a rare condition elicited by hypo- or hyper - natraemia , especially when an inappropriate ( i.e. too fast ) correction occurs . magnetic resonance imaging might show a characteristic lesion , namely a trident - shaped signal change within the pons with sparing of the tegmentum ( pontine myelinolysis ) or symmetrical bilateral signal changes of the middle cerebellar peduncles , of the cortico - spinal tract and of the splenium of the corpus callosum ( extrapontine myelinolysis ) ( fig . in fact , several case reports of pontine stroke present with signal abnormalities in the middle cerebellar peduncles . however , in wallerian degeneration signal abnormalities are usually fainter compared with those of extrapontine myelinolysis . furthermore , an associated pontine lesion is the key to a definitive diagnosis of wallerian degeneration of the ponto - cerebellar tracts .fig . 8axial diffusion - weighted ( a ) , adc maps ( b ) and t2-weighted ( c ) images at the level of the pons ( left images ) and basal ganglia ( right images ) obtained in a 54-year - old man who developed impaired consciousness after rapid correction of severe hyponatraemia . diffusion - weighted and t2-weighted images revealed white matter hyperintensities that were more evident in the middle cerebellar peduncles ( arrows ) and in the cortico - spinal tract ( arrowheads ) consistent with osmotic extrapontine myelinolysis . adc values were mildly decreased axial diffusion - weighted ( a ) , adc maps ( b ) and t2-weighted ( c ) images at the level of the pons ( left images ) and basal ganglia ( right images ) obtained in a 54-year - old man who developed impaired consciousness after rapid correction of severe hyponatraemia . diffusion - weighted and t2-weighted images revealed white matter hyperintensities that were more evident in the middle cerebellar peduncles ( arrows ) and in the cortico - spinal tract ( arrowheads ) consistent with osmotic extrapontine myelinolysis . adc values were mildly decreased despite the term myelinolysis , several cases of bilateral basal ganglia involvement have been reported . dwi may show a variable decrease in adc values , which probably depends on the timing of the mr examination and on the severity of white matter involvement . following an initial stage of diffuse encephalopathy , the neurological sequelae are usually characterised by diffuse cognitive deficits and extrapyramidal or cortico - bulbo - spinal signs ; however , a complete remission of neurological symptoms can also be achieved . variable t2 hyperintense lesions with increased adc values are usually found on follow - up examinations , although complete regression of the mr lesions is possible . methotrexate ( mtx ) is a cycle - specific antimetabolite that inhibits the enzyme dihydrofolate reductase and is used for the treatment of oncological disorders . mtx crosses the blood - brain barrier and can be administered intravenously as well as intrathecally to eradicate leukaemic cells from the cns and to prevent cns recurrence in acute lymphocytic leukaemia . acute mtx neurotoxicity has been described in 310% of cases and varies with dose , frequency and route of administration [ 2729 ] . the temporal relationship between acute mtx neurotoxicity and induction ranges from 2 to 127 weeks , but it is most often seen 1011 days after intrathecal administrations of mtx . from a clinical point of view acute mtx neurotoxicity may mimic a stroke and present with aphasia or hemiparesis . conventional mr imaging shows bilateral t2 hyperintense lesions within the deep fronto - parietal white matter . 9 ) and reveals a variable decrease of adc values probably due to the derangement of folate and homocysteine metabolism . dwi abnormalities and clinical symptoms usually resolve within a few days even when t2 abnormalities persist.fig . 9this 14-year - old girl affected by acute lymphocytic leukaemia and treated with intrathecal mtx acutely developed hemiparesis and aphasia . axial diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) showed bilateral t2 hyperintense lesions within the deep fronto - parietal white matter ( arrows ) . adc maps ( b ) presented reduced diffusivity . the neurological deficits and the dwi / adc abnormalities resolved completely within 1 week , while slight t2 hyperintensities persisted on follow - up examinations this 14-year - old girl affected by acute lymphocytic leukaemia and treated with intrathecal mtx acutely developed hemiparesis and aphasia . axial diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) showed bilateral t2 hyperintense lesions within the deep fronto - parietal white matter ( arrows ) . the neurological deficits and the dwi / adc abnormalities resolved completely within 1 week , while slight t2 hyperintensities persisted on follow - up examinations inhalation of heroin vapour , a practice known as chasing the dragon the pathophysiological mechanism seems to be selectively activated by substances originating during the heating of heroin . symptoms usually occur within some days after the last heroin consumption and are first dominated by cerebellar deficits . characteristically , heroin - induced leukoencephalopathy appears as symmetrical t2 and dwi hyperintense lesions involving the cerebellar white matter , the posterior limbs of the internal capsule , the splenium of the corpus callosum and the posterior cerebral white matter . a decrease in the adc values and the absence of contrast enhancement further characterise the affected areas , whereas a spongiform demyelination is considered the histological substrate . clinical and neuroradiological pictures may slowly improve if the patients are treated promptly ; otherwise manifest severe neurological impairment and death occur . diffuse axonal injury ( dai ) accounts for 50% of all primary intra - axial traumatic brain injuries . these lesions can be both haemorrhagic and non - haemorrhagic , and are caused by differential linear and rotational accelerations of the cortex in relation to the underlying deep brain structures . besides multi - focal punctuate haemorrhages at the fronto - temporal cortico - medullary junction , deep grey matter and upper brainstem , isolated dwi signal abnormalities of the corpus callosum may also be found . the splenium and the undersurface of the posterior body are mainly involved ( fig . 10axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury signal abnormalities are thought to be due to traumatic axonal stretching with consequent depolarisation and cellular swelling . adc abnormalities may also represent wallerian degeneration secondary to proximal neuronal death or axonal disconnection and may therefore appear in the subacute phase . most dai lesions ( 80% ) are microscopic so that those visible on ct or mri are just the tip of the iceberg : this fact might explain the poor correlation between neuroimaging and the clinical picture after a head trauma . multiple sclerosis ( ms ) is a chronic , multifocal , immunomediated inflammatory demyelinating and degenerative disorder of the central nervous system of unknown aetiology . ms lesions , known as plaques , typically appear as periventricular , ovoid white matter abnormalities perpendicular to the ventricles , along the path of the deep medullary veins . on mri dwi usually discloses high adc values in the plaque core due to prevailing inflammatory changes with vasogenic oedema or gliosis with myelin and axonal loss . electron microscopy studies have shown the presence of inflammatory infiltrates of lymphocytes and lipid - engulfing macrophages , swollen oligodendroglia , which could account for the adc decrease . very rarely , acute plaques might present a homogeneous reduction in adc values , which could suggest a diagnosis of stroke rather than an acute episode of ms . however , according to our experience , concomitant lesions with increased adc values are typically detected in these cases and the cytotoxic - like lesions usually present components of increased adc values as well [ 33 , 34].fig . 11a 30-year - old female patient affected by relapsing - remitting multiple sclerosis ( ms ) presenting with a large demyelinating plaque in the right centrum semiovale . this atypical ms lesion appears hyperintense on diffusion - weighted ( a ) and t2-weighted ( c ) axial images with predominant adc restriction ( b ) a 30-year - old female patient affected by relapsing - remitting multiple sclerosis ( ms ) presenting with a large demyelinating plaque in the right centrum semiovale . this atypical ms lesion appears hyperintense on diffusion - weighted ( a ) and t2-weighted ( c ) axial images with predominant adc restriction ( b ) focal reversible lesions of the splenium ( rsl ) of the corpus callosum ( fig . 12 ) have been recently reported in several miscellaneous conditions such as viral encephalitis , encephalopathies , seizures , antiepileptic and anticancer drug administration [ 35 , 36 ] . a few dwi studies [ 36 , 37 ] have shown transitory restricted diffusion , but its aetiology and pathophysiology remain enigmatic . as a matter of fact , the proposed excitotoxic mechanism due to increased extracellular glutamate with consequent intramyelinic oedema does not fully explain its detection in poorly myelinated brain such as in newborns with mitochondrial genetic disorders .fig . 12a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . axial flair ( superior row ) and sagittal t2-weighted images ( inferior row ) . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . axial flair ( superior row ) and sagittal t2-weighted images ( inferior row ) . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared clinically , rsl can present with seizures , confusion , ataxia , drowsiness , coma , headache and delirium , while the expected , specifically splenium - related deficits ( visual simultanagnosia , hemialexia , unilateral agraphia and unilateral apraxia ) are often difficult to evaluate . isolated corpus callosum lesions without concomitant parenchymal lesions usually regress rapidly , leading to complete clinical and neuroradiological recovery regardless of their cause [ 35 , 36 ] . x - linked charcot - marie - tooth disease ( cmtx ) is the second most common form of inherited motor and sensory demyelinating neuropathy next to the cmt type 1a ; it accounts for approximately 1020% of all hereditary demyelinating neuropathies . it is associated with different mutations in the protein connexin 32 ( cx32 ) , a gap junction protein widely expressed in schwann cells , oligodendrocytes and neurons [ 1 , 2 ] . even if this disease is considered a disorder of the peripheral nervous system , some cx32-mutated subjects might present with acute onset of central nervous system dysfunctions such as hemiparesis , ataxia or aphasia associated with transient white matter changes in the brain . in particular , neuroradiological mr findings in the acute phase show symmetrical , largely confluent cerebral deep white matter signal abnormalities , predominantly in the posterior centrum semiovale , the posterior limb of the internal capsule , the splenium of the corpus callosum ( fig . 1 ) and sometimes in the middle cerebellar peduncles [ 13 ] . bilateral t2 and dwi signal abnormalities might disappear within 1 week or might persist for several months , unrelated to the clinical picture that often appears clearly asymmetric and usually improves rapidly.fig . 1a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . apparent diffusion coefficient ( adc ) maps ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas as adc reduction might reverse after a few months , signal abnormalities can not be interpreted as simple cytotoxic oedema , which usually lasts less than 2 weeks , while the increase in the magnetisation transfer ratios is not consistent with demyelination or oedema . as gap junction dysfunctions may disrupt the diffusion of small molecules and ions across myelin sheaths , the more likely explanation of the reversible white matter lesions remains myelin splitting and intra - myelin oedema with compression of the extracellular spaces . maple syrup urine disease ( msud ) is a rare autosomic recessive disorder caused by a deficiency of the branched - chain -keto acid dehydrogenase mitochondrial multi - enzyme complex . a sudden metabolic decompensation with encephalopathy occurs spontaneously or in concomitance with minor illnesses ; high concentrations of upstream metabolites are thought to exert the neurotoxic effect . clinical presentation and outcome are heterogeneous ; in the classical phenotype the prognosis is very poor , and untreated surviving children are left with psychomotor retardation , spasticity , generalised dystonia and cerebral blindness . in the classical neonatal form , which accounts for about 80% of cases , brain mri may provide a highly suggestive pattern of lesions with a generalised cerebral oedema and a striking increase in dwi and t2 signal intensity in all areas that are already myelinated at birth ( fig . 2 ) . these peculiar histological changes are thought to explain the observed reduction in adc values and its reversibility . early diagnosis and treatment may normalise clinical and neuroradiological abnormalities , although inter - current illnesses throughout life may lead to further metabolic derangements .fig . 2diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . the myelinated areas shown as symmetrical , markedly high intensity signal on a rather homogeneous brain parenchyma depict the cortico - spinal tract ( a , b ) and the cerebello - pontine white matter ( c ) . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . the myelinated areas shown as symmetrical , markedly high intensity signal on a rather homogeneous brain parenchyma depict the cortico - spinal tract ( a , b ) and the cerebello - pontine white matter ( c ) . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) menkes disease is an x - linked disorder caused by defective copper - transporting adenosine triphosphatase , which results in low levels of intracellular copper . male children are normal in the first 24 months of life , and then present with loss of developmental milestones , hypotonia , seizures and failure to thrive ; these patients usually have short , hypopigmented , kinked hair ; death often occurs early in childhood . brain mri might disclose diffuse brain atrophy , subdural effusion , or haematoma and vascular tortuosity . asymmetrical localised white matter lesions involving lobar white matter have been described [ 5 , 6 ] . recently , lee et al . reported that white matter lesions are bright on dwi and present decreased adc values consistent with cytotoxic oedema ( fig . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . adc maps ( b ) of the lesions showed low intensity signal an 8-month - old child affected by menkes disease . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . adc maps ( b ) of the lesions showed low intensity signal as in cmt and msud disease , the pathogenesis of these findings is uncertain . according to hsich et al . , the tortuosity of the vessels might produce erratic and turbulent blood flow , thus predisposing to deep white matter ischaemia . alternatively , brain tissue abnormalities may be related to a deficiency in atp7a mitochondrial copper - containing enzyme with consequent energy failure . glutaric aciduria type 1 ( ga-1 ) is a rare autosomal recessive disorder caused by deficiency of the mitochondrial enzyme glutaryl coa dehydrogenase , necessary for the metabolism of three essential amino acids : lysine , hydroxylysine and tryptophan . the metabolic blocking of these amino acids results in increased levels of glutaric acid and 3-hydroxy glutaric acid . because excessive glutaric acid is probably toxic to the striatal cells , atrophy in these nuclei occurs with age . patients may be asymptomatic or present with acute metabolic decompensation and striatal necrosis , which often leads to severe dyskinesia and cognitive impairment . magnetic resonance imaging findings in ga-1 include wide cerebrospinal fluid spaces anterior to the temporal lobes and within the sylvian fissure , subdural collections and brain atrophy . oedema and increased t2 signal intensity within both caudate heads and lentiform nuclei are seen during metabolic derangement followed by necrosis and shrinking of these structures . in older children as well as in asymptomatic patients , a peculiar and persistent dwi hyperintensity of white matter with a peculiar strip - like involvement of the corpus callosum has been noted . within white matter lesions , 4axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . adc maps ( b ) revealed mildly reduced values in the affected areas axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . adc maps ( b ) revealed mildly reduced values in the affected areas no convincing , definitive explanation has yet been found for the long persistence of these signal abnormalities . phenylketonuria ( pku ) is a severe autosomal recessive disorder due to phenylalanine hydroxylase deficiency [ 4 , 12 , 13 ] that strongly benefits from early diagnosis and dietary treatment . brain mr examination of adolescents and adults discloses white matter abnormalities both in untreated pku patients and those treated early . typically , white matter lesions are occipital - parietal periventricular signal abnormalities on t2 sequences and are related to phenylalanine blood concentrations without significant correlation with the neurological impairment . recently , restricted diffusion of water molecules has been reported in the areas of white matter abnormalities [ 4 , 12 , 14 , 15 ] , appearing hyperintense on dwi in an otherwise rather homogeneously grey brain parenchyma ( fig . these findings have been interpreted as cytotoxic oedema because of a reduction in the na+k+-atpase activity or alternatively as dysmyelination with intra - myelin oedema , but the long persistence of the signal abnormalities does not support these hypotheses .fig . 5magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol / l n.v . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol / l n.v . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) watershed infarctions account for 0.73.2% of all strokes and may be confined to the deep white matter . in the acute phase , mr examination may reveal peri - ventricular round foci of restricted diffusion , with a string of pearls appearance within the deep white matter ( fig . , all these lesions functionally represent a single vascular territory between superficial branches and deep perforators of cerebral arteries . this territory is particularly vulnerable in conditions of severely impaired cerebral blood flow such as prolonged hypotensive events , cardiac arrest with resuscitation and moyamoya disease.fig . 6a 53-year - old male patient affected by dissection of the left internal carotid artery . axial diffusion ( a ) and t2-weighted images ( c ) revealed hyperintense foci in the subcortical watershed territories in a characteristic parasagittal chain - like distribution ( arrows ) . adc maps disclosed the cytotoxic nature of the lesions ( b ) a 53-year - old male patient affected by dissection of the left internal carotid artery . axial diffusion ( a ) and t2-weighted images ( c ) revealed hyperintense foci in the subcortical watershed territories in a characteristic parasagittal chain - like distribution ( arrows ) . adc maps disclosed the cytotoxic nature of the lesions ( b ) on mri lesions might also be unilateral in the case of an ipsilateral internal carotid artery occlusion or tight stenosis or main intracranial artery stenosis , especially when intracranial collateral flow is not compensatory ( e.g. because of the incompleteness of the circle of willis ) . contrary to patients with territorial infarction , patients with wi often do have a history of transient ischaemic attacks , generally have better outcomes , and usually do not benefit from thrombolysis . grey matter is known to be more vulnerable to diffuse hypoxic - ischaemic injury than neighbouring white matter , but recent reports have highlighted that white matter is more sensitive to ischaemia than previously thought and may be primarily involved , sometimes in an isolated fashion [ 19 , 20 ] . global hypoxic - ischaemic encephalopathy of the adult is generally secondary to prolonged cardiac arrest , cardiac arrhythmias , drug overdose , respiratory failure or carbon monoxide poisoning 7 ) have been described in the early stages of diffuse hypoxic - ischaemic injury , white matter abnormalities typically appear in the late - subacute period ( 1420 days ) and may normalise thereafter.fig . 7magnetic resonance images obtained during the subacute stage of co intoxication in a 47-year - old man with a history of heroin abuse found unconscious at home . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . on adc maps ( b ) , white matter lesions appeared moderately hypointense due to reduced water diffusion . as co intoxication and heroin - associated spongiform leukoencephalopathy may produce similar mri findings , both events could have resulted in this peculiar mr picture in our patient magnetic resonance images obtained during the subacute stage of co intoxication in a 47-year - old man with a history of heroin abuse found unconscious at home . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . on adc maps ( b ) , as co intoxication and heroin - associated spongiform leukoencephalopathy may produce similar mri findings , both events could have resulted in this peculiar mr picture in our patient histologically , diffuse white matter abnormalities on mr imaging have correlated with deep white matter vacuolar degeneration and with adjacent axonal injury in normal - appearing white matter . the outcome is generally poor with only a minority of patients regaining independent function . pontine and extrapontine myelinolysis is a rare condition elicited by hypo- or hyper - natraemia , especially when an inappropriate ( i.e. too fast ) correction occurs . magnetic resonance imaging might show a characteristic lesion , namely a trident - shaped signal change within the pons with sparing of the tegmentum ( pontine myelinolysis ) or symmetrical bilateral signal changes of the middle cerebellar peduncles , of the cortico - spinal tract and of the splenium of the corpus callosum ( extrapontine myelinolysis ) ( fig . in fact , several case reports of pontine stroke present with signal abnormalities in the middle cerebellar peduncles . however , in wallerian degeneration signal abnormalities are usually fainter compared with those of extrapontine myelinolysis . furthermore , an associated pontine lesion is the key to a definitive diagnosis of wallerian degeneration of the ponto - cerebellar tracts .fig . 8axial diffusion - weighted ( a ) , adc maps ( b ) and t2-weighted ( c ) images at the level of the pons ( left images ) and basal ganglia ( right images ) obtained in a 54-year - old man who developed impaired consciousness after rapid correction of severe hyponatraemia . diffusion - weighted and t2-weighted images revealed white matter hyperintensities that were more evident in the middle cerebellar peduncles ( arrows ) and in the cortico - spinal tract ( arrowheads ) consistent with osmotic extrapontine myelinolysis . adc values were mildly decreased axial diffusion - weighted ( a ) , adc maps ( b ) and t2-weighted ( c ) images at the level of the pons ( left images ) and basal ganglia ( right images ) obtained in a 54-year - old man who developed impaired consciousness after rapid correction of severe hyponatraemia . diffusion - weighted and t2-weighted images revealed white matter hyperintensities that were more evident in the middle cerebellar peduncles ( arrows ) and in the cortico - spinal tract ( arrowheads ) consistent with osmotic extrapontine myelinolysis . adc values were mildly decreased despite the term myelinolysis , several cases of bilateral basal ganglia involvement have been reported . dwi may show a variable decrease in adc values , which probably depends on the timing of the mr examination and on the severity of white matter involvement . following an initial stage of diffuse encephalopathy , the neurological sequelae are usually characterised by diffuse cognitive deficits and extrapyramidal or cortico - bulbo - spinal signs ; however , a complete remission of neurological symptoms can also be achieved . variable t2 hyperintense lesions with increased adc values are usually found on follow - up examinations , although complete regression of the mr lesions is possible . methotrexate ( mtx ) is a cycle - specific antimetabolite that inhibits the enzyme dihydrofolate reductase and is used for the treatment of oncological disorders . mtx crosses the blood - brain barrier and can be administered intravenously as well as intrathecally to eradicate leukaemic cells from the cns and to prevent cns recurrence in acute lymphocytic leukaemia . acute mtx neurotoxicity has been described in 310% of cases and varies with dose , frequency and route of administration [ 2729 ] . the temporal relationship between acute mtx neurotoxicity and induction ranges from 2 to 127 weeks , but it is most often seen 1011 days after intrathecal administrations of mtx . from a clinical point of view acute mtx neurotoxicity may mimic a stroke and present with aphasia or hemiparesis . conventional mr imaging shows bilateral t2 hyperintense lesions within the deep fronto - parietal white matter . 9 ) and reveals a variable decrease of adc values probably due to the derangement of folate and homocysteine metabolism . dwi abnormalities and clinical symptoms usually resolve within a few days even when t2 abnormalities persist.fig . 9this 14-year - old girl affected by acute lymphocytic leukaemia and treated with intrathecal mtx acutely developed hemiparesis and aphasia . axial diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) showed bilateral t2 hyperintense lesions within the deep fronto - parietal white matter ( arrows ) . the neurological deficits and the dwi / adc abnormalities resolved completely within 1 week , while slight t2 hyperintensities persisted on follow - up examinations this 14-year - old girl affected by acute lymphocytic leukaemia and treated with intrathecal mtx acutely developed hemiparesis and aphasia . axial diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) showed bilateral t2 hyperintense lesions within the deep fronto - parietal white matter ( arrows ) . the neurological deficits and the dwi / adc abnormalities resolved completely within 1 week , while slight t2 hyperintensities persisted on follow - up examinations inhalation of heroin vapour , a practice known as chasing the dragon , can cause a specific leukoencephalopathy . the pathophysiological mechanism seems to be selectively activated by substances originating during the heating of heroin . symptoms usually occur within some days after the last heroin consumption and are first dominated by cerebellar deficits . characteristically , heroin - induced leukoencephalopathy appears as symmetrical t2 and dwi hyperintense lesions involving the cerebellar white matter , the posterior limbs of the internal capsule , the splenium of the corpus callosum and the posterior cerebral white matter . a decrease in the adc values and the absence of contrast enhancement further characterise the affected areas , whereas a spongiform demyelination is considered the histological substrate . clinical and neuroradiological pictures may slowly improve if the patients are treated promptly ; otherwise manifest severe neurological impairment and death occur . diffuse axonal injury ( dai ) accounts for 50% of all primary intra - axial traumatic brain injuries . these lesions can be both haemorrhagic and non - haemorrhagic , and are caused by differential linear and rotational accelerations of the cortex in relation to the underlying deep brain structures . besides multi - focal punctuate haemorrhages at the fronto - temporal cortico - medullary junction , deep grey matter and upper brainstem , isolated dwi signal abnormalities of the corpus callosum may also be found . the splenium and the undersurface of the posterior body are mainly involved ( fig . 10axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury signal abnormalities are thought to be due to traumatic axonal stretching with consequent depolarisation and cellular swelling . adc abnormalities may also represent wallerian degeneration secondary to proximal neuronal death or axonal disconnection and may therefore appear in the subacute phase . most dai lesions ( 80% ) are microscopic so that those visible on ct or mri are just the tip of the iceberg : this fact might explain the poor correlation between neuroimaging and the clinical picture after a head trauma . multiple sclerosis ( ms ) is a chronic , multifocal , immunomediated inflammatory demyelinating and degenerative disorder of the central nervous system of unknown aetiology . ms lesions , known as plaques , typically appear as periventricular , ovoid white matter abnormalities perpendicular to the ventricles , along the path of the deep medullary veins . on mri dwi usually discloses high adc values in the plaque core due to prevailing inflammatory changes with vasogenic oedema or gliosis with myelin and axonal loss . electron microscopy studies have shown the presence of inflammatory infiltrates of lymphocytes and lipid - engulfing macrophages , swollen oligodendroglia , which could account for the adc decrease . very rarely , acute plaques might present a homogeneous reduction in adc values , which could suggest a diagnosis of stroke rather than an acute episode of ms . however , according to our experience , concomitant lesions with increased adc values are typically detected in these cases and the cytotoxic - like lesions usually present components of increased adc values as well [ 33 , 34].fig . 11a 30-year - old female patient affected by relapsing - remitting multiple sclerosis ( ms ) presenting with a large demyelinating plaque in the right centrum semiovale . this atypical ms lesion appears hyperintense on diffusion - weighted ( a ) and t2-weighted ( c ) axial images with predominant adc restriction ( b ) a 30-year - old female patient affected by relapsing - remitting multiple sclerosis ( ms ) presenting with a large demyelinating plaque in the right centrum semiovale . this atypical ms lesion appears hyperintense on diffusion - weighted ( a ) and t2-weighted ( c ) axial images with predominant adc restriction ( b ) focal reversible lesions of the splenium ( rsl ) of the corpus callosum ( fig . 12 ) have been recently reported in several miscellaneous conditions such as viral encephalitis , encephalopathies , seizures , antiepileptic and anticancer drug administration [ 35 , 36 ] . a few dwi studies [ 36 , 37 ] have shown transitory restricted diffusion , but its aetiology and pathophysiology remain enigmatic . as a matter of fact , the proposed excitotoxic mechanism due to increased extracellular glutamate with consequent intramyelinic oedema does not fully explain its detection in poorly myelinated brain such as in newborns with mitochondrial genetic disorders .fig . 12a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . axial flair ( superior row ) and sagittal t2-weighted images ( inferior row ) . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . axial flair ( superior row ) and sagittal t2-weighted images ( inferior row ) . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared clinically , rsl can present with seizures , confusion , ataxia , drowsiness , coma , headache and delirium , while the expected , specifically splenium - related deficits ( visual simultanagnosia , hemialexia , unilateral agraphia and unilateral apraxia ) are often difficult to evaluate . isolated corpus callosum lesions without concomitant parenchymal lesions usually regress rapidly , leading to complete clinical and neuroradiological recovery regardless of their cause [ 35 , 36 ] . the literature ripens with diversified causes of areas of adc restriction in the brain parenchyma : a few genetic , metabolic , iatrogenic , traumatic , infectious and vascular conditions can present with selective involvement of white matter with dwi hyperintense lesions and decreased adc values . the neuroradiologist is often unnerved by the diagnostic challenges suggested by the clinician , as clinical history and course are not always disease - specific . in this setting , some suggestive or specific dwi mr patterns may provide powerful clues that can help in addressing the diagnosis and in tailoring treatment ( table 1).table 1dwi mr patterns that can help with the diagnosis . cci : corpus callosum involvement , csti : cortico - spinal tract involvement , csi : involvement of the centra semiovaliadiseaseageccicsticsiiwmineuroradiologic featuresother featuresevolutioncmt x - linkedyoung adult++-+++moose - horn appearancestroke - like onsetreversiblemsudnewborn-++++++already myelinated areassevere metabolic decompensationvariable recoverymdinfant--++-oval / drop - shaped lesionssevere metabolic decompensationpersistent t2 lesionsga-1child / adult++-++-+strip - like appearance of corpus callosumalso in asymptomatic patientspersistent on t2 and dwipkuchild / adult+-++-+prominent parieto - occipital involvementalso in asymptomatic patientspersistent on t2 and dwiwielderly--++-chain - like appearanceipsilateral severe carotid stenosispersistent t2 lesionsgcaadult / elderly-+-+++-extensive bilateral involvement of centrum semiovaleevocative clinical historylate necrotic evolutionextrapontine myelinolysisadult / elderly+-++++natrium serum level abnormalitiesvariable recoverymtxchild--++-posterior part of centrum semiovaleclinical history of mtx administrationreversibleinhalation of heroin vapouryoung adult-+++++extensive bilateral involvement of centrum semiovaleclinical history of drug abusepersistent t2 lesionsdiffuse axonal injuryany++-+-+-+splenium and undersurface of corpus callosumclinical history of head traumapersistent t2 lesionsreversible lesions of the spleniumany++-+-spleniummiscellaneous conditionsreversible dwi mr patterns that can help with the diagnosis . cci : corpus callosum involvement , csti : cortico - spinal tract involvement , csi : involvement of the centra semiovalia

backgroundreduction of apparent diffusion coefficient ( adc ) values in white matter is not always ischaemic in nature.methodswe retrospectively analysed our mri records featuring reduced adc values in the centrum semiovale without grey matter involvement or significant vasogenic oedema.resultsseveral conditions showed the aforementioned mr findings : moose - horn lesions on coronal images in x - linked charcot - marie - tooth disease ; small fronto - parietal lesions in menkes disease ; marked signal abnormalities in the myelinised regions in the acute neonatal form of maple syrup urine disease ; strip - like involvement of the corpus callosum in glutaric aciduria type 1 ; persistent periventricular parieto - occipital abnormalities in phenylketonuria ; diffuse signal abnormalities with necrotic evolution in global cerebral anoxia or after heroin vapour inhalation ; almost completely reversible symmetric fronto - parietal lesions in methotrexate neurotoxicity ; chain - like lesions in watershed ischaemia ; splenium involvement that normalises in reversible splenial lesions or leads to gliosis in diffuse axonal injury.conclusionneuroradiologists must be familiar with these features , thereby preventing misdiagnosis and inappropriate management .

Introduction Genetic and metabolic disorders Vascular conditions Iatrogenic conditions Non-iatrogenic toxicity Traumatic condition Demyelinating disorders Other conditions Conclusions

the reduction of the apparent diffusion coefficient ( adc ) values depends on the disease studied , and it might involve an increase in cell debris , cellularity , intramyelin and axonal oedema . x - linked charcot - marie - tooth disease ( cmtx ) is the second most common form of inherited motor and sensory demyelinating neuropathy next to the cmt type 1a ; it accounts for approximately 1020% of all hereditary demyelinating neuropathies . even if this disease is considered a disorder of the peripheral nervous system , some cx32-mutated subjects might present with acute onset of central nervous system dysfunctions such as hemiparesis , ataxia or aphasia associated with transient white matter changes in the brain . in particular , neuroradiological mr findings in the acute phase show symmetrical , largely confluent cerebral deep white matter signal abnormalities , predominantly in the posterior centrum semiovale , the posterior limb of the internal capsule , the splenium of the corpus callosum ( fig . 1a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . maple syrup urine disease ( msud ) is a rare autosomic recessive disorder caused by a deficiency of the branched - chain -keto acid dehydrogenase mitochondrial multi - enzyme complex . 2diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . glutaric aciduria type 1 ( ga-1 ) is a rare autosomal recessive disorder caused by deficiency of the mitochondrial enzyme glutaryl coa dehydrogenase , necessary for the metabolism of three essential amino acids : lysine , hydroxylysine and tryptophan . in older children as well as in asymptomatic patients , a peculiar and persistent dwi hyperintensity of white matter with a peculiar strip - like involvement of the corpus callosum has been noted . 4axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . ( b ) revealed mildly reduced values in the affected areas axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . 5magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol / l n.v . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) watershed infarctions account for 0.73.2% of all strokes and may be confined to the deep white matter . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . dwi may show a variable decrease in adc values , which probably depends on the timing of the mr examination and on the severity of white matter involvement . characteristically , heroin - induced leukoencephalopathy appears as symmetrical t2 and dwi hyperintense lesions involving the cerebellar white matter , the posterior limbs of the internal capsule , the splenium of the corpus callosum and the posterior cerebral white matter . besides multi - focal punctuate haemorrhages at the fronto - temporal cortico - medullary junction , deep grey matter and upper brainstem , isolated dwi signal abnormalities of the corpus callosum may also be found . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury signal abnormalities are thought to be due to traumatic axonal stretching with consequent depolarisation and cellular swelling . on mri dwi usually discloses high adc values in the plaque core due to prevailing inflammatory changes with vasogenic oedema or gliosis with myelin and axonal loss . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared clinically , rsl can present with seizures , confusion , ataxia , drowsiness , coma , headache and delirium , while the expected , specifically splenium - related deficits ( visual simultanagnosia , hemialexia , unilateral agraphia and unilateral apraxia ) are often difficult to evaluate . x - linked charcot - marie - tooth disease ( cmtx ) is the second most common form of inherited motor and sensory demyelinating neuropathy next to the cmt type 1a ; it accounts for approximately 1020% of all hereditary demyelinating neuropathies . in particular , neuroradiological mr findings in the acute phase show symmetrical , largely confluent cerebral deep white matter signal abnormalities , predominantly in the posterior centrum semiovale , the posterior limb of the internal capsule , the splenium of the corpus callosum ( fig . 1a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . apparent diffusion coefficient ( adc ) maps ( b ) disclosed a restricted diffusion of water molecules in the corresponding areas a 23-year - old male patient affected by genetically proven x - linked charcot - marie - tooth disease presenting with acute onset of aphasia and right hemiparesis . diffusion - weighted ( a ) and t2-weighted images ( c ) at admission showed symmetrical confluent hyperintensities in the posterior centrum semiovale and in the splenium of the corpus callosum , with a coronal image resembling moose horns . maple syrup urine disease ( msud ) is a rare autosomic recessive disorder caused by a deficiency of the branched - chain -keto acid dehydrogenase mitochondrial multi - enzyme complex . 2diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) diffusion - weighted axial images ( superior row ) and adc axial images ( inferior row ) obtained in a newborn with a biochemically confirmed classical form of maple syrup urine disease . restricted diffusion of water molecules is well demonstrated by adc images ( d , e , f ) menkes disease is an x - linked disorder caused by defective copper - transporting adenosine triphosphatase , which results in low levels of intracellular copper . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . axial diffusion- ( a ) and t2-weighted images ( c ) disclosed bilateral asymmetric oval bright white matter lesions in the centrum semiovalis ( arrows ) . glutaric aciduria type 1 ( ga-1 ) is a rare autosomal recessive disorder caused by deficiency of the mitochondrial enzyme glutaryl coa dehydrogenase , necessary for the metabolism of three essential amino acids : lysine , hydroxylysine and tryptophan . in older children as well as in asymptomatic patients , a peculiar and persistent dwi hyperintensity of white matter with a peculiar strip - like involvement of the corpus callosum has been noted . within white matter lesions , 4axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . adc maps ( b ) revealed mildly reduced values in the affected areas axial diffusion- ( a ) and t2-weighted images ( c ) of an asymptomatic 26-year - old patient with type 1 glutaric aciduria demonstrating the peculiar strip - like involvement of the corpus callosum due to signal abnormalities in the median genu of the corpus callosum ( arrows ) and in periventricular white matter ( arrowhead ) . adc maps ( b ) revealed mildly reduced values in the affected areas no convincing , definitive explanation has yet been found for the long persistence of these signal abnormalities . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) magnetic resonance images obtained in an asymptomatic , phenylketonuric adult who was treated early ( plasmatic phenylalanine levels of 729 mol / l n.v . axial diffusion - weighted ( a ) adc maps ( b ) and t2-weighted images ( c ) showed periventricular white matter signal abnormalities predominantly in the parieto - occipital regions ( arrows ) watershed infarctions account for 0.73.2% of all strokes and may be confined to the deep white matter . adc maps disclosed the cytotoxic nature of the lesions ( b ) on mri lesions might also be unilateral in the case of an ipsilateral internal carotid artery occlusion or tight stenosis or main intracranial artery stenosis , especially when intracranial collateral flow is not compensatory ( e.g. grey matter is known to be more vulnerable to diffuse hypoxic - ischaemic injury than neighbouring white matter , but recent reports have highlighted that white matter is more sensitive to ischaemia than previously thought and may be primarily involved , sometimes in an isolated fashion [ 19 , 20 ] . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) disclosed prominent , bilateral and symmetrical confluent hyperintensities in the centrum semiovalis that were more evident in the fronto - parietal lobes ( arrows ) . dwi may show a variable decrease in adc values , which probably depends on the timing of the mr examination and on the severity of white matter involvement . conventional mr imaging shows bilateral t2 hyperintense lesions within the deep fronto - parietal white matter . axial diffusion - weighted ( a ) , flair ( c ) and t2-weighted images ( d ) showed bilateral t2 hyperintense lesions within the deep fronto - parietal white matter ( arrows ) . characteristically , heroin - induced leukoencephalopathy appears as symmetrical t2 and dwi hyperintense lesions involving the cerebellar white matter , the posterior limbs of the internal capsule , the splenium of the corpus callosum and the posterior cerebral white matter . besides multi - focal punctuate haemorrhages at the fronto - temporal cortico - medullary junction , deep grey matter and upper brainstem , isolated dwi signal abnormalities of the corpus callosum may also be found . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury axial mr images obtained in a child with a head injury at the level of the lateral ventricles . the midline of the splenium of the corpus callosum presented diffusion - weighted ( a ) and t2-weighted ( c ) hyperintensity ( arrows ) with reduced adc values on adc maps ( b ) consistent with diffuse axonal injury signal abnormalities are thought to be due to traumatic axonal stretching with consequent depolarisation and cellular swelling . on mri dwi usually discloses high adc values in the plaque core due to prevailing inflammatory changes with vasogenic oedema or gliosis with myelin and axonal loss . however , according to our experience , concomitant lesions with increased adc values are typically detected in these cases and the cytotoxic - like lesions usually present components of increased adc values as well [ 33 , 34].fig . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared a 32-year - old male patient admitted with partial seizures secondary to antiepileptic drug withdrawal . at onset mri examination ( a ) revealed increased signal intensity in the central area of the splenium of the corpus callosum ( white arrowheads ) ; 1 month later ( b ) the lesion had disappeared clinically , rsl can present with seizures , confusion , ataxia , drowsiness , coma , headache and delirium , while the expected , specifically splenium - related deficits ( visual simultanagnosia , hemialexia , unilateral agraphia and unilateral apraxia ) are often difficult to evaluate . the literature ripens with diversified causes of areas of adc restriction in the brain parenchyma : a few genetic , metabolic , iatrogenic , traumatic , infectious and vascular conditions can present with selective involvement of white matter with dwi hyperintense lesions and decreased adc values . cci : corpus callosum involvement , csti : cortico - spinal tract involvement , csi : involvement of the centra semiovaliadiseaseageccicsticsiiwmineuroradiologic featuresother featuresevolutioncmt x - linkedyoung adult++-+++moose - horn appearancestroke - like onsetreversiblemsudnewborn-++++++already myelinated areassevere metabolic decompensationvariable recoverymdinfant--++-oval / drop - shaped lesionssevere metabolic decompensationpersistent t2 lesionsga-1child / adult++-++-+strip - like appearance of corpus callosumalso in asymptomatic patientspersistent on t2 and dwipkuchild / adult+-++-+prominent parieto - occipital involvementalso in asymptomatic patientspersistent on t2 and dwiwielderly--++-chain - like appearanceipsilateral severe carotid stenosispersistent t2 lesionsgcaadult / elderly-+-+++-extensive bilateral involvement of centrum semiovaleevocative clinical historylate necrotic evolutionextrapontine myelinolysisadult / elderly+-++++natrium serum level abnormalitiesvariable recoverymtxchild--++-posterior part of centrum semiovaleclinical history of mtx administrationreversibleinhalation of heroin vapouryoung adult-+++++extensive bilateral involvement of centrum semiovaleclinical history of drug abusepersistent t2 lesionsdiffuse axonal injuryany++-+-+-+splenium and undersurface of corpus callosumclinical history of head traumapersistent t2 lesionsreversible lesions of the spleniumany++-+-spleniummiscellaneous conditionsreversible dwi mr patterns that can help with the diagnosis .

multifactorial hypocalcemia , calcitriol and vitamin d deficiency , p retention and fibroblast growth factor ( fgf)-23 dysregulation , resistance to the action of pth and vitamin d , and decreased activation , decreased number , or downregulation of several related receptors ( vdr , ca - sensing receptor , fgf-23/klotho ) , among other factors , can lead to prolonged and excessive synthesis and secretion of pth , eventually leading to the development of shpt and different forms of renal osteodystrophy . bone is currently considered an active endocrine ' organ , as demonstrated by the osteocyte product fgf-23 regulating p balance , vitamin d metabolism , and pth production . furthermore , the effect of pth on fgf-23 has been recently described , completing a bone parathyroid endocrine feedback loop in which shpt has been shown to be essential for the high fgf-23 levels in early ckd . the role of fgf-23 and klotho in ckd is widely covered by vervloet and larsson in this issue . beyond parathyroid and bone diseases , numerous studies have shown clearly the association between disorders of mineral metabolism ( and the therapies used to correct these abnormalities ) and fractures , extraskeletal and vascular calcification ( vc ) , progression of renal failure , cv disease , and mortality . these studies have prompted the development of the new term ckd mineral and bone disorder ( mbd ) to acknowledge its systemic consequences and damage to organs beyond bone , primarily vc . thus , ckd mbd is a real syndrome , currently defined by one or a combination of the following : ( 1 ) abnormalities of ca , p , pth , or vitamin d metabolism ; ( 2 ) abnormalities in bone turnover , mineralization , volume , linear growth , or strength ; and/or ( 3 ) vascular or other soft - tissue calcifications ( figure 1 ) . the old general term of renal osteodystrophy is currently restricted to an alteration of bone morphology in patients with ckd , representing one measure of the skeletal component of ckd mbd that is quantifiable by histomorphometry of bone biopsy . although bone biopsy is invasive and thus can not be performed easily , it is the gold standard for the diagnosis of renal osteodystrophy , as biochemical parameters do not adequately predict the underlying bone histology . this is a field with a particular need for improvement , especially taking into account the great variability of pth assays and the current scarcity of information available on bone behavior and bone disease beyond the so - called intact pth thus , a significant proportion of patients with intermediate pth levels ( that is , two to nine times the upper limit of normality for a particular assay ) present quite different rates of bone formation , from adynamic bone disease ( abd ) to normality and hyperparathyroid bone disease . it is well known that the traditional types of renal osteodystrophy have been defined on the basis of turnover and mineralization , with substantial differences in pathophysiology and treatment . a recent kidney disease : improving global outcomes ( kdigo ) report has suggested that bone biopsies in patients with ckd should be characterized by determining bone turnover ( t ) , mineralization ( m ) , and volume ( v ) ( the tmv system ) . six types of bone disorder are distinguished in the ckd mbd complex : hyperparathyroid bone disease ( high turnover , normal mineralization , and any bone volume ) ; mixed bone disease ( high turnover with mineralization defect and normal bone volume ) ; osteomalacia ( low turnover with abnormal mineralization and low - to - medium bone volume ) ; abd ( low turnover with normal mineralization and low or normal bone volume ) ; and two distinct disorders due to specific causative agents , namely , amyloid bone disease and aluminum bone disease . according to old studies , bone abnormalities are found almost universally in patients with ckd requiring dialysis ( stage 5d ) and in the majority of patients with ckd stages 35 . moreover , the distribution of histological types in patients with ckd stage 5 has been changing over the years . abd is increasing in prevalence and in many ckd populations now represents the most frequent type of bone lesion . steadily growing proportions of elderly , diabetic , and malnourished patients may , at least partially , explain this finding . the term aplastic ' or this type of disorder is characterized by a low bone turnover with normal mineralization represented by an absence of osteoid accumulation . in adynamic bone , the number of osteoblasts , the bone formation rate , and the activation frequency are markedly diminished . bone histomorphometry cutoff levels in different studies are inconsistent , and the reported prevalence of abd varies from 5 to 49% in ckd stages 34 ( refs . 17 , 21 , 27 , 28 , 29 ) and from 10 to 71% in ckd stage 5d . the regulation of osteoblast apoptosis is now recognized to be a major mechanism in determining rates of bone formation . increased osteoblast apoptosis and decreased osteoblastogenesis are important determinants of decreased bone formation rate and activation frequency . a variant of abd ( so - called abd - v ) , characterized by high osteoclastic resorption , has also been described . in the past , aluminum overload was the predominant cause of low - turnover bone disease in dialysis patients . a chronic low - dose exposure with concomitant high doses of vitamin d probably favoring mineralization thus , it is important to distinguish between aluminum - induced and non - aluminum - induced forms of abd . advanced age and diabetes ( through elevated levels of advanced glycation end - products ) , glucocorticoid - induced osteoporosis , and hypoparathyroidism are associated with low bone turnover . growth hormone resistance or insulin - like growth factor - i insufficiency , excess of proinflammatory cytokines , and low serum calcitriol levels likely have a role in suppressing bone formation by inhibiting the number of osteoblasts that can form bone . low pth levels or bone resistance to the action of pth in ckd ( relative hypoparathyroidism ) are also regarded as important risk factors for abd , and many factors from p loading , decreased calcitriol , or uremia itself to antagonistic pth fragments , downregulation of pth receptors , or decreased pulsatility of pth have been related to skeletal or calcemic resistance to pth . recently , the bone formation rate has also been shown to be associated with resistance to pth . several other factors have been linked to abd , such as ca loading ( via p binders or ca in the dialysate ) , vitamin d treatment ( children with high doses , high dialysate ca content , or high incidences of hypercalcemia ) , peritoneal dialysis ( dialysate ca , increased glucose , low albumin ) , acidosis , hypogonadism , inflammation , or the use of bisphosphonates in ckd patients . the importance of abd is that it diminishes the ability to repair microdamage , resulting in an increased fracture risk , according to observational evidence using bone markers but not bone biopsies . curiously enough , patients with idiopathic hypoparathyroidism have preserved or increased bone mass during therapy with ca and vitamin d , and patients with total parathyroidectomy do not have an associated increase in the risk of fracture . low pth is also associated with low protein intake and may be a risk factor for malnutrition . as will be mentioned later , of more importance is the existence of a relationship between abd , abnormal ca balance , calcific arteriolopathy , and vc , and the fact that a u - shaped curve depicts pth levels and mortality . thus , the causal relationship between abd and vascular disease may , at least in part , explain why not only high but also low intact pth plasma levels , as well as low pth and high serum ca levels ( a combination typical for abd ) , are associated with increased mortality . multiple circulating factors contribute to bone activity by interfering with pth receptor , fgf-23/klotho , or vdr pathways . deficiency in vitamin d and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization . it is also well known that vitamin d not only indirectly decreases bone formation rate in hyperparathyroid bone disease , but also directly increases both bone turnover and bone growth . recent studies in vdr - knockout mice have identified the vdr as being crucial in maintaining normal bone formation and bone mineralization by directly enhancing osteoblast differentiation . in this model , ca and vitamin d had both independent and interdependent effects on skeletal and mineral homeostasis . mineralization of bone reflected ambient ca levels rather than the calcitriol / vdr system , whereas increased ca absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by calcitriol / vdr . vdr activation of the osteoblast also functions to prevent apoptosis , similarly to the effect of estrogen and androgen receptors in preventing apoptosis of osteoblasts and osteocytes . intermittent injections of pth also serve an antiapoptotic function to increase bone formation and bone mass in contrast to continuous pth infusions , which cause bone loss . in addition , osteoblasts contain 1-hydroxylase , presumably to function in an autocrine / paracrine manner to maintain bone formation . it is necessary to remember that vitamin d upregulates its own receptor , whereas pth decreases vdr and blocks homologous upregulation . as expected , it has been demonstrated that the actions of vdr activators on bone are more important than those expected just for the inhibition of pth , and thus the effects of active vitamin d compounds and calcimimetics on bone differ under certain experimental conditions in vivo . moreover , some of the improvement in ca and skeletal homeostasis seen in vdr - knockout mice may occur through alternative vitamin d signaling pathways . interestingly , other actions of vitamin d are explained by mitochondrial non - genomic activity of vdr , making way for challenging future studies on vdr physiology . in an in vitro study , nakane et al . found that paricalcitol caused less ca release from mouse calvariae than did calcitriol and doxercalciferol ; furthermore , paricalcitol was the most effective preparation in stimulating collagen synthesis . the authors concluded that this bone effect of paricalcitol may contribute to its lower calcemic profile and that paricalcitol may be more effective in stimulating bone formation . importantly , opposite effects of calcitriol and paricalcitol on the pth ratio ( whole pth(184)/carboxy - terminal pth ) have been described in patients with ckd stage 5d . the relatively higher pth 184 levels with paricalcitol is in agreement with data obtained in a rat model of renal failure showing that paricalcitol can prevent and treat shpt without a major decrease in bone turnover . there are no data on the effects of new activators of the vdr on human bone . finally , with regard to renal osteodystrophy , it is necessary to consider that an early diagnosis of ckd , through the generalized use of estimated glomerular filtration rate laboratory reports , will allow earlier diagnosis of biochemical shpt , abnormalities in vitamin d metabolism , and ckd mbd . efforts should be made to improve our knowledge of bone histology and its relationship with new biochemical parameters . earlier reports suggesting that the use of the pth ratio improves the prediction of bone histology have not been corroborated in subsequent studies . bone alkaline phosphatase as well as new parameters may improve the predictive value of intact pth or the pth ratio . it is also of particular importance to learn the effect of different treatments on bone outcomes , particularly the normalization of bone histomorphometry ( as stated in the recent kdigo clinical practice guidelines ) and bone buffering capacity , as it is becoming increasingly clear that there is a link between abnormal vascular processes ( that is , vc ) and bone metabolism ( the so - called bone vascular axis ) , and this may also have an impact on survival . the old general term of renal osteodystrophy is currently restricted to an alteration of bone morphology in patients with ckd , representing one measure of the skeletal component of ckd mbd that is quantifiable by histomorphometry of bone biopsy . although bone biopsy is invasive and thus can not be performed easily , it is the gold standard for the diagnosis of renal osteodystrophy , as biochemical parameters do not adequately predict the underlying bone histology . this is a field with a particular need for improvement , especially taking into account the great variability of pth assays and the current scarcity of information available on bone behavior and bone disease beyond the so - called intact pth thus , a significant proportion of patients with intermediate pth levels ( that is , two to nine times the upper limit of normality for a particular assay ) present quite different rates of bone formation , from adynamic bone disease ( abd ) to normality and hyperparathyroid bone disease . it is well known that the traditional types of renal osteodystrophy have been defined on the basis of turnover and mineralization , with substantial differences in pathophysiology and treatment . a recent kidney disease : improving global outcomes ( kdigo ) report has suggested that bone biopsies in patients with ckd should be characterized by determining bone turnover ( t ) , mineralization ( m ) , and volume ( v ) ( the tmv system ) . six types of bone disorder are distinguished in the ckd mbd complex : hyperparathyroid bone disease ( high turnover , normal mineralization , and any bone volume ) ; mixed bone disease ( high turnover with mineralization defect and normal bone volume ) ; osteomalacia ( low turnover with abnormal mineralization and low - to - medium bone volume ) ; abd ( low turnover with normal mineralization and low or normal bone volume ) ; and two distinct disorders due to specific causative agents , namely , amyloid bone disease and aluminum bone disease . according to old studies , bone abnormalities are found almost universally in patients with ckd requiring dialysis ( stage 5d ) and in the majority of patients with ckd stages 35 . moreover , the distribution of histological types in patients with ckd stage 5 has been changing over the years . abd is increasing in prevalence and in many ckd populations now represents the most frequent type of bone lesion . steadily growing proportions of elderly , diabetic , and malnourished patients may , at least partially , explain this finding . the term aplastic ' or adynamic ' bone disease was introduced in the early 1980s . this type of disorder is characterized by a low bone turnover with normal mineralization represented by an absence of osteoid accumulation . in adynamic bone , the number of osteoblasts , the bone formation rate , and the activation frequency are markedly diminished . bone histomorphometry cutoff levels in different studies are inconsistent , and the reported prevalence of abd varies from 5 to 49% in ckd stages 34 ( refs . 17 , 21 , 27 , 28 , 29 ) and from 10 to 71% in ckd stage 5d . the regulation of osteoblast apoptosis is now recognized to be a major mechanism in determining rates of bone formation . increased osteoblast apoptosis and decreased osteoblastogenesis are important determinants of decreased bone formation rate and activation frequency . a variant of abd ( so - called abd - v ) , characterized by high osteoclastic resorption , has also been described . in the past , aluminum overload was the predominant cause of low - turnover bone disease in dialysis patients . a chronic low - dose exposure with concomitant high doses of vitamin d probably favoring mineralization thus , it is important to distinguish between aluminum - induced and non - aluminum - induced forms of abd . independently of ckd , advanced age and diabetes ( through elevated levels of advanced glycation end - products ) , glucocorticoid - induced osteoporosis , and hypoparathyroidism are associated with low bone turnover . growth hormone resistance or insulin - like growth factor - i insufficiency , excess of proinflammatory cytokines , and low serum calcitriol levels likely have a role in suppressing bone formation by inhibiting the number of osteoblasts that can form bone . low pth levels or bone resistance to the action of pth in ckd ( relative hypoparathyroidism ) are also regarded as important risk factors for abd , and many factors from p loading , decreased calcitriol , or uremia itself to antagonistic pth fragments , downregulation of pth receptors , or decreased pulsatility of pth have been related to skeletal or calcemic resistance to pth . recently , the bone formation rate has also been shown to be associated with resistance to pth . several other factors have been linked to abd , such as ca loading ( via p binders or ca in the dialysate ) , vitamin d treatment ( children with high doses , high dialysate ca content , or high incidences of hypercalcemia ) , peritoneal dialysis ( dialysate ca , increased glucose , low albumin ) , acidosis , hypogonadism , inflammation , or the use of bisphosphonates in ckd patients . the importance of abd is that it diminishes the ability to repair microdamage , resulting in an increased fracture risk , according to observational evidence using bone markers but not bone biopsies . curiously enough , patients with idiopathic hypoparathyroidism have preserved or increased bone mass during therapy with ca and vitamin d , and patients with total parathyroidectomy do not have an associated increase in the risk of fracture . low pth is also associated with low protein intake and may be a risk factor for malnutrition . as will be mentioned later , of more importance is the existence of a relationship between abd , abnormal ca balance , calcific arteriolopathy , and vc , and the fact that a u - shaped curve depicts pth levels and mortality . thus , the causal relationship between abd and vascular disease may , at least in part , explain why not only high but also low intact pth plasma levels , as well as low pth and high serum ca levels ( a combination typical for abd ) , are associated with increased mortality . multiple circulating factors contribute to bone activity by interfering with pth receptor , fgf-23/klotho , or vdr pathways . deficiency in vitamin d and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization . it is also well known that vitamin d not only indirectly decreases bone formation rate in hyperparathyroid bone disease , but also directly increases both bone turnover and bone growth . recent studies in vdr - knockout mice have identified the vdr as being crucial in maintaining normal bone formation and bone mineralization by directly enhancing osteoblast differentiation . in this model , ca and vitamin d had both independent and interdependent effects on skeletal and mineral homeostasis . mineralization of bone reflected ambient ca levels rather than the calcitriol / vdr system , whereas increased ca absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by calcitriol / vdr . vdr activation of the osteoblast also functions to prevent apoptosis , similarly to the effect of estrogen and androgen receptors in preventing apoptosis of osteoblasts and osteocytes . intermittent injections of pth also serve an antiapoptotic function to increase bone formation and bone mass in contrast to continuous pth infusions , which cause bone loss . in addition , osteoblasts contain 1-hydroxylase , presumably to function in an autocrine / paracrine manner to maintain bone formation . it is necessary to remember that vitamin d upregulates its own receptor , whereas pth decreases vdr and blocks homologous upregulation . as expected , it has been demonstrated that the actions of vdr activators on bone are more important than those expected just for the inhibition of pth , and thus the effects of active vitamin d compounds and calcimimetics on bone differ under certain experimental conditions in vivo . moreover , some of the improvement in ca and skeletal homeostasis seen in vdr - knockout mice may occur through alternative vitamin d signaling pathways . interestingly , other actions of vitamin d are explained by mitochondrial non - genomic activity of vdr , making way for challenging future studies on vdr physiology . in an in vitro study , nakane et al . found that paricalcitol caused less ca release from mouse calvariae than did calcitriol and doxercalciferol ; furthermore , paricalcitol was the most effective preparation in stimulating collagen synthesis . the authors concluded that this bone effect of paricalcitol may contribute to its lower calcemic profile and that paricalcitol may be more effective in stimulating bone formation . importantly , opposite effects of calcitriol and paricalcitol on the pth ratio ( whole pth(184)/carboxy - terminal pth ) have been described in patients with ckd stage 5d . the relatively higher pth 184 levels with paricalcitol is in agreement with data obtained in a rat model of renal failure showing that paricalcitol can prevent and treat shpt without a major decrease in bone turnover . there are no data on the effects of new activators of the vdr on human bone . finally , with regard to renal osteodystrophy , it is necessary to consider that an early diagnosis of ckd , through the generalized use of estimated glomerular filtration rate laboratory reports , will allow earlier diagnosis of biochemical shpt , abnormalities in vitamin d metabolism , and ckd mbd . efforts should be made to improve our knowledge of bone histology and its relationship with new biochemical parameters . earlier reports suggesting that the use of the pth ratio improves the prediction of bone histology have not been corroborated in subsequent studies . bone alkaline phosphatase as well as new parameters may improve the predictive value of intact pth or the pth ratio . it is also of particular importance to learn the effect of different treatments on bone outcomes , particularly the normalization of bone histomorphometry ( as stated in the recent kdigo clinical practice guidelines ) and bone buffering capacity , as it is becoming increasingly clear that there is a link between abnormal vascular processes ( that is , vc ) and bone metabolism ( the so - called bone vascular axis ) , and this may also have an impact on survival . vc is a common feature in both prevalent and incident dialysis patients , as well as before entering dialysis . vc has been classically associated with both atheromatosis and atherosclerosis , as well as poor outcomes . uremia is a calcifying environment and ckd has been described as the perfect storm ' for cv calcification . the presence of vc in patients with ckd is multifactorial , and several biochemical abnormalities ( excessive p loading , hyperphosphatemia , hypercalcemia , high ca p product , fgf-23 , and high and low pth levels ) have been related to clinically significant vc . it is not clear which aspect of bone pathology is most closely associated with vc . not only severe forms of shpt but also low levels of pth are associated with vc . as mentioned previously , low pth levels seem to increase the prevalence of both abd and vc , likely by preventing the ca - buffering ability of bone and the handling of any extra - ca load . using bone biopsies , tomiyama et al . found an association of vc with bone density , volume , and formation rate in ckd patients who are not yet in dialysis treatment . similarly , london et al . found an association between vc and indices of low bone turnover in the aorta and systemic arteries of dialysis patients . in an expanded cohort , london et al . reported a significant interaction between the dose of ca - containing p binders , bone activity , vc , and stiffening . of relevance was the fact that ca load had a significant impact on vc and stiffness in patients with abd when compared with patients with active bone . on the other hand , barreto et al . did not observe an association between the type of bone disease and coronary calcification on cross - sectional analysis . in a prospective study , the same authors found a negative correlation between coronary calcifications and trabecular bone volume , and low - turnover bone status at the 12-month bone biopsy was the only independent predictor for progression of coronary artery calcification . completed these results by describing an association between low bone volume , but not bone turnover , and coronary calcifications . this effect was not observed in patients who were on dialysis for more than 6 years . finally , asci et al . recently reported that bone formation rate is positively correlated with calcification scores as well as lower bone volume . when only patients with coronary calcifications were included , there was a u - shaped relationship between coronary calcifications and bone turnover , whereas the association with bone volume was lost . the discrepancies among these different studies may be due to the different vascular zones analyzed , different aluminum exposures in the past , number of parathyroidectomized patients , dialysis vintage , age , gender , diabetes , or other factors . have also shown that femoral bone density reflects histologically determined cortical bone volume , as well as associations between measurement of bone mineral density and vc or arterial stiffness . in any case , bone is currently considered an important variable when evaluating predisposing factors for vc . frequent associations of osteoporosis and atherosclerotic vc or cv disease have also been observed in postmenopausal women , as have associations between the progression of vc and bone demineralization , underlining the importance of this relationship beyond ckd . other multiple risk factors , passive and active regulated processes , have also been associated with the occurrence and/or progression of vc and , as mentioned before , they are comprehensively reviewed elsewhere in this supplement . it is important to emphasize that different vitamin d derivatives functioning on the same vdr ( selective vdr activators such as paricalcitol or maxacalcitol ) seem to have different effects on ca and p intestinal absorption at the same degree of pth suppression . thus , the affinity of paricalcitol for the vdr is three times less than that of calcitriol , but its calcemic and phosphatemic effects have been shown to be 10 times lower . the affinity of paricalcitol for vitamin d - binding protein is also three times less than that of calcitriol . beyond the effect on ca and p levels , for instance , paricalcitol or maxacalcitol appear to have fewer in vivo experimental cv or renal procalcifying effects than doxercalciferol or equivalent doses of calcitriol despite comparable serum ca p products . whether the described differential effects on bone , such as relatively lower bone suppression , contribute to this observation is not known . considering all the previous observations on biochemical parameters and the striking distinct effects on vc , compounds that selectively activate vdr might enhance cv and renal protection , providing a significant clinical benefit due to the known relationship between ca and p levels and vc and mortality . the key discoveries regarding the conversion of vitamin d to its hormonal form and its regulation , as well as the evolving picture of its molecular mechanisms of action , have been described elsewhere . nevertheless , the recognition of its role beyond regulation of ca homeostasis and bone metabolism represents a recent development . vitamin d deficiency is an increasingly recognized public health problem in the general population and in chronic diseases such as ckd . impaired vdr activation and signaling results in cellular dysfunction in several organs and biological systems , leading to an increased risk for disturbances of immunity , cancer , cell differentiation , infection , diabetes , and , in ckd , for arteriosclerosis , arterial dysfunction , and vc . the relationship between vitamin d deficiency and heart disease has been extensively reviewed by pilz et al . in this issue . vdr is ubiquitous and both vdr and 1-hydroxylase ( cyp27b1 ) are present in the cv system . the action of vitamin d in these tissues is implicated in the regulation of endothelial , vsmc , heart function , inflammatory , and fibrotic pathways and immune responses . failure of cv vdr activation is associated with many cv problems , as well as the progression of kidney and cv disease . in view of the foregoing , it seems likely that the relationship between vdr activation and hard outcomes , both in ckd and the general population , extends at least partially beyond vc and bone activity , as discussed previously . furthermore , recent epidemiological evidence suggests that there is a narrow range of vitamin d levels at which vascular function is optimized . levels above or below this range seem to confer a significant increase in risk for cv disease , and thus a u - shaped curve or a reversed j curve may describe quite well the relationship between adverse systemic or vascular effects and the vitamin d status . in figure 2 in addition to pilz et al . in this issue , the narrow relationship of the heart , cv system , and kidney with the vitamin d system has recently been reviewed . in both dialysis and non - dialysis patients , vitamin d deficiency affects survival , and activation of vdr is associated with improved survival . this observation may not be surprising , considering that shpt is associated with higher mortality even in moderate non - dialysis ckd . nevertheless , these survival benefits are observed even beyond mineral metabolism control as they are significant over a wide range of biochemical mineral metabolism parameters ( that is , higher and lower quartiles of ca , p , and pth serum levels ) , vc scores , and measures of stiffness . thus , pleiotropic effects of vitamin d likely have a role . in dialysis patients , a huge retrospective study suggested that intravenous paricalcitol may provide additional survival benefits as compared with intravenous calcitriol ; however , although consistency in the beneficial effects of vitamin d in ckd patients is important , differences among the specific vitamin d compounds are still a matter of controversy . it also has to be admitted that the overall exposure to vitamin d over time may be higher with selective vdr activators , as intermittent drug withdrawal due to hypercalcemia or hyperphosphatemia is less frequent owing to their wider therapeutic window . as therapy with vdr activators appears to be useful in preventing bone loss and in decreasing morbidity and mortality , across different biochemical mineral metabolism parameters , some consider vitamin d and/or selective vdr activation to be essential therapy in ckd regardless of bone turnover status . it is conceivable that other beneficial effects of activation of the vdr beyond bone result in some benefit even in the presence of abd . however , the current kidney disease outcomes quality initiative and kdigo recommendations limit the administration of vdr activators for the treatment of hyperparathyroidism only . moreover , if vitamin d therapy is planned in patients with ckd for reasons beyond ckd mbd , such as reduction of albuminuria in patients with type 2 diabetes mellitus , only a selective vdr activator has shown this ability in a recent prospective short - term study . in this setting , selectivity is probably desirable considering the long - term potential consequences , such as the impact on ca levels , the development of abd , or the potential effects on vc of different vdr activators . in conclusion , vdr can be activated by vitamin d or selective vdr activators . selective vdr activation is defined by different effects at the tissue and molecular ( gene ) levels . a selective effect of paricalcitol in mineral metabolism is identified by its low calcemic , low phosphatemic profile . on the basis of gene expression profiles , there are other non - mineral metabolism effects and tissue - specific differences between vitamin d and selective vdr activation . thus , treating individual components of ckd mbd is no longer recommended , and a wider perspective encompassing skeletal , renal , and cv effects is required . in this regard , selective vdr activation has been associated with survival improvement at least in dialysis patients , and new vdr activators should be considered a novel and interesting approach to enhance the standard of care in these high - risk patients .

progressive loss of kidney function leads to reduced production of calcitriol ( 1,25-dihydroxyvitamin d ; active vitamin d ) and an imbalance in serum calcium ( ca ) and phosphorus ( p ) levels , which are associated with progression of renal failure as well as increased rates of cardiovascular ( cv ) events and mortality . in addition , multifactorial hypocalcemia and resistance to parathyroid hormone ( pth ) can lead to prolonged and excessive synthesis and secretion of pth , eventually leading to development of secondary hyperparathyroidism and renal osteodystrophy . these changes associated with chronic kidney disease ( ckd ) , extending beyond bone and related biochemical abnormalities , have prompted the development of the term ckd mineral and bone disorder to describe its systemic nature . excessive p loading , among other factors , will promote vascular calcification ( vc ) , and pth production will affect bone remodeling . although administration of calcitriol increases serum ca levels and decreases pth , it is also associated with elevated ca p product . therefore , compounds that selectively activate vitamin d receptors ( vdr activators ) , potentially reducing ca p toxicity and distinctly affecting pathogenic mechanisms of vc , might enhance cv and renal protection , increase the vitamin d therapeutic window , and thus provide a significant clinical benefit . moreover , selective vdr activators have been associated with improvement in survival , at least among dialysis patients . thus , selective vdr activators should be considered a novel and interesting approach to enhance the standard of care in ckd patients .

CHRONIC KIDNEY DISEASEMINERAL AND BONE DISORDER RENAL OSTEODYSTROPHIES AND VITAMIN D Renal osteodystrophy The unsolved problem of ABD VDR activation is essential for bone formation VC, BONE ACTIVITY, AND VDR ACTIVATION VDR ACTIVATION AND VASCULAR DYSFUNCTION

multifactorial hypocalcemia , calcitriol and vitamin d deficiency , p retention and fibroblast growth factor ( fgf)-23 dysregulation , resistance to the action of pth and vitamin d , and decreased activation , decreased number , or downregulation of several related receptors ( vdr , ca - sensing receptor , fgf-23/klotho ) , among other factors , can lead to prolonged and excessive synthesis and secretion of pth , eventually leading to the development of shpt and different forms of renal osteodystrophy . bone is currently considered an active endocrine ' organ , as demonstrated by the osteocyte product fgf-23 regulating p balance , vitamin d metabolism , and pth production . beyond parathyroid and bone diseases , numerous studies have shown clearly the association between disorders of mineral metabolism ( and the therapies used to correct these abnormalities ) and fractures , extraskeletal and vascular calcification ( vc ) , progression of renal failure , cv disease , and mortality . these studies have prompted the development of the new term ckd mineral and bone disorder ( mbd ) to acknowledge its systemic consequences and damage to organs beyond bone , primarily vc . thus , ckd mbd is a real syndrome , currently defined by one or a combination of the following : ( 1 ) abnormalities of ca , p , pth , or vitamin d metabolism ; ( 2 ) abnormalities in bone turnover , mineralization , volume , linear growth , or strength ; and/or ( 3 ) vascular or other soft - tissue calcifications ( figure 1 ) . the old general term of renal osteodystrophy is currently restricted to an alteration of bone morphology in patients with ckd , representing one measure of the skeletal component of ckd mbd that is quantifiable by histomorphometry of bone biopsy . although bone biopsy is invasive and thus can not be performed easily , it is the gold standard for the diagnosis of renal osteodystrophy , as biochemical parameters do not adequately predict the underlying bone histology . this is a field with a particular need for improvement , especially taking into account the great variability of pth assays and the current scarcity of information available on bone behavior and bone disease beyond the so - called intact pth thus , a significant proportion of patients with intermediate pth levels ( that is , two to nine times the upper limit of normality for a particular assay ) present quite different rates of bone formation , from adynamic bone disease ( abd ) to normality and hyperparathyroid bone disease . it is well known that the traditional types of renal osteodystrophy have been defined on the basis of turnover and mineralization , with substantial differences in pathophysiology and treatment . a recent kidney disease : improving global outcomes ( kdigo ) report has suggested that bone biopsies in patients with ckd should be characterized by determining bone turnover ( t ) , mineralization ( m ) , and volume ( v ) ( the tmv system ) . six types of bone disorder are distinguished in the ckd mbd complex : hyperparathyroid bone disease ( high turnover , normal mineralization , and any bone volume ) ; mixed bone disease ( high turnover with mineralization defect and normal bone volume ) ; osteomalacia ( low turnover with abnormal mineralization and low - to - medium bone volume ) ; abd ( low turnover with normal mineralization and low or normal bone volume ) ; and two distinct disorders due to specific causative agents , namely , amyloid bone disease and aluminum bone disease . steadily growing proportions of elderly , diabetic , and malnourished patients may , at least partially , explain this finding . a chronic low - dose exposure with concomitant high doses of vitamin d probably favoring mineralization thus , it is important to distinguish between aluminum - induced and non - aluminum - induced forms of abd . advanced age and diabetes ( through elevated levels of advanced glycation end - products ) , glucocorticoid - induced osteoporosis , and hypoparathyroidism are associated with low bone turnover . low pth levels or bone resistance to the action of pth in ckd ( relative hypoparathyroidism ) are also regarded as important risk factors for abd , and many factors from p loading , decreased calcitriol , or uremia itself to antagonistic pth fragments , downregulation of pth receptors , or decreased pulsatility of pth have been related to skeletal or calcemic resistance to pth . several other factors have been linked to abd , such as ca loading ( via p binders or ca in the dialysate ) , vitamin d treatment ( children with high doses , high dialysate ca content , or high incidences of hypercalcemia ) , peritoneal dialysis ( dialysate ca , increased glucose , low albumin ) , acidosis , hypogonadism , inflammation , or the use of bisphosphonates in ckd patients . curiously enough , patients with idiopathic hypoparathyroidism have preserved or increased bone mass during therapy with ca and vitamin d , and patients with total parathyroidectomy do not have an associated increase in the risk of fracture . low pth is also associated with low protein intake and may be a risk factor for malnutrition . as will be mentioned later , of more importance is the existence of a relationship between abd , abnormal ca balance , calcific arteriolopathy , and vc , and the fact that a u - shaped curve depicts pth levels and mortality . thus , the causal relationship between abd and vascular disease may , at least in part , explain why not only high but also low intact pth plasma levels , as well as low pth and high serum ca levels ( a combination typical for abd ) , are associated with increased mortality . it is also well known that vitamin d not only indirectly decreases bone formation rate in hyperparathyroid bone disease , but also directly increases both bone turnover and bone growth . intermittent injections of pth also serve an antiapoptotic function to increase bone formation and bone mass in contrast to continuous pth infusions , which cause bone loss . as expected , it has been demonstrated that the actions of vdr activators on bone are more important than those expected just for the inhibition of pth , and thus the effects of active vitamin d compounds and calcimimetics on bone differ under certain experimental conditions in vivo . moreover , some of the improvement in ca and skeletal homeostasis seen in vdr - knockout mice may occur through alternative vitamin d signaling pathways . importantly , opposite effects of calcitriol and paricalcitol on the pth ratio ( whole pth(184)/carboxy - terminal pth ) have been described in patients with ckd stage 5d . finally , with regard to renal osteodystrophy , it is necessary to consider that an early diagnosis of ckd , through the generalized use of estimated glomerular filtration rate laboratory reports , will allow earlier diagnosis of biochemical shpt , abnormalities in vitamin d metabolism , and ckd mbd . it is also of particular importance to learn the effect of different treatments on bone outcomes , particularly the normalization of bone histomorphometry ( as stated in the recent kdigo clinical practice guidelines ) and bone buffering capacity , as it is becoming increasingly clear that there is a link between abnormal vascular processes ( that is , vc ) and bone metabolism ( the so - called bone vascular axis ) , and this may also have an impact on survival . the old general term of renal osteodystrophy is currently restricted to an alteration of bone morphology in patients with ckd , representing one measure of the skeletal component of ckd mbd that is quantifiable by histomorphometry of bone biopsy . although bone biopsy is invasive and thus can not be performed easily , it is the gold standard for the diagnosis of renal osteodystrophy , as biochemical parameters do not adequately predict the underlying bone histology . this is a field with a particular need for improvement , especially taking into account the great variability of pth assays and the current scarcity of information available on bone behavior and bone disease beyond the so - called intact pth thus , a significant proportion of patients with intermediate pth levels ( that is , two to nine times the upper limit of normality for a particular assay ) present quite different rates of bone formation , from adynamic bone disease ( abd ) to normality and hyperparathyroid bone disease . it is well known that the traditional types of renal osteodystrophy have been defined on the basis of turnover and mineralization , with substantial differences in pathophysiology and treatment . a recent kidney disease : improving global outcomes ( kdigo ) report has suggested that bone biopsies in patients with ckd should be characterized by determining bone turnover ( t ) , mineralization ( m ) , and volume ( v ) ( the tmv system ) . six types of bone disorder are distinguished in the ckd mbd complex : hyperparathyroid bone disease ( high turnover , normal mineralization , and any bone volume ) ; mixed bone disease ( high turnover with mineralization defect and normal bone volume ) ; osteomalacia ( low turnover with abnormal mineralization and low - to - medium bone volume ) ; abd ( low turnover with normal mineralization and low or normal bone volume ) ; and two distinct disorders due to specific causative agents , namely , amyloid bone disease and aluminum bone disease . steadily growing proportions of elderly , diabetic , and malnourished patients may , at least partially , explain this finding . a chronic low - dose exposure with concomitant high doses of vitamin d probably favoring mineralization thus , it is important to distinguish between aluminum - induced and non - aluminum - induced forms of abd . independently of ckd , advanced age and diabetes ( through elevated levels of advanced glycation end - products ) , glucocorticoid - induced osteoporosis , and hypoparathyroidism are associated with low bone turnover . low pth levels or bone resistance to the action of pth in ckd ( relative hypoparathyroidism ) are also regarded as important risk factors for abd , and many factors from p loading , decreased calcitriol , or uremia itself to antagonistic pth fragments , downregulation of pth receptors , or decreased pulsatility of pth have been related to skeletal or calcemic resistance to pth . several other factors have been linked to abd , such as ca loading ( via p binders or ca in the dialysate ) , vitamin d treatment ( children with high doses , high dialysate ca content , or high incidences of hypercalcemia ) , peritoneal dialysis ( dialysate ca , increased glucose , low albumin ) , acidosis , hypogonadism , inflammation , or the use of bisphosphonates in ckd patients . low pth is also associated with low protein intake and may be a risk factor for malnutrition . as will be mentioned later , of more importance is the existence of a relationship between abd , abnormal ca balance , calcific arteriolopathy , and vc , and the fact that a u - shaped curve depicts pth levels and mortality . thus , the causal relationship between abd and vascular disease may , at least in part , explain why not only high but also low intact pth plasma levels , as well as low pth and high serum ca levels ( a combination typical for abd ) , are associated with increased mortality . it is also well known that vitamin d not only indirectly decreases bone formation rate in hyperparathyroid bone disease , but also directly increases both bone turnover and bone growth . intermittent injections of pth also serve an antiapoptotic function to increase bone formation and bone mass in contrast to continuous pth infusions , which cause bone loss . as expected , it has been demonstrated that the actions of vdr activators on bone are more important than those expected just for the inhibition of pth , and thus the effects of active vitamin d compounds and calcimimetics on bone differ under certain experimental conditions in vivo . moreover , some of the improvement in ca and skeletal homeostasis seen in vdr - knockout mice may occur through alternative vitamin d signaling pathways . importantly , opposite effects of calcitriol and paricalcitol on the pth ratio ( whole pth(184)/carboxy - terminal pth ) have been described in patients with ckd stage 5d . finally , with regard to renal osteodystrophy , it is necessary to consider that an early diagnosis of ckd , through the generalized use of estimated glomerular filtration rate laboratory reports , will allow earlier diagnosis of biochemical shpt , abnormalities in vitamin d metabolism , and ckd mbd . it is also of particular importance to learn the effect of different treatments on bone outcomes , particularly the normalization of bone histomorphometry ( as stated in the recent kdigo clinical practice guidelines ) and bone buffering capacity , as it is becoming increasingly clear that there is a link between abnormal vascular processes ( that is , vc ) and bone metabolism ( the so - called bone vascular axis ) , and this may also have an impact on survival . vc is a common feature in both prevalent and incident dialysis patients , as well as before entering dialysis . vc has been classically associated with both atheromatosis and atherosclerosis , as well as poor outcomes . the presence of vc in patients with ckd is multifactorial , and several biochemical abnormalities ( excessive p loading , hyperphosphatemia , hypercalcemia , high ca p product , fgf-23 , and high and low pth levels ) have been related to clinically significant vc . not only severe forms of shpt but also low levels of pth are associated with vc . as mentioned previously , low pth levels seem to increase the prevalence of both abd and vc , likely by preventing the ca - buffering ability of bone and the handling of any extra - ca load . found an association of vc with bone density , volume , and formation rate in ckd patients who are not yet in dialysis treatment . reported a significant interaction between the dose of ca - containing p binders , bone activity , vc , and stiffening . frequent associations of osteoporosis and atherosclerotic vc or cv disease have also been observed in postmenopausal women , as have associations between the progression of vc and bone demineralization , underlining the importance of this relationship beyond ckd . other multiple risk factors , passive and active regulated processes , have also been associated with the occurrence and/or progression of vc and , as mentioned before , they are comprehensively reviewed elsewhere in this supplement . it is important to emphasize that different vitamin d derivatives functioning on the same vdr ( selective vdr activators such as paricalcitol or maxacalcitol ) seem to have different effects on ca and p intestinal absorption at the same degree of pth suppression . thus , the affinity of paricalcitol for the vdr is three times less than that of calcitriol , but its calcemic and phosphatemic effects have been shown to be 10 times lower . the affinity of paricalcitol for vitamin d - binding protein is also three times less than that of calcitriol . beyond the effect on ca and p levels , for instance , paricalcitol or maxacalcitol appear to have fewer in vivo experimental cv or renal procalcifying effects than doxercalciferol or equivalent doses of calcitriol despite comparable serum ca p products . considering all the previous observations on biochemical parameters and the striking distinct effects on vc , compounds that selectively activate vdr might enhance cv and renal protection , providing a significant clinical benefit due to the known relationship between ca and p levels and vc and mortality . the key discoveries regarding the conversion of vitamin d to its hormonal form and its regulation , as well as the evolving picture of its molecular mechanisms of action , have been described elsewhere . impaired vdr activation and signaling results in cellular dysfunction in several organs and biological systems , leading to an increased risk for disturbances of immunity , cancer , cell differentiation , infection , diabetes , and , in ckd , for arteriosclerosis , arterial dysfunction , and vc . failure of cv vdr activation is associated with many cv problems , as well as the progression of kidney and cv disease . in view of the foregoing , it seems likely that the relationship between vdr activation and hard outcomes , both in ckd and the general population , extends at least partially beyond vc and bone activity , as discussed previously . levels above or below this range seem to confer a significant increase in risk for cv disease , and thus a u - shaped curve or a reversed j curve may describe quite well the relationship between adverse systemic or vascular effects and the vitamin d status . in this issue , the narrow relationship of the heart , cv system , and kidney with the vitamin d system has recently been reviewed . in both dialysis and non - dialysis patients , vitamin d deficiency affects survival , and activation of vdr is associated with improved survival . nevertheless , these survival benefits are observed even beyond mineral metabolism control as they are significant over a wide range of biochemical mineral metabolism parameters ( that is , higher and lower quartiles of ca , p , and pth serum levels ) , vc scores , and measures of stiffness . in dialysis patients , a huge retrospective study suggested that intravenous paricalcitol may provide additional survival benefits as compared with intravenous calcitriol ; however , although consistency in the beneficial effects of vitamin d in ckd patients is important , differences among the specific vitamin d compounds are still a matter of controversy . it also has to be admitted that the overall exposure to vitamin d over time may be higher with selective vdr activators , as intermittent drug withdrawal due to hypercalcemia or hyperphosphatemia is less frequent owing to their wider therapeutic window . as therapy with vdr activators appears to be useful in preventing bone loss and in decreasing morbidity and mortality , across different biochemical mineral metabolism parameters , some consider vitamin d and/or selective vdr activation to be essential therapy in ckd regardless of bone turnover status . it is conceivable that other beneficial effects of activation of the vdr beyond bone result in some benefit even in the presence of abd . however , the current kidney disease outcomes quality initiative and kdigo recommendations limit the administration of vdr activators for the treatment of hyperparathyroidism only . moreover , if vitamin d therapy is planned in patients with ckd for reasons beyond ckd mbd , such as reduction of albuminuria in patients with type 2 diabetes mellitus , only a selective vdr activator has shown this ability in a recent prospective short - term study . in this setting , selectivity is probably desirable considering the long - term potential consequences , such as the impact on ca levels , the development of abd , or the potential effects on vc of different vdr activators . in conclusion , vdr can be activated by vitamin d or selective vdr activators . on the basis of gene expression profiles , there are other non - mineral metabolism effects and tissue - specific differences between vitamin d and selective vdr activation . in this regard , selective vdr activation has been associated with survival improvement at least in dialysis patients , and new vdr activators should be considered a novel and interesting approach to enhance the standard of care in these high - risk patients .

it affects 200300 million people and leads to a million deaths annually , thereby posing a global threat . the clinical symptoms of the infection are manifested during the blood stage of the parasite life cycle in the human host . in acute stages of the disease , more than one tissue types are known to be affected [ 2 , 3 ] . it was shown that during this stage of the disease , the parasitized rbcs show sequestration thereby affecting the microvasculature of heart , kidney , liver , intestines , adipose tissues , and eyes [ 46 ] . several complications arise , possibly due to the inflammatory immune response of the host which result in complications such as liver damage , renal failure , cerebral malaria , hypoglycemia and acidosis , some of which may lead to death [ 79 ] . although , the transition into these conditions is poorly understood , all of them are associated with different metabolic complications . thus , one approach towards understanding the disease progression is to understand the metabolic alterations that occur as the disease progresses towards severe stage . these alterations can be monitored at single tissue or biofluid level and/or at the level of the intertissue and/or tissue - biofluid correlation , where one focuses on more than one tissue / biofluid simultaneously . h nmr spectroscopy of tissues and biofluids followed by multivariate statistical analyses in a systems biological approach has been widely used in understanding metabolic changes . h nmr spectra provide an unbiased profile of metabolites present in the tissue or biofluids thereby providing a good platform for the study of different conditions like breast cancer , diabetes , coronary artery disease , and high blood pressure [ 1114 ] . metabolic response towards parasitic infections has also been reported in the mouse models of trypanosome and schistosome infection [ 15 , 16 ] . recently , our group has shown relevant metabolic links and distinct sexual dimorphism with the progression of malaria in the mouse model by nmr spectroscopy and multivariate statistical analysis on urine , sera , and brain . h nmr spectra from a biofluid like serum or urine at a single time point provide the snapshot of the molecular phenotype or metabotype of the organism at a global level . this , in principle , arises from the complex exchanges of the metabolites among different compartments . therefore , the understanding of metabolic processes of multicellular organisms at a global level requires the study of metabolic correlations among the tissue and biofluid metabolic profiles . montoliu et al . compared different unsupervised chemometric techniques in order to study intercompartmental metabolic analysis . in this contribution , we report a nmr - based metabolite profiling followed by the multivariate statistical analysis strategy to delineate a distinct sexual dimorphism in the liver metabolic profile in the mouse model ( balb / c mouse infected with plasmodium berghei anka ) of malaria parasite infection . furthermore , employing an unsupervised chemometric strategy ( multiway principal component analysis- mpca ) , we show the correlation of brain and liver metabolic profile with that of serum metabolic profile . the animals in the study were treated in accordance with the prescribed guidelines of the institutional animal ethics committee . briefly , 24 inbred balb / c mice ( 12 males and 12 females ) aged 6 to 8 weeks were used for the study . four infected males , four infected females and four control mice ( two males and two females ) were sacrificed on day 5 after infection . the liver samples were snap frozen and kept frozen until the preparation of perchloric acid extract as described earlier . the sera and brain sample collection and extraction were done by the method described in . h nmr spectra were acquired on 700 mhz spectrometer ( bruker biospin , germany ) equipped with broad band inverse probe using 2-dimethyl-2-silapentane-5-sulfonic acid ( dss ) as an internal standard and the d2o as the field frequency lock at 300 k. the pulse sequence used included an excitation sculpting routine for the suppression of the water signal . 1024 transients were collected into 32,768 data points using a spectral width of 12.01 ppm resulting in an acquisition time of 1.94 s. a relaxation delay of 1 s was used between consecutive pulses . the fids so obtained were subjected to an exponential multiplication leading to an additional line broadening of 0.2 hz . 2d cosy spectra were obtained for the identification of the metabolites in the following manner . in the direct dimension , a qsine function was used for processing with 2048 and 1024 data points in direct and indirect dimension , respectively . two different data reduction procedures were followed . for the purpose of single tissue analysis ( liver , for our case ) , the spectral region 9.5 ppm to 0.5 ppm was segmented in frequency regions of 0.04 ppm , and each bin was integrated using mestrec 4.7.0 . the region corresponding to water and urea ( 4.5 ppm to 6.5 ppm ) was excluded to avoid artifacts due to the water suppression and highly variable urea . certain regions of the spectra are known to have high concentration metabolites with high variance that may mask the important features in the peaks with lower concentration . the resulting integrals were normalized to total intensity and pareto scaled to generate the working data matrix . for the purpose of the intercompartmental correlation analysis , the binning procedure was done using amix 5.0 . here , a binning width of 0.003 ppm was used , and only the water region was excluded ( 4.5 ppm to 5.1 ppm ) . the bins were integrated and normalized with respect to the total integral to generate the working data matrix . the liver spectra were subjected to the principal component analysis ( pca ) and orthogonal partial least square - discriminant analysis ( opls - da ) , which were performed using simca - p 12.0 ( umetrics , sweden ) . briefly , pca is an unsupervised method which is used for revealing any inherent trend or pattern in the data set whereas , opls - da is a supervised method . here , the class entity towards the sample set is provided a priori , thereby showing the class - specific distinction . q(cum ) is the diagnostic parameter for the opls - da model which represents the separation of the classes assigned . the models were set up in the following ways : ( 1 ) using data from uninfected males and day 5 post - infection males , ( 2 ) using data from uninfected males and day 13 post - infection males , ( 3 ) same as ( 1 ) for females , and ( 4 ) same as ( 3 ) for females . initially , pca was employed ( data not shown ) in order to find out any hidden trend in the data and to find outliers . the visualization was aided by two - dimensional scores plot that shows the sample clustering in a two - dimensional space . the variables contributing towards the clustering in the scores plot were extracted using loadings s - plot and vip plot . the metabolites were identified using human metabolome database ( hmdb ) along with the two - dimensional nmr spectral profiles . after extracting the metabolites from the multivariate analytical techniques ( pca and opls - da ) , individual peaks from the metabolites were selected in the one - dimensional nmr spectra , and they were integrated using topspin 2.1 . the crowding in the nmr spectral profile sometimes resulted in overlap of the peaks . in those cases , the peak intensities of individual metabolites were calculated with respect to the intensity of internal standard dss and normalized to the total tissue weight . significance test for comparison early- and late - stage infection time points were compared with uninfected controls of the same sex . if the relative peak intensities of a metabolite were comparable ( p > .15 ) between male and female uninfected samples , then a t - test was conducted to compare males and females at each post - infection time point . if the levels of the metabolite were different ( p < .15 ) between uninfected males and females , then from each post - infection data point the value of the corresponding control average was subtracted . an average of these normalized values was taken and males and females were now compared for each post - infection time point to determine if the deviation from corresponding controls was significantly different between them . multiway pca ( mpca ) as described elsewhere was used for the intercompartmental correlation analysis . initially a multivariate curve resolution - alternating least square algorithm ( mcr - als ) was employed to check the relative contribution of the two relevant compartments . ( 1 ) liver and serum and ( 2 ) brain and serum . in each of these categories , three different models were made for males and females , ( a ) controls , ( b ) day 5 after infection , and ( c ) day 13 after infection . this helps in the visualization of how the intercompartmental correlation changes as the disease progresses . for this purpose , a 3d matrix was prepared from the spectral integrals mentioned earlier using matlab 7.0.1 . in this matrix , the rows are the samples / animals , the columns are the spectral variables , and the third mode is the tissues / biofluids . results for the serum and brain analysis have already been discussed elsewhere . here , we report the alteration of metabolic profile of liver and the intercompartmental correlation of brain and liver with sera . the liver extracts for the males showed no perturbation when the early infection time points were compared with uninfected animals ( q(cum ) = 0.882 ) . however , in the early infection time points , females showed a large change from that of uninfected controls ( q(cum ) = 0.988 ) . when the late - stage animals were compared with the uninfected controls , drastic changes were observed irrespective of the sex ( q(cum for males = 0.975 and q(cum ) for females = 0.993 ) . the representative scores plot of the respective opls - da models is shown in the figure 1 . the vip plots ( see supplementary information 1 available at doi:10.1155/2011/901854 ) and loadings s - plot ( supplementary information 2 ) were further investigated in order to extract the most significant spectral variables / bins . these bins were further analyzed by the help of hmdb and two - dimensional nmr spectral profiles to generate the significant metabolites that contribute towards the pattern seen in the opls - da models . table 1 shows a list of the metabolites perturbed in the liver as the disease progresses in both the sexes . however , as the early infection stage of the males showed insignificant variation ( q(cum)=0.882 , figure 1(a ) ) from the control animals , hence , metabolites corresponding to that stage are not investigated . further , the specific peaks from the one - dimensional nmr profiles were integrated with respect to the total intensity of the internal standard ( dss ) . many of the metabolites identified from the opls - da model showed statistically significant difference as the disease progresses ( figure 2 ) . moreover , analysis was done to compare some of the metabolites in the two post - infection stages which were different in the uninfected males and females to check whether they deviate significantly from the same sex control . opls - da analysis of liver spectra showed some of the metabolites ( table 1 ) which were not observed in other tissues or biofluids . however , although the opls - da analysis showed a large difference in the early infection stage of females and uninfected controls ( table 1 ) , some of the metabolites that were expected to be increased in the infected animals showed a large deviation in the vip ( supplementary information 1 ) . therefore , opls - da was unable to predict the dramatic change in the early infected females with certainty ; hence , the early - stage changes in the female liver remain to be characterized . in the late stage , the liver extracts showed an increase in 2-hydroxy-2-methylbutyrate and beta - alanine ( figures 2(a ) and 2(e ) ) and a decrease in an unidentified compound at 3.82 ppm ( figure 2(f ) ) in both males and females . along with these , females showed increase in kynurenic acid in the late stage ( figure 2(h ) ) . increase in two unidentified peaks at 2.34 and 3.86 ppm was also shown by the females ( figures 2(b ) and 2(g ) ) . in spite of the poor difference shown in the opls - da between the male control and early - stage infected animals ( figure 1 ) , some of the metabolites showed significant changes . these include asparagine , dmg , and creatine ( figures 2(c ) and 2(d ) ) , all of which are significantly decreased from the control animals in the early stage . comparisons of the individual metabolite level of the animals were also made with that of the controls ( figure 3 ) , and several interesting results were obtained . this was done for the fact that several metabolites in the liver showed significant difference in their levels between male and female control animals ( data not shown ) . therefore , we investigated whether the post - infection alterations are different between males and females . several of the changes observed in such analysis were often only in the early - stage . females showed a slight increase from the corresponding controls , while males showed a decrease in the carnitine level at this stage ( figure 3(b ) ) . kynurenic acid levels in females did not change much from their controls whereas the males showed a decrease ( figure 3(f ) ) . asparagine , dmg and o - phosphoethanolamine levels were seen to be reduced significantly more in the early stage infected male liver samples as compared to females ( figures 3(c ) and 3(e ) ) . for example , an unidentified peak at 2.34 ppm showed decrease in the males while slight increase in the females compared to the corresponding controls ( figure 3(a ) ) . similar trend was observed for carnitine ( figure 3(b ) ) , serine and dmg ( figure 3(d ) ) . interesting here to note that is although the global metabolic profile of the male controls does not differ much from the male early infection animals , some of the individual metabolites show a significant alteration ( figures 2(c ) , 2(d ) , and 3 ) . however , these alterations are possibly not enough to have an impact on the differential global metabolic profile ( figures 1(a ) and 1(b ) ) . mpca has been established as useful tool for correlation analysis between tissues and circulating biofluids . in this paper , we have used the technique to delineate the effect of disease progression on the correlation of metabolites across tissues / biofluids . we used earlier data of serum and brain and the liver data described here to investigate the correlations of metabolites in brain and liver to that of serum with the disease progression in both the sexes . earlier studies made by our group showed that the brain metabolic profile is significantly altered in both the sexes . although this model of malaria is not a cerebral variety , it is important to understand how the brain metabolic profile varies with the circulating biofluid . initially , mcr - als algorithm ( data not shown ) was employed that showed appreciable contribution of one compartment on the spectrotype of the other . further , mpca models were built using the nmr metabolite profiles of the sera and the relevant tissue from a set of animals ( e.g. , 4 female controls , sera , and brain nmr metabolite profile ) . 12 such models were generated ( control , early- and late - stage infection for males and females ) for both types of correlations ( brain - sera and liver - sera ) to be addressed . the first two principal components ( pcs ) were calculated for each of them . the loadings in pc2 described the correlation of the nmr spectral peaks across the relevant tissue ( either liver or brain ) and serum . in female control animals , high concentration of branched chain amino acids ( bcaa ) ( 1 ) leucine , isoleucine , valine , choline ( 2 ) and a low concentration of glucose ( 3 ) in the liver were associated with low lactate ( 4 ) , glucose ( 3 ) and high concentration of lysine ( 5 ) in serum ( figure 4(a ) ) . there was a peak at 3.20 ppm ( close to choline peak ) which could not be identified . the females at early - stage infection showed essentially a similar serum profile to that of control animals , with an exception of a high dimethylamine ( dma ) ( 9 ) in the serum ( figure 4(b ) ) . however , the liver profile of these animals showed high branched chain amino acids ( 1 ) , alanine ( 6 ) , trimethylamine - n - oxide ( tmao ) ( 7 ) , glycine ( 8) and lactate ( 4 ) ( figure 4(b ) ) . in the late - stage infection of the females , the liver showed a high choline ( 2 ) and glucose ( 3 ) which were associated with low lactate ( 4 ) and high glucose ( 3 ) level in the serum ( figure 4(c ) ) . male control animals showed a different profile than that of the females . in the liver of the male control animals isoleucine ( 1 as bcaa ) , leucine ( 1 as bcaa ) , lactate ( 4 ) , choline ( 2 ) and , tmao ( 7 ) were low and glucose ( 3 ) was high . these were correlated to a low lactate ( 4 ) and high dma ( 9 ) in the serum ( figure 4(d ) ) . when the early - stage infection for the male animals was considered , the liver profile showed increase in bcaa ( 1 ) , alanine ( 6 ) , choline ( 2 ) , and lactate ( 4 ) compared to the control animals , with the contribution of tmao ( 7 ) becoming insignificant . the serum profile for this group of animals showed a high dma ( 9 ) and low choline ( 2 ) level with respect to the liver profile ( figure 4(e ) ) . the late - stage infection for the males was also relatively simple with low glucose ( 3 ) in liver being associated to high glucose ( 3 ) , lactate ( 4 ) and low branched chain amino acids ( 1 ) in the serum ( figure 4(f ) ) . our earlier studies ( 15 ) showed a significant deviation of the brain metabolic profile in the early - stage infection as well as the late - stage infection from the control animals . thus , it would be interesting to see whether the serum- brain intercompartmental correlation is also affected with the disease progression . therefore , the correlation of the brain metabolic profile with that of serum was assessed in order to delineate the crosstalk between these two biological compartments . the female control animals showed a high lactate ( 4 ) , n - acetyl - aspartate ( naa , 10 ) , sarcosine ( 11 ) , and low choline ( 2 ) in the brain that correlated with high lysine ( 5 ) and low lactate ( 4 ) and glucose ( 3 ) in the serum ( figure 5(a ) ) . in the early infection , the brain profile showed high lactate ( 4 ) , dma ( 9 ) , creatine ( 12 ) , and betaine ( 13 ) . however , the serum profile remained similar to that of controls , although it showed a high dma ( 9 ) ( figure 5(b ) ) . in the late - stage , the brain metabolic profile showed high values of lactate ( 4 ) , naa ( 10 ) , dma ( 9 ) , creatine ( 12 ) , and choline ( 2 ) . these were found to be significantly correlated with the high glucose ( 3 ) and low lactate ( 4 ) in the serum ( figure 5(c ) ) . they showed a low lactate ( 4 ) , naa ( 10 ) , dma ( 9 ) , choline ( 2 ) , and creatine ( 12 ) in the brain profile associated to a low lactate ( 4 ) and high dma ( 9 ) in the serum ( figure 5(d ) ) . the males showed change in the serum profile only in terms of a high lactate and low choline level , and the rest of the profile remained quite similar at the early - stage infection . at this stage , they showed a low creatine ( 12 ) and high dma ( 9 ) in the brain ( figure 5(e ) ) . the late infection stage showed a low lactate ( 4 ) , high creatine ( 12 ) , and dma ( 9 ) in the brain and high lactate ( 4 ) , glucose ( 3 ) and low bcaa in the serum ( figure 5(f ) ) . figure 4 ( liver - serum correlation ) and figure 5 ( brain - serum correlation ) show a comprehensive visualization of all the increased and decreased metabolites in different compartments during the various stages of the disease . important to note here is that the set of metabolites that were found to be significant from the opls - da ( table 1 , figures 2 and 3 for liver metabolites and for brain metabolites ) study are different from those found from mpca ( figures 4 and 5 ) . it is important to realize at this point that opls - da showed a set of metabolites that were significantly altered among the infected and control sets in a single tissue or biofluid . however , mpca resulted in a set of metabolites which are significant in terms of the intercompartmental correlation . in this paper , we report our results on the alterations of the liver metabolism during the progress of malaria as well as the change in the correlation of the metabolic profile of the liver and brain with that of blood serum as the disease progresses . murine model ( balb / c mice infected with p. berghei anka ) using animals from both the sexes used for this purpose . earlier results from our group suggested that the host response towards the infection exhibits sexual dimorphism in the urine and serum metabolic profile . the liver profile in the female mice was altered in the early stage of the disease as compared to the males ( figure 1 ) . liver being the regulatory organ for metabolic activities , a drastic change in its metabolic profile in the early stage of the disease raises the possibility that the female hosts are able to respond to the infection by maintaining the homeostasis within them . this is supported by our earlier result that the female hosts show no early changes of the metabolic profile of the serum , while the males do . at the late stage , drastic changes in the metabolic alteration are observed irrespective of the sex of the host ( figure 1 ) , which is anticipated because of the fact that the animals are very ill at this time point . our earlier findings indicated several alterations in the metabolic pathways during the progression of the disease . among them , glucose metabolism , amino acid metabolism , kynurenine pathway , uracil degradation , and fatty acid metabolism were found to be significant . these observations were based on the alteration on the urine , sera , and brain metabolic profile . glucose metabolism is known to be perturbed during malaria parasite infection largely due to the enhanced rate of glycolysis [ 1921 ] . our earlier results in the serum and brain metabolic profile showed a sexual dimorphism in lactate levels of those tissues . however , the comparison made with the liver metabolic profile does not show a pronounced increase in the lactate level . instead , in the late - stage the lactate in the liver is shown to be decreased in males as well as females ( table 1 ) . enhancement of rate of glycolysis leads to an increased formation of pyruvate which may be transaminated to alanine . alanine did not show the trend like lactate ( table 1 ) . in the early - stage female animals the change in the alanine level is an indicator of the alteration of circulating amino acid levels . . liver being the metabolic regulatory organ , alteration in the level of the amino acids in the liver is expected . along with alanine , the level of serine is also seen to be altered in the liver extracts . in the males , it is decreased , and in the females , the level is increased marginally when compared to the corresponding control thereby showing a sexual dimorphism ( figure 3(d ) ) . asparagine level also showed a significantly greater decrease in the males compared to the females with respect to the corresponding controls ( figure 3(c ) ) . are known to incorporate disproportionately a large amount of asparagine in their proteome in the ubiquitous low - complexity region . along with the amino acid mentioned , leucine levels was also increased in both males and females in the late stage ( table 1 ) . however , alteration in the branched chain amino acids and aromatic amino acids were reported in context of trypanosome infection also [ 23 , 24 ] . we were the first to observe the increased excretion of kynurenic acid ( ka ) and quinolinic acid in the male infected mice . in the sera and brain when liver extracts are considered , ka exhibited a sexual dimorphism in the early - stage liver that its level in the infected male liver extracts decreased , while those in female did not ( figure 2(h ) ) . the kynurenine pathway is activated by the immunological factor ifn- ; therefore ; it is systematically upregulated during the activation of the immune response under multiple disease condition . therefore , an interesting question that remains to be asked is whether the sexual dimorphism that we see in the pathway is a cause for the differential immunological activation between males and females . several components of fatty acid metabolic pathway are indicated to be perturbed in the liver extracts . a rise in fatty acid concentration in the male and female liver is indicated by the 0.9 ppm peak ( table 1 ) . oxidation of these fatty acids would entail generation of carnitine which we reported to be decreased in the urine of female mice . the liver levels of carnitine also showed a sexual dimorphism in the early stage ( figure 3(b ) ) . whereas the males showed a slight decrease , the females demonstrated a slight increase from the corresponding controls . along with this , choline was indicated to be increased in the early - stage infected females ( table 1 ) . o - phosphoethanolamine was detected in the liver sample to show a sexual dimorphism in the late stage ( figure 3(e ) ) along with 2-hydroxy-2-methylbutyrate which was increased significantly in both the sexes in the late stage ( figure 2(a ) ) . the crosstalk ( correlation ) of metabolites across organs and biofluids is an important parameter in their changing dynamical interaction during disease progression . for this purpose , we analyzed the correlation of the liver and brain metabolic profile with that of blood serum . previously , we delineated the effect of disease progression on the brain metabolic profile individually . important to note is that at the late stage of infection , the correlation profiles look drastically different than that of the controls ( figures 4(c ) , 4(f ) , 5(c ) , and 5(f ) ) . therefore , we would concentrate more on the early - stage correlation profile . as noted earlier the liver serum correlation of the female animal is distinct from that of the males in the control animals ( figures 4(a ) and 4(d ) ) . initial study on the liver profile of the control animals showed no distinction between the two sexes ( data not shown ) , and similar results were observed for the serum profile . thus , it is evident that the dynamic intercompartmental correlation is maintained differently in the two sexes which result in similar global metabolic profile of the individual compartment . for example , in both of them , the amino acids ( 1 ) in the liver are anticorrelated to the glucose ( 3 ) in the liver . males showed a low amino acid concentration to be correlated to a high glucose concentration ( figure 4(d ) ) whereas females showed the opposite ( figure 4(a ) ) . in addition , males showed a low serum lactate ( 4 ) to be correlated with high liver glucose ( 3 ) ( figure 4(d ) ) , and females showed a low serum lactate ( 4 ) to be correlated with low liver glucose ( 3 ) . this suggests a complex glycolytic - gluconeogenetic relationship which keeps the global metabolic profile of the individual tissue and/or biofluid more or less similar between the two sexes . the data presented here also suggests that this correlation is compromised during the course of the disease in some way or another . for the females in the early stage , the serum compartment of the correlation profile looks more or less the same except for an increased contribution from the dma ( 9 ) , whereas there are notable changes in the liver segments of the profile ( figures 4(a ) and 4(b ) ) . however , in the males , both serum and liver segment showed notable changes ( figures 4(d ) and 4(e ) ) . therefore , we observe a sexual dimorphism even in the intercompartmental correlation status when the liver and serum are considered . this may implicate that the amount of circulating metabolites in the females remain under homeostasis , while their metabolism in the liver is altered drastically . this supports the hypothesis that there is significant amount of change in the early - stage female liver in order to maintain the homeostasis in the circulating fluid in response to the pathogen . in the female early - stage infection an increased alanine ( 6 ) , tmao ( 7 ) , glycine ( 8) , and lactate ( 4 ) in the liver were correlated with an increased dma ( 9 ) in the serum . dma is a metabolic product of tmao , so an increased dma in the serum can be correlated with an increased tmao in the liver . tmao has been reported to be associated with kidney disease and is used by the body as an osmolyte to counter - act the accumulation of urea due to renal failure . this refers to the fact that the females can or at least try to maintain other organs under functional state at the early stage of the disease . as noted earlier , lactate did not show a significant contribution towards the differentiation of infected and control animals ; however , in the correlation analysis , we observe an inverse relation of the liver and serum lactate for the early - stage females ( figure 4(b ) ) . here , a relative buildup in the liver lactate is associated with the depletion of the serum lactate . it is possible that the heavy utilization of glucose in the blood is counteracted by the host by enhancing the rate of gluconeogenesis in the liver . however , if that is the case , then one would expect an opposite relation between the liver and serum lactate to that we see here . our result , therefore , suggests a more intricate functional and spatiotemporal relationship between the liver and serum lactate . for example , it might be possible that an alteration in the cori cycle pathway changes the uptake of lactate from serum into liver . however , the details of this process remain to be investigated . in males , however , both the liver and serum profile changes in the early stage of infection ( figure 4(e ) ) , referring to the fact that the males are unable to maintain the blood homeostasis . moreover , the liver metabolism of these animals is altered drastically . here , the choline of liver is found to be anticorrelated to the choline of serum . precisely , an increased choline in the liver is associated with a decreased choline in the serum . this may be due to the fact that the intraerythrocytic parasite causes a 5-fold increase in the phospholipid content of the parasitized cell , which may eventually lead to a phospholipid degradation in the liver of the host , thereby generating more of the phosphocholine components . as far as the brain to serum correlation analysis is concerned , for the females , the contribution from the serum towards the brain - serum crosstalk does not alter much ; however , the brain profile did change notably ( figures 5(a ) and 5(b ) ) . for the males , both the brain and serum profile was altered ( figures 5(d ) and 5(e ) ) , although the early - stage brain profile looks similar to that of control animals . earlier report showed that the brain profile in the early - stage males and females did not change significantly . however , this data suggests that the brain profile is maintained by the aid of a complex relationship with the serum . in the females , as noted earlier , the homeostasis of the serum is maintained , while for the males , the brain profile is maintained at the cost of serum homeostasis . in females , the brain showed a relative increase in the lactate and creatine , whereas the males showed a relative decrease of creatine . this indicates that the energy metabolism of the brain is altered in the early stage of the infection in both sexes . however , the alteration in the energy metabolism probably bears different meanings in the two sexes . in the males , this is an effect of the maintenance of brain homeostasis at the cost of serum homeostasis , whereas , for the females it is the other way around . this paper presents the initial understanding of the complex interplay of the various biological compartments during the progression of malaria in mice . this , therefore , opens up the possibility of a greater insight into the changes in the metabolic pathways as a response to malarial parasite infection .

1h nmr - based metabonomics was used to investigate the multimodal response of mice to malarial parasite infection by p. berghei anka . liver metabolism was followed by nmr spectroscopy through the course of the disease in both male and female mice . our results showed alterations in the level of several metabolites as a result of the infection . metabolites like kynurenic acid , alanine , carnitine , and -alanine showed significant alteration in the liver , suggesting altered kynurenic acid , glucose , fatty acid and amino acid pathways . distinct sexual dimorphism was also observed in the global analysis of the liver metabolic profiles . multiway principal component analysis ( mpca ) was carried out on the liver , brain , and serum metabolic profile in order to explore the correlation of liver and brain metabolic profile to the metabolite profile of serum . changes in such correlation profile also indicated distinct sexual dimorphism at the early stage of the disease . indications are that the females are able to regulate their metabolism in the liver in such a way to maintain homeostasis in the blood . in males , however , choline in liver showed anticorrelation to choline content of serum indicating a higher phospholipid degradation process . the brain - serum correlation profile showed an altered energy metabolism in both the sexes . the differential organellar responses during disease progression have implications in malaria management .

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

the clinical symptoms of the infection are manifested during the blood stage of the parasite life cycle in the human host . it was shown that during this stage of the disease , the parasitized rbcs show sequestration thereby affecting the microvasculature of heart , kidney , liver , intestines , adipose tissues , and eyes [ 46 ] . several complications arise , possibly due to the inflammatory immune response of the host which result in complications such as liver damage , renal failure , cerebral malaria , hypoglycemia and acidosis , some of which may lead to death [ 79 ] . these alterations can be monitored at single tissue or biofluid level and/or at the level of the intertissue and/or tissue - biofluid correlation , where one focuses on more than one tissue / biofluid simultaneously . h nmr spectra provide an unbiased profile of metabolites present in the tissue or biofluids thereby providing a good platform for the study of different conditions like breast cancer , diabetes , coronary artery disease , and high blood pressure [ 1114 ] . recently , our group has shown relevant metabolic links and distinct sexual dimorphism with the progression of malaria in the mouse model by nmr spectroscopy and multivariate statistical analysis on urine , sera , and brain . in this contribution , we report a nmr - based metabolite profiling followed by the multivariate statistical analysis strategy to delineate a distinct sexual dimorphism in the liver metabolic profile in the mouse model ( balb / c mouse infected with plasmodium berghei anka ) of malaria parasite infection . furthermore , employing an unsupervised chemometric strategy ( multiway principal component analysis- mpca ) , we show the correlation of brain and liver metabolic profile with that of serum metabolic profile . for the purpose of single tissue analysis ( liver , for our case ) , the spectral region 9.5 ppm to 0.5 ppm was segmented in frequency regions of 0.04 ppm , and each bin was integrated using mestrec 4.7.0 . the liver spectra were subjected to the principal component analysis ( pca ) and orthogonal partial least square - discriminant analysis ( opls - da ) , which were performed using simca - p 12.0 ( umetrics , sweden ) . the models were set up in the following ways : ( 1 ) using data from uninfected males and day 5 post - infection males , ( 2 ) using data from uninfected males and day 13 post - infection males , ( 3 ) same as ( 1 ) for females , and ( 4 ) same as ( 3 ) for females . initially , pca was employed ( data not shown ) in order to find out any hidden trend in the data and to find outliers . multiway pca ( mpca ) as described elsewhere was used for the intercompartmental correlation analysis . here , we report the alteration of metabolic profile of liver and the intercompartmental correlation of brain and liver with sera . table 1 shows a list of the metabolites perturbed in the liver as the disease progresses in both the sexes . however , as the early infection stage of the males showed insignificant variation ( q(cum)=0.882 , figure 1(a ) ) from the control animals , hence , metabolites corresponding to that stage are not investigated . opls - da analysis of liver spectra showed some of the metabolites ( table 1 ) which were not observed in other tissues or biofluids . however , although the opls - da analysis showed a large difference in the early infection stage of females and uninfected controls ( table 1 ) , some of the metabolites that were expected to be increased in the infected animals showed a large deviation in the vip ( supplementary information 1 ) . therefore , opls - da was unable to predict the dramatic change in the early infected females with certainty ; hence , the early - stage changes in the female liver remain to be characterized . in the late stage , the liver extracts showed an increase in 2-hydroxy-2-methylbutyrate and beta - alanine ( figures 2(a ) and 2(e ) ) and a decrease in an unidentified compound at 3.82 ppm ( figure 2(f ) ) in both males and females . in spite of the poor difference shown in the opls - da between the male control and early - stage infected animals ( figure 1 ) , some of the metabolites showed significant changes . these include asparagine , dmg , and creatine ( figures 2(c ) and 2(d ) ) , all of which are significantly decreased from the control animals in the early stage . comparisons of the individual metabolite level of the animals were also made with that of the controls ( figure 3 ) , and several interesting results were obtained . this was done for the fact that several metabolites in the liver showed significant difference in their levels between male and female control animals ( data not shown ) . several of the changes observed in such analysis were often only in the early - stage . asparagine , dmg and o - phosphoethanolamine levels were seen to be reduced significantly more in the early stage infected male liver samples as compared to females ( figures 3(c ) and 3(e ) ) . for example , an unidentified peak at 2.34 ppm showed decrease in the males while slight increase in the females compared to the corresponding controls ( figure 3(a ) ) . interesting here to note that is although the global metabolic profile of the male controls does not differ much from the male early infection animals , some of the individual metabolites show a significant alteration ( figures 2(c ) , 2(d ) , and 3 ) . however , these alterations are possibly not enough to have an impact on the differential global metabolic profile ( figures 1(a ) and 1(b ) ) . in this paper , we have used the technique to delineate the effect of disease progression on the correlation of metabolites across tissues / biofluids . we used earlier data of serum and brain and the liver data described here to investigate the correlations of metabolites in brain and liver to that of serum with the disease progression in both the sexes . earlier studies made by our group showed that the brain metabolic profile is significantly altered in both the sexes . initially , mcr - als algorithm ( data not shown ) was employed that showed appreciable contribution of one compartment on the spectrotype of the other . the loadings in pc2 described the correlation of the nmr spectral peaks across the relevant tissue ( either liver or brain ) and serum . in female control animals , high concentration of branched chain amino acids ( bcaa ) ( 1 ) leucine , isoleucine , valine , choline ( 2 ) and a low concentration of glucose ( 3 ) in the liver were associated with low lactate ( 4 ) , glucose ( 3 ) and high concentration of lysine ( 5 ) in serum ( figure 4(a ) ) . the females at early - stage infection showed essentially a similar serum profile to that of control animals , with an exception of a high dimethylamine ( dma ) ( 9 ) in the serum ( figure 4(b ) ) . however , the liver profile of these animals showed high branched chain amino acids ( 1 ) , alanine ( 6 ) , trimethylamine - n - oxide ( tmao ) ( 7 ) , glycine ( 8) and lactate ( 4 ) ( figure 4(b ) ) . in the late - stage infection of the females , the liver showed a high choline ( 2 ) and glucose ( 3 ) which were associated with low lactate ( 4 ) and high glucose ( 3 ) level in the serum ( figure 4(c ) ) . in the liver of the male control animals isoleucine ( 1 as bcaa ) , leucine ( 1 as bcaa ) , lactate ( 4 ) , choline ( 2 ) and , tmao ( 7 ) were low and glucose ( 3 ) was high . when the early - stage infection for the male animals was considered , the liver profile showed increase in bcaa ( 1 ) , alanine ( 6 ) , choline ( 2 ) , and lactate ( 4 ) compared to the control animals , with the contribution of tmao ( 7 ) becoming insignificant . our earlier studies ( 15 ) showed a significant deviation of the brain metabolic profile in the early - stage infection as well as the late - stage infection from the control animals . therefore , the correlation of the brain metabolic profile with that of serum was assessed in order to delineate the crosstalk between these two biological compartments . the female control animals showed a high lactate ( 4 ) , n - acetyl - aspartate ( naa , 10 ) , sarcosine ( 11 ) , and low choline ( 2 ) in the brain that correlated with high lysine ( 5 ) and low lactate ( 4 ) and glucose ( 3 ) in the serum ( figure 5(a ) ) . in the early infection , the brain profile showed high lactate ( 4 ) , dma ( 9 ) , creatine ( 12 ) , and betaine ( 13 ) . in the late - stage , the brain metabolic profile showed high values of lactate ( 4 ) , naa ( 10 ) , dma ( 9 ) , creatine ( 12 ) , and choline ( 2 ) . they showed a low lactate ( 4 ) , naa ( 10 ) , dma ( 9 ) , choline ( 2 ) , and creatine ( 12 ) in the brain profile associated to a low lactate ( 4 ) and high dma ( 9 ) in the serum ( figure 5(d ) ) . the males showed change in the serum profile only in terms of a high lactate and low choline level , and the rest of the profile remained quite similar at the early - stage infection . the late infection stage showed a low lactate ( 4 ) , high creatine ( 12 ) , and dma ( 9 ) in the brain and high lactate ( 4 ) , glucose ( 3 ) and low bcaa in the serum ( figure 5(f ) ) . figure 4 ( liver - serum correlation ) and figure 5 ( brain - serum correlation ) show a comprehensive visualization of all the increased and decreased metabolites in different compartments during the various stages of the disease . in this paper , we report our results on the alterations of the liver metabolism during the progress of malaria as well as the change in the correlation of the metabolic profile of the liver and brain with that of blood serum as the disease progresses . murine model ( balb / c mice infected with p. berghei anka ) using animals from both the sexes used for this purpose . earlier results from our group suggested that the host response towards the infection exhibits sexual dimorphism in the urine and serum metabolic profile . the liver profile in the female mice was altered in the early stage of the disease as compared to the males ( figure 1 ) . liver being the regulatory organ for metabolic activities , a drastic change in its metabolic profile in the early stage of the disease raises the possibility that the female hosts are able to respond to the infection by maintaining the homeostasis within them . this is supported by our earlier result that the female hosts show no early changes of the metabolic profile of the serum , while the males do . at the late stage , drastic changes in the metabolic alteration are observed irrespective of the sex of the host ( figure 1 ) , which is anticipated because of the fact that the animals are very ill at this time point . our earlier findings indicated several alterations in the metabolic pathways during the progression of the disease . among them , glucose metabolism , amino acid metabolism , kynurenine pathway , uracil degradation , and fatty acid metabolism were found to be significant . these observations were based on the alteration on the urine , sera , and brain metabolic profile . our earlier results in the serum and brain metabolic profile showed a sexual dimorphism in lactate levels of those tissues . however , the comparison made with the liver metabolic profile does not show a pronounced increase in the lactate level . instead , in the late - stage the lactate in the liver is shown to be decreased in males as well as females ( table 1 ) . in the early - stage female animals the change in the alanine level is an indicator of the alteration of circulating amino acid levels . liver being the metabolic regulatory organ , alteration in the level of the amino acids in the liver is expected . along with alanine , the level of serine is also seen to be altered in the liver extracts . in the males , it is decreased , and in the females , the level is increased marginally when compared to the corresponding control thereby showing a sexual dimorphism ( figure 3(d ) ) . asparagine level also showed a significantly greater decrease in the males compared to the females with respect to the corresponding controls ( figure 3(c ) ) . along with the amino acid mentioned , leucine levels was also increased in both males and females in the late stage ( table 1 ) . in the sera and brain when liver extracts are considered , ka exhibited a sexual dimorphism in the early - stage liver that its level in the infected male liver extracts decreased , while those in female did not ( figure 2(h ) ) . therefore , an interesting question that remains to be asked is whether the sexual dimorphism that we see in the pathway is a cause for the differential immunological activation between males and females . several components of fatty acid metabolic pathway are indicated to be perturbed in the liver extracts . a rise in fatty acid concentration in the male and female liver is indicated by the 0.9 ppm peak ( table 1 ) . the liver levels of carnitine also showed a sexual dimorphism in the early stage ( figure 3(b ) ) . along with this , choline was indicated to be increased in the early - stage infected females ( table 1 ) . o - phosphoethanolamine was detected in the liver sample to show a sexual dimorphism in the late stage ( figure 3(e ) ) along with 2-hydroxy-2-methylbutyrate which was increased significantly in both the sexes in the late stage ( figure 2(a ) ) . for this purpose , we analyzed the correlation of the liver and brain metabolic profile with that of blood serum . previously , we delineated the effect of disease progression on the brain metabolic profile individually . important to note is that at the late stage of infection , the correlation profiles look drastically different than that of the controls ( figures 4(c ) , 4(f ) , 5(c ) , and 5(f ) ) . therefore , we would concentrate more on the early - stage correlation profile . as noted earlier the liver serum correlation of the female animal is distinct from that of the males in the control animals ( figures 4(a ) and 4(d ) ) . initial study on the liver profile of the control animals showed no distinction between the two sexes ( data not shown ) , and similar results were observed for the serum profile . thus , it is evident that the dynamic intercompartmental correlation is maintained differently in the two sexes which result in similar global metabolic profile of the individual compartment . for example , in both of them , the amino acids ( 1 ) in the liver are anticorrelated to the glucose ( 3 ) in the liver . this suggests a complex glycolytic - gluconeogenetic relationship which keeps the global metabolic profile of the individual tissue and/or biofluid more or less similar between the two sexes . the data presented here also suggests that this correlation is compromised during the course of the disease in some way or another . for the females in the early stage , the serum compartment of the correlation profile looks more or less the same except for an increased contribution from the dma ( 9 ) , whereas there are notable changes in the liver segments of the profile ( figures 4(a ) and 4(b ) ) . however , in the males , both serum and liver segment showed notable changes ( figures 4(d ) and 4(e ) ) . therefore , we observe a sexual dimorphism even in the intercompartmental correlation status when the liver and serum are considered . this may implicate that the amount of circulating metabolites in the females remain under homeostasis , while their metabolism in the liver is altered drastically . this supports the hypothesis that there is significant amount of change in the early - stage female liver in order to maintain the homeostasis in the circulating fluid in response to the pathogen . in the female early - stage infection an increased alanine ( 6 ) , tmao ( 7 ) , glycine ( 8) , and lactate ( 4 ) in the liver were correlated with an increased dma ( 9 ) in the serum . this refers to the fact that the females can or at least try to maintain other organs under functional state at the early stage of the disease . as noted earlier , lactate did not show a significant contribution towards the differentiation of infected and control animals ; however , in the correlation analysis , we observe an inverse relation of the liver and serum lactate for the early - stage females ( figure 4(b ) ) . here , a relative buildup in the liver lactate is associated with the depletion of the serum lactate . it is possible that the heavy utilization of glucose in the blood is counteracted by the host by enhancing the rate of gluconeogenesis in the liver . however , if that is the case , then one would expect an opposite relation between the liver and serum lactate to that we see here . our result , therefore , suggests a more intricate functional and spatiotemporal relationship between the liver and serum lactate . in males , however , both the liver and serum profile changes in the early stage of infection ( figure 4(e ) ) , referring to the fact that the males are unable to maintain the blood homeostasis . here , the choline of liver is found to be anticorrelated to the choline of serum . this may be due to the fact that the intraerythrocytic parasite causes a 5-fold increase in the phospholipid content of the parasitized cell , which may eventually lead to a phospholipid degradation in the liver of the host , thereby generating more of the phosphocholine components . as far as the brain to serum correlation analysis is concerned , for the females , the contribution from the serum towards the brain - serum crosstalk does not alter much ; however , the brain profile did change notably ( figures 5(a ) and 5(b ) ) . for the males , both the brain and serum profile was altered ( figures 5(d ) and 5(e ) ) , although the early - stage brain profile looks similar to that of control animals . earlier report showed that the brain profile in the early - stage males and females did not change significantly . however , this data suggests that the brain profile is maintained by the aid of a complex relationship with the serum . in the females , as noted earlier , the homeostasis of the serum is maintained , while for the males , the brain profile is maintained at the cost of serum homeostasis . this indicates that the energy metabolism of the brain is altered in the early stage of the infection in both sexes . however , the alteration in the energy metabolism probably bears different meanings in the two sexes . in the males , this is an effect of the maintenance of brain homeostasis at the cost of serum homeostasis , whereas , for the females it is the other way around . this , therefore , opens up the possibility of a greater insight into the changes in the metabolic pathways as a response to malarial parasite infection .

more than 200 million people suffer from chronic obstructive pulmonary disease ( copd ) worldwide , and three million of them die each year.1 thereby , copd is the fourth most common cause of death globally . copd consists of both bronchial disease and emphysema , with the latter leading to enlargement of the airspaces distal to the terminal bronchioles . clinical evaluation of peripheral airspaces is difficult , since this region is not readily accessible for analysis.2 the current standard diagnostic test for copd is spirometry , which can detect airflow obstruction but has limited sensitivity to changes in the small airways.3 thus , early - stage emphysema may be present without being diagnosed if only spirometry is used.4,5 a noninvasive technique with the ability to detect morphological changes in the small airways , including changes due to emphysema , is aerosol - derived airway morphometry ( adam).611 adam provides information on the airway dimensions by comparing inhaled and exhaled concentrations of monodisperse aerosol particles , usually in the range of 0.81.0 m . in still air , particles of this size deposit primarily by gravitational settling , with the deposition efficiency depending on the vertical distance to the airway surfaces.12 based on this notion , the effective airway diameters ( eads ) are derived with a simple deposition model.7,13,14 adam has been shown to provide detailed information on airway diameters , especially in healthy subjects.6,7,1520 the method has also been validated with excised lungs of humans,21 donkeys,22 and dogs,23 as well as with a bed of packed glass beads.24 furthermore , adam was found to be sufficiently sensitive to detect the increase in airspace dimensions with age.17,18,25 for patients or animal models with respiratory disease , adam generally provides more ambiguous data , with the most promising findings related to enlarged airspaces in emphysema.6,2639 although adam has several advantages in comparison to other diagnostic techniques , it has not become a common clinical practice for diagnosing copd or other airway diseases . in fact , research of adam and its application as a diagnostic tool has ceased during the last decade . two difficulties with adam are its requirement of a repeatable ( and low ) breathing flow rate and uncertainties concerning particle penetration into the diseased lungs.6,7,39 the need for a highly controlled breathing maneuver at a low flow rate makes it difficult to measure the peripheral airspace dimensions at larger volumetric lung depths and at maximum inflation of the lungs.17 hence , adam has often been used to measure recovery at a fraction of the total lung capacity ( tlc ) , which requires a preceding determination of lung volumes . it is hypothesized that these difficulties can be reduced by using nanoparticles < 0.1 m instead of micrometer - sized particles . as further detailed in this study , particles in this size range have a higher ability to penetrate into the peripheral airways , and they are less sensitive to obstructions and disturbances to the airflow in the tracheobronchial tree than micron - sized particles . nanoparticles with neutral electrical charge deposit almost exclusively by brownian diffusion.40 deposition by diffusion depends on the residence time and on the distance to the airway wall , but not on the airway orientation . similar to particle deposition by gravitational settling in adam , particle loss due to diffusion in the alveolar region during breath - hold is related to the size of the airspaces.41 the objective of this work is to provide a basic theoretical background to the method of airspace dimension assessment by nanoparticles ( hereafter named aida ) . in the following , previous findings on nanoparticle deposition in subjects with respiratory disease are summarized . suitable breathing maneuvers are discussed for aida , an optimal particle size for the method is estimated , and the assumptions about aida are compared with the results obtained for adam . the relationship between the airspace dimensions and the recovery of exhaled nanoparticles is approximated and finally the potential use of aida for copd diagnosis is briefly elaborated . experimental data on deposition probabilities of inhaled nanoparticles in patients with copd are scarce and inconsistent ( figure 1 ) . apart from a few studies on asthmatics , the deposition of nanoparticles has only been investigated in 40 subjects with respiratory disease : 32 with copd , five with obstructive lung disease , and three with restrictive lung disease.4246 different diagnostic methods were used , with only one study separating the copd patients into those with predominantly airways disease ( n=7 ) and those with predominantly emphysema ( n=3).43 the exposure procedures varied , with three studies using spontaneous breathing,43,44,46 one using bolus inhalation,45 and one a predetermined breathing cycle.42 the particle types also differed : monodisperse technetium-99 m - labeled ultrafine carbon aerosol,43,45 polydisperse di - ethyl - hexyl - sebacate,42 emissions from biomass combustion,46 and diesel exhaust particles.44 hence , it is difficult to draw any solid conclusions from the available data . all the experimental studies found deviations in deposition of nanoparticles between diseased subjects and healthy controls , although differences were both positive and negative ( figure 1 ) . however , group differences are uncertain , since the number of subjects with respiratory disease was limited to 310 in each study . since only one of the studies reported a similar breathing pattern for all subjects , it is also difficult to assess if differences were due to varying breathing flows or due to lung morphology . a comparison of experimental data and the icrp model ( international commission on radiological protection ) for a group of 17 subjects ( 10 with copd ) who were exposed to diesel exhaust particles reveals that breathing pattern changes can not explain the decrease in deposition of particles < 40 nm in the copd patients , nor the increase in deposition of particles > 400 nm.44,47 similar results were obtained for biomass combustion particles.46 experimental data with imaging techniques also show that nanoparticles , unlike larger - sized particles , do not have a decreased peripheral deposition in patients with airway obstructions.43,45,4853 mller et al45 measured the central / peripheral deposition ratio during bolus inhalation that targeted the peripheral lung region or the central airways . for the peripheral lung , no differences were found between copd patients or smokers and the nonsmoking controls . for boluses targeted to the central airways , the fraction of nanoparticles reaching further into the peripheral lung was significantly higher in copd ( p<0.01 , 100 nm particles , central / peripheral ratio 2.01 and 1.22 for healthy and copd subjects , respectively ) . in contrast , brown et al43 found a small increase in central deposition for patients with copd ( p<0.05 , 61 nm particles , central / peripheral ratio 1.01 and 1.11 for healthy and copd subjects , respectively ) . for particles > 450 nm , measurements consistently showed an increase in the central deposition in copd or asthma patients even at moderate flow rates.4854 there are few model calculations on deposition of nanoparticles in diseased lungs , and often these are limited to confined regions of the respiratory tract . in general , modeling of particle deposition in diseased lungs is difficult and requires numerous assumptions and simplifications of both the lung morphology and the airflow patterns , since the morphology of diseased lungs is more complex and heterogeneous compared to normal lungs . computational lung deposition models can be divided into two classes : local deposition models that are normally based on computational fluid dynamics ( cfd ) and whole - lung deposition models.55,56 cfd models are computationally demanding and usually restricted to the upper airways or specific regions of the tracheobronchial tree . these models calculate flow rates and deposition patterns of aerosol particles at high spatial resolution . however , simplifications are often made to the structure of respiratory tree , for example , regarding cartilaginous rings , carinal ridges , and the complex alveolar region and its dynamic change in dimensions during the breathing cycle.55,57 in addition , isolating local regions of the respiratory tract entails uncertainties due to the unknown flow fields at the entrance to the computational domain.58 the calculations are also difficult to validate with experimental data , especially for the narrow airspaces . whole - lung models on the other hand are semiempirical and do not provide detailed information on changes in deposition due to local abnormalities in the airways . typically , the deposition is modeled as the sum of particle losses in a series of filters or pipes with simple characteristics . few cfd models investigate diseased lungs , and almost all of these models are restricted to calculating flow fields or the deposition of micrometer - sized particles . one study reported that particle deposition in the central airways was significantly altered by sidewall or carinal tumors and airway constrictions or blockage with higher deposition of large particles ( > 1 m ) when tumors were present.59 the opposite , that is , decreased deposition , was estimated for nanoparticles . it is noteworthy that cfd models for normal lungs show a nonuniform deposition of micron - sized particles , with increased particle deposition at specific sites , such as the carinal ridges , while nanoparticles tend to coat the surfaces more uniformly.58,6062 the deposition enhancement factor ( def ) , which is high at deposition hot spots , is often more than two orders of magnitude larger for micron - sized particles compared to nanoparticles.6163 for instance , zhang et al62 calculated that def in generation 03 of the bronchial tree was up to 4002,400 for particles in the range of 110 m , while only 210 for nanoparticles in the range of 10100 nm.62 based on the computational model results for micron - sized particles , def and local deposition are expected to be increased in patients with respiratory airway disease . whole - lung models estimated increased deposition of nanoparticles in bronchitic patients and decreased deposition in emphysematic patients.64,65 deposition was also predicted to vary depending on the emphysema type the lowest deposition was predicted for bullous emphysema , since particles need to pass larger distances before depositing on the alveoli walls . yet , this result awaits experimental verification , since it is known that the bullae , that is , large air pockets in the lungs , are poorly ventilated , and it is thus unclear to what extent the inhaled aerosol actually enters these airspaces . interestingly , particles in the range of 3050 nm were suggested to have a substantially decreased deposition in emphysema patients , and thus be most suitable for reaching the outermost region of the lung.65 in contrast to the few inconclusive studies on nanoparticle deposition patterns in diseased lungs , micron - sized particles repeatedly demonstrate an increased deposition during tidal breathing for patients with copd.6670 this is an effect of the altered flow field in the obstructed and constricted airways , as the micron - sized particles are mainly affected by inertial and gravitational losses . in contrast , deposition by diffusion , as is the case for nanoparticles , is less affected by disturbances of the airflows . the only exception to the increase in deposition of large particles in copd is the measurements made with adam , where deposition is studied in still air from the respiratory zone . there are three versions of adam : single - breath recovery , bolus recovery , and the particle concentration technique.9,13 the single - breath technique provides a volume - weighted ead for the whole lung by determining the particle half - life from different breath - holding periods.6,7 the bolus recovery technique probes different volumetric lung depths with small ( 30100 ml ) boluses to obtain information of ead as a function of volumetric lung depth , but at the expense of an extensive measurement protocol with several breath - holding periods for each studied lung depth.15,16 the particle recovery technique combines the more detailed information from the bolus recovery technique with the simple execution of the single - breath technique through a continuous measurement of the recovery in the exhaled air . the recovery data is then divided into consecutive bins that correspond to eads at different volumetric lung depths.17,18 the fundamental difference between the proposed new method aida and aerosol - derived airway morphometry ( adam ) is the particle deposition mode . the three main particle deposition mechanisms in the respiratory tract are diffusion , gravitational settling , and inertial impaction . figure 2 shows the displacement velocity due to brownian motion , and the settling velocity due to gravity for unit density particles.71 as seen , the diffusion displacement speed of 0.1 m particles is approximately similar to the settling velocity of 1 m particles . deposition by impaction occurs foremost in the proximal airways and has important contribution to the total deposition only when particle inertia is substantial . hence , significant contribution by inertial deposition to the total lung deposition in healthy people during normal breathing conditions is expected only for particles > 2 m . however , inertial deposition can be substantially altered in patients with airflow obstruction , due to the velocity field in partially blocked airways , and affect the deposition of particles > 200 nm.49,72 apart from the distinct particle size , the breathing pattern of adam and aida is also different . the breathing procedure used in aida is similar to the well - established maneuver performed during measurement of the diffusion capacity for carbon monoxide , dl , co.73 a measurement of dl , co begins with a period of spontaneous tidal breathing at a mouthpiece to adjust to the equipment . thereafter follows an exhalation to the residual volume ( rv ) , a fast inhalation to tlc , a 105 seconds breath - hold and finally exhalation . in adam , a comparable breathing pattern is used , but the flow rate during inhalation and exhalation is held constant , usually at 250 ml / s , by the subjects following a signal on a screen . because of the low airflow , inhalation normally begins at the functional residual capacity ( frc ) , or between the rv and frc , and ends at around 70%85% of the tlc . next follows a breath - hold of 210 seconds , and finally expiration at a similar flow rate as during inspiration . the low and controlled airflow in adam is required for achieving constant particle losses during the inspiration and expiration phases for all the breath - holding periods . thereby , the recovery can be obtained only from the breath - hold periods , when particles settle in presumably still air . the fast and deep inhalation , as used in dl , co , could result in substantial uncertainties if it was applied in adam , with the high airflow rates leading to increased micron - sized particle losses due to inertial impaction which are harder to measure with high precision , especially in copd patients.6 in addition , deposition during inhalation and exhalation may be enhanced if a patient has an unsteady flow rate.74 it is also notable that particle losses differ between inspiration and expiration.75,76 the breathing procedure in adam , with controlled breathing flow rate , is possible to achieve by most subjects after some training , but has been reported to be challenging for some groups of patients and for children.8,39 in clinical practice , difficulties may be larger as time is more limited and as volunteers in research studies may be more approving and adaptive than patients in general . for aida , high flows during inspiration and expiration are not expected to cause difficulties , as they will increase rather than decrease the fraction of the nanoparticles that deposit in the pulmonary region ( figures 3 and 4 ) . figure 3 illustrates the particle deposition fraction in the pulmonary region for a breathing cycle with 2,000 ml inhalation , 10-second breath - hold and exhalation at a constant flow rate . figure 4 shows the deposition fraction in each generation along the respiratory tree during inhalation and exhalation ( without breath - hold ) for a healthy adult breathing through the mouth in an upright position ( based on the multiple path particle dosimetry model version 2.1177 ) . as seen , deposition of nanoparticles ( 50100 nm ) in the pulmonary region is relatively insensitive to the breathing rate ( figure 3 ) , whereas increased deposition of larger particles in the extra - thoracic and tracheobronchial regions is evident at higher flows . the advantage of being able to use higher flow rates in aida is that a breathing pattern similar to that used in dl , co measurements can be used . this means an easier breathing protocol for the patient and that the airspace dimensions can be measured at tlc , when the alveoli are maximally inflated . airspace dimensions are almost exclusively determined at a fixed percentage of tlc , which entails additional time to measure tlc and preferably also frc a process that requires a body plethysmograph or instruments for gas dilution measurements , and may give rise to uncertainties . a number of studies develop the theoretical framework for the relationship between pulmonary airway dimensions and recovery of monodisperse particles that deposit predominantly by gravitational settling.7,10,11,14,41,7881 two approaches were presented : the tube model , which can be used for both the airways and the acini , and the chord - length model , which is specific for the acini . in the tube model , the airspace dimensions are approximated assuming that particles deposit in a system of identical , yet randomly oriented , cylindrical tubes . the airway radius , r , is related to the recovery , r(t ) , of particles after a breath - hold as r(t)=e2vstr(1)where vs is the particle terminal settling velocity.7,78 thus , the tube radius can be calculated from the particle half - life time , t : r=2vt1/2in2.(2 ) this model , however , does not account for diffusion . for unit density spheres , the root mean square ( r.m.s . ) brownian displacement and the terminal settling velocity are approximately equal for 0.5 m particles ( figure 2 ) . hence , particles with this diameter are considered to be the smallest particles suitable for adam.82 for a 100 nm particle , the r.m.s . brownian displacement is 45 times larger than the terminal settling velocity of unit density spheres , and on timescales smaller than a few minutes , nanoparticles can be considered to deposit exclusively by diffusion . for longer times , gravitational settling may become significant , because it is directly proportional to the time , whereas deposition by diffusion scales with the square root of time . the theory behind the estimation of airway radii from diffusion losses is based on solution of the diffusion equation in circular tubes,79,83,84 n(x , t)td2n(x , t)=0(3)where n(x , t ) is the number concentration of particles at location x and time t , and d is the diffusion coefficient given by the stokes einstein relation d = ktcc3dp.(4)here , k is the boltzmann coefficient , t is the temperature , cc is the cunningham slip correction factor , is the air viscosity and dp is the particle diameter ( all in si units ) . 3 ) for a cylindrical tube with axisymmetric boundary conditions can be solved easily by separation of variables . assuming homogenous initial concentration , the fraction of recovered particles is given by r(t)=4m=11k0,m2e(k0,m2dtr2)(5)where k0,m is the mth root of the bessel function of the first kind , j0(x ) = 0 ( ie , k0,1 = 2.41 , k0,2 = 5.52 , k0,3 = 8.65 , k0,4 = 11.79 , etc ) . as long as the initial concentration profile is axisymmetric , the solutions to r(t ) will be a sum of a series of exponential functions.83 after a certain time , a single exponential function will dominate the summation ( normally for m=1 ) . the time needed to reach the single exponential phase depends on the initial concentration profile , the particle size ( via its diffusion coefficient , eq . 4 ) and the tube radius . for nanoparticles ( < 0.1 m ) , the typical time for this to occur in the peripheral airspaces is expected to be < 5 seconds . hence , the measured half - life time , t , of the particles is assumed to correspond to only the first term in the infinite exponential series . thus , the representative radius is estimated from 12=e(k0,12dt1/2r2)(6)which leads to r=2.89dt1/2.(7 ) since adam reports the eads , sticking to the same terminology , ead = 2r . the recovery without breath - hold is denoted by r0 . in general , it depends on the initial concentration profile and on the particle losses during inhalation and exhalation . when taking only the first exponential term into account , r0 can be determined from the measured r(t ) values as r0=r(t)e(k0,12dtr2)=r(t)e(tin2t1/2)(8 ) eq . 2 reveals that when the airway radius is assessed by recovery of diffusive nanoparticles , it is proportional to the square root of the half - life of the diffusional deposition process and not to the half - life itself , as when the assessment is based on deposition of gravitationally settling particles ( eq . hence , a change in the airway radii has a larger effect on the half - life of nanoparticles than of micron - sized particles . this is a notable advantage of aida , since based on this model , changes in the lung morphology will lead to more sizeable variations , which can be measured more easily . 7 is expected to be less sensitive to ( indirect ) measurement errors of t ( which is estimated by regressing the exponential function against time series of measured data ) . 7 is clearly more supported when diffusional deposition is predominant , which according to goldberg and smith,83 corresponds to dp<1.17107r3.(9 ) figure 5 illustrates estimated half - life times for different tube radii and particle sizes . as can be seen , t ranges between about 1 and 60 seconds for particles with size between 40 and 150 nm being in the lower end of this size range has the advantages of less interference by gravitational settling and a shorter time to reach the single - exponential behavior . a consequence of the nonlinear relationship between the airspace radius and the half - life for aida ( eq . 7 ) is that the derived radius , rmean , of a collection of airspaces of different dimensions , ri , will be weighted toward the larger dimensions . assuming equal volumes of all airspaces , rmean=2.89dt1/2,mean=2.89d1ni=1nt1/2,i=1ni=1nri2.(10 ) thus , for aida , the rmean is the r.m.s . of all the measured radii , whereas for adam , it is the arithmetic mean . from a diagnostic perspective , it is an advantage that a small number of enlarged airspaces have a disproportional effect on rmean . uncertainties in the analysis of airflow and particle deposition data in recovery experiments of both nanoparticles and micrometer - sized particles stem from the numerous assumptions used , including that the airways are tubular and uniform . in reality , the airways geometry is much more complex , and the physical meaning of the estimated airway radius is unclear.85 it is also well known that many lung diseases , such as pulmonary emphysema and asthma , are heterogeneously distributed in the lungs . the effects of this morphological heterogeneity on the measured recovery can not be easily addressed theoretically , especially for late stages of emphysema where the diseased parts of the lungs may be poorly ventilated . possibly , the most important difference between aida and adam is the potential for higher penetration of nanoparticles into the peripheral regions of diseased lungs . the optimal particle size for reaching the most distal parts of the acinus is a compromise between particle with minimal deposition characteristics , which can be carried to the deep lungs ( convective transport ) during inhalation , and a large displacement velocity during the subsequent breath - hold to reach remote areas . the former is achieved by large nanoparticles , while the latter increases for small nanoparticles.47,8688 based on the analysis explained , the optimal particle size range for aida is 40100 nm , but experiments and detailed model calculations , especially for diseased lungs , are necessary to confirm these theoretical predictions . in addition to the information on the airway radii obtained from t , the recovery without breath - hold as derived from the single exponential function , r0 , also reflects the lung morphology . the factor r0 depends on the initial concentration profile in the airway tubes , the time for the inhalation and exhalation and particle losses during transport of the aerosol . assuming that all the lungs have similar initial concentration profiles , variations in r0 reflect particle loss during the inhalation and exhalation phases of the breathing maneuver . for nanoparticles in the range of 40100 nm , most of these losses occur distal to generation 15 in the respiratory tree , corresponding to the terminal bronchioles and the gas - exchange region ( figure 4 ) . thus , r0 is likely to reveal either changes in the deeper parts of the lungs or abnormal particle concentration profiles , for instance , due to local turbulence or distorted airways . variations in r0 , therefore , may provide additional clinical information about possible abnormalities in these regions . there is a need for techniques to assess abnormalities in the peripheral lung , not least in diagnosis of copd . the diagnosis of copd is based on long - standing cough and/or dyspnea on exertion in combination with airflow limitation after bronchodilatation , usually demonstrated by spirometry and a history of exposure to noxious inhalants.3 but as spirometry has poor sensitivity to changes in small airways , emphysema may remain unidentified if no other techniques are used.4,5 emphysema is an indicator of poor prognosis , and since the most effective treatment is to reduce harmful exposure , primarily by smoking cessation , an early diagnosis is crucial to motivate patients to stop smoking.89,90 current techniques with high sensitivity for emphysema are x - ray computed tomography ( ct ) and magnetic resonance imaging ( mri ) , where hyperpolarized and poorly soluble gases , normally helium-3 or xenon-129 , are used as contrast agents.73,9194 ct is expensive compared to spirometry and dl , co , and also entails a radiation dose . mri with hyperpolarized gases is inaccessible and generally too costly to be used other than for research purposes . dl , co , although a sensitive indicator of abnormal lung function , is affected by blood flow , hemoglobin concentration or alterations of the alveolar membranes and is therefore not specific for emphysema.73 in the absence of airflow limitation , reduced dl , co can therefore not be taken to indicate emphysema . thus , there is a need for simple , sensitive and reliable method for diagnosing emphysema . compared to ct and mri with hyperpolarized gases , lung diagnosis with an aerosol - based method , such as aida , is likely to be less time consuming , considerably cheaper , simpler to use , and easier to interpret ( a single value rather than an image that has to be analyzed ) . corresponding to carbon monoxide molecules in dl , co , nanoparticles move by diffusion , and thus , a breathing maneuver similar to that of dl , co could be utilized . in contrast to carbon monoxide in dl , co , the uptake of nanoparticles in the lung is not affected by other variables than morphology . the major challenge of aida , in comparison to adam , is the need for a more advanced experimental technique . particles larger than about 0.2 m can be detected by light scattering directly at the mouthpiece . for smaller particles , more sophisticated methods , such as condensation particle counters or instruments based on electrometers , are needed these instruments are on the other hand more accurate than optical particle counters , which are sensitive to scattering properties ( for instance , caused by changes in humidity ) , particle shape and mal - detection from multiple particles in the measurement zone . however , over the last decade , such techniques have improved rapidly due to an increased interest in nanotechnology and health aspects of nanoparticles . for instance , kim and jaques95 performed fast sampling of nanoparticles directly at the mouthpiece through bypassing of the averaging circuit in an ultrafine condensation particle counter . another option is to use fast valves for sampling of specific volumes of the exhaled air before analysis.96 the use of inhaled particles for diagnosis has implications for approval and regulation of aida as a medical device . insoluble nanoparticles can be more toxic than larger particles at equal mass concentrations due to a higher total surface area.97 on the other hand , the mass concentration of nanoparticles in aida is expected to be very low . an optimal inhaled number concentration is below 100,000 particles / cm to avoid coincidence in the detection instruments and particle losses by coagulation.98,99 this concentration is comparable to normal urban background of nanoparticles , and over a single breath procedure , the deposited amount of particulate material will be insignificant compared to real - world particle exposures . in addition , the particle substance used in aida can be of a material with low toxicity which ideally is biodegradable . due to the small particle size , the total inhaled particle mass in aida will be orders of magnitude less than for adam . the usage of nanoparticles for assessment of distal airspace morphology and detection of emphysema may have several advantages in comparison to previous aerosol - based methods with larger particles : 1 ) high breathing flow rates do not increase deposition and a breathing maneuver similar to the clinically well - established dl , co could most likely be used , which considerably would facilitate the procedure for the patient and reduces overall examination time as no training is needed prior to measurement . 2 ) a higher breathing flow rate makes it possible to inhale from the rv to tlc , and consequently , the peripheral airspace dimensions can be measured at maximum inflation which eliminates the need for determination of lung volumes for interpretation of data . 3 ) a change in airway radius has a larger effect on the measured parameter , the particle half - life time t , for nanoparticles than for micrometer - sized particles , and thus , the sensitivity is expected to increase . 4 ) interpretation of clinical data is expected to be simpler as the deposition of nanoparticles is almost exclusively determined by diffusion , while the deposition of micron - sized particles is a complex combination of settling and diffusion . 5 ) the mass concentration of the inhaled particles is likely to be reduced by two to three orders of magnitude , which may be of importance for acceptance by patients or regulatory authorities . 6 ) finally , but perhaps most importantly , the penetration of inhaled particles into diseased or poorly ventilated regions of the lung is probably superior for nanoparticles of certain sizes to micron - sized particles . in summary , aida has several potential benefits which are still unexplored , and the technique could be a way forward to simplify diagnosis of pulmonary diseases , such as emphysema . based on the available experimental data and model calculations , the preferable particle size range is likely to be within 40100 nm . development of new instruments for rapid and cheap detection of nanoparticles has opened a new window for such studies .

there is a need for efficient techniques to assess abnormalities in the peripheral regions of the lungs , for example , for diagnosis of pulmonary emphysema . considerable scientific efforts have been directed toward measuring lung morphology by studying recovery of inhaled micron - sized aerosol particles ( 0.41.5 m ) . in contrast , it is suggested that the recovery of inhaled airborne nanoparticles may be more useful for diagnosis . the objective of this work is to provide a theoretical background for the use of nanoparticles in measuring lung morphology and to assess their applicability based on a review of the literature . using nanoparticles for studying distal airspace dimensions is shown to have several advantages over other aerosol - based methods . 1 ) nanoparticles deposit almost exclusively by diffusion , which allows a simpler breathing maneuver with minor artifacts from particle losses in the oropharyngeal and upper airways . 2 ) a higher breathing flow rate can be utilized , making it possible to rapidly inhale from residual volume to total lung capacity ( tlc ) , thereby eliminating the need to determine the tlc before measurement . 3 ) recent studies indicate better penetration of nanoparticles than micron - sized particles into poorly ventilated and diseased regions of the lungs ; thus , a stronger signal from the abnormal parts is expected . 4 ) changes in airspace dimensions have a larger impact on the recovery of nanoparticles . compared to current diagnostic techniques with high specificity for morphometric changes of the lungs , computed tomography and magnetic resonance imaging with hyperpolarized gases , an aerosol - based method is likely to be less time consuming , considerably cheaper , simpler to use , and easier to interpret ( providing a single value rather than an image that has to be analyzed ) . compared to diagnosis by carbon monoxide ( dl , co ) , the uptake of nanoparticles in the lung is not affected by blood flow , hemoglobin concentration or alterations of the alveolar membranes , but relies only on lung morphology .

Introduction Deposition of nanoparticles in diseased lungs Breathing maneuver in ADAM and AiDA Assessment of airspace dimensions from particle recovery Relationships between particle loss and the morphology of terminal bronchioles and gas-exchange region AiDA as a diagnostic tool Summary and conclusion

clinical evaluation of peripheral airspaces is difficult , since this region is not readily accessible for analysis.2 the current standard diagnostic test for copd is spirometry , which can detect airflow obstruction but has limited sensitivity to changes in the small airways.3 thus , early - stage emphysema may be present without being diagnosed if only spirometry is used.4,5 a noninvasive technique with the ability to detect morphological changes in the small airways , including changes due to emphysema , is aerosol - derived airway morphometry ( adam).611 adam provides information on the airway dimensions by comparing inhaled and exhaled concentrations of monodisperse aerosol particles , usually in the range of 0.81.0 m . in still air , particles of this size deposit primarily by gravitational settling , with the deposition efficiency depending on the vertical distance to the airway surfaces.12 based on this notion , the effective airway diameters ( eads ) are derived with a simple deposition model.7,13,14 adam has been shown to provide detailed information on airway diameters , especially in healthy subjects.6,7,1520 the method has also been validated with excised lungs of humans,21 donkeys,22 and dogs,23 as well as with a bed of packed glass beads.24 furthermore , adam was found to be sufficiently sensitive to detect the increase in airspace dimensions with age.17,18,25 for patients or animal models with respiratory disease , adam generally provides more ambiguous data , with the most promising findings related to enlarged airspaces in emphysema.6,2639 although adam has several advantages in comparison to other diagnostic techniques , it has not become a common clinical practice for diagnosing copd or other airway diseases . two difficulties with adam are its requirement of a repeatable ( and low ) breathing flow rate and uncertainties concerning particle penetration into the diseased lungs.6,7,39 the need for a highly controlled breathing maneuver at a low flow rate makes it difficult to measure the peripheral airspace dimensions at larger volumetric lung depths and at maximum inflation of the lungs.17 hence , adam has often been used to measure recovery at a fraction of the total lung capacity ( tlc ) , which requires a preceding determination of lung volumes . it is hypothesized that these difficulties can be reduced by using nanoparticles < 0.1 m instead of micrometer - sized particles . as further detailed in this study , particles in this size range have a higher ability to penetrate into the peripheral airways , and they are less sensitive to obstructions and disturbances to the airflow in the tracheobronchial tree than micron - sized particles . nanoparticles with neutral electrical charge deposit almost exclusively by brownian diffusion.40 deposition by diffusion depends on the residence time and on the distance to the airway wall , but not on the airway orientation . similar to particle deposition by gravitational settling in adam , particle loss due to diffusion in the alveolar region during breath - hold is related to the size of the airspaces.41 the objective of this work is to provide a basic theoretical background to the method of airspace dimension assessment by nanoparticles ( hereafter named aida ) . suitable breathing maneuvers are discussed for aida , an optimal particle size for the method is estimated , and the assumptions about aida are compared with the results obtained for adam . the relationship between the airspace dimensions and the recovery of exhaled nanoparticles is approximated and finally the potential use of aida for copd diagnosis is briefly elaborated . apart from a few studies on asthmatics , the deposition of nanoparticles has only been investigated in 40 subjects with respiratory disease : 32 with copd , five with obstructive lung disease , and three with restrictive lung disease.4246 different diagnostic methods were used , with only one study separating the copd patients into those with predominantly airways disease ( n=7 ) and those with predominantly emphysema ( n=3).43 the exposure procedures varied , with three studies using spontaneous breathing,43,44,46 one using bolus inhalation,45 and one a predetermined breathing cycle.42 the particle types also differed : monodisperse technetium-99 m - labeled ultrafine carbon aerosol,43,45 polydisperse di - ethyl - hexyl - sebacate,42 emissions from biomass combustion,46 and diesel exhaust particles.44 hence , it is difficult to draw any solid conclusions from the available data . since only one of the studies reported a similar breathing pattern for all subjects , it is also difficult to assess if differences were due to varying breathing flows or due to lung morphology . a comparison of experimental data and the icrp model ( international commission on radiological protection ) for a group of 17 subjects ( 10 with copd ) who were exposed to diesel exhaust particles reveals that breathing pattern changes can not explain the decrease in deposition of particles < 40 nm in the copd patients , nor the increase in deposition of particles > 400 nm.44,47 similar results were obtained for biomass combustion particles.46 experimental data with imaging techniques also show that nanoparticles , unlike larger - sized particles , do not have a decreased peripheral deposition in patients with airway obstructions.43,45,4853 mller et al45 measured the central / peripheral deposition ratio during bolus inhalation that targeted the peripheral lung region or the central airways . for particles > 450 nm , measurements consistently showed an increase in the central deposition in copd or asthma patients even at moderate flow rates.4854 there are few model calculations on deposition of nanoparticles in diseased lungs , and often these are limited to confined regions of the respiratory tract . in general , modeling of particle deposition in diseased lungs is difficult and requires numerous assumptions and simplifications of both the lung morphology and the airflow patterns , since the morphology of diseased lungs is more complex and heterogeneous compared to normal lungs . computational lung deposition models can be divided into two classes : local deposition models that are normally based on computational fluid dynamics ( cfd ) and whole - lung deposition models.55,56 cfd models are computationally demanding and usually restricted to the upper airways or specific regions of the tracheobronchial tree . however , simplifications are often made to the structure of respiratory tree , for example , regarding cartilaginous rings , carinal ridges , and the complex alveolar region and its dynamic change in dimensions during the breathing cycle.55,57 in addition , isolating local regions of the respiratory tract entails uncertainties due to the unknown flow fields at the entrance to the computational domain.58 the calculations are also difficult to validate with experimental data , especially for the narrow airspaces . whole - lung models on the other hand are semiempirical and do not provide detailed information on changes in deposition due to local abnormalities in the airways . few cfd models investigate diseased lungs , and almost all of these models are restricted to calculating flow fields or the deposition of micrometer - sized particles . it is noteworthy that cfd models for normal lungs show a nonuniform deposition of micron - sized particles , with increased particle deposition at specific sites , such as the carinal ridges , while nanoparticles tend to coat the surfaces more uniformly.58,6062 the deposition enhancement factor ( def ) , which is high at deposition hot spots , is often more than two orders of magnitude larger for micron - sized particles compared to nanoparticles.6163 for instance , zhang et al62 calculated that def in generation 03 of the bronchial tree was up to 4002,400 for particles in the range of 110 m , while only 210 for nanoparticles in the range of 10100 nm.62 based on the computational model results for micron - sized particles , def and local deposition are expected to be increased in patients with respiratory airway disease . yet , this result awaits experimental verification , since it is known that the bullae , that is , large air pockets in the lungs , are poorly ventilated , and it is thus unclear to what extent the inhaled aerosol actually enters these airspaces . interestingly , particles in the range of 3050 nm were suggested to have a substantially decreased deposition in emphysema patients , and thus be most suitable for reaching the outermost region of the lung.65 in contrast to the few inconclusive studies on nanoparticle deposition patterns in diseased lungs , micron - sized particles repeatedly demonstrate an increased deposition during tidal breathing for patients with copd.6670 this is an effect of the altered flow field in the obstructed and constricted airways , as the micron - sized particles are mainly affected by inertial and gravitational losses . in contrast , deposition by diffusion , as is the case for nanoparticles , is less affected by disturbances of the airflows . there are three versions of adam : single - breath recovery , bolus recovery , and the particle concentration technique.9,13 the single - breath technique provides a volume - weighted ead for the whole lung by determining the particle half - life from different breath - holding periods.6,7 the bolus recovery technique probes different volumetric lung depths with small ( 30100 ml ) boluses to obtain information of ead as a function of volumetric lung depth , but at the expense of an extensive measurement protocol with several breath - holding periods for each studied lung depth.15,16 the particle recovery technique combines the more detailed information from the bolus recovery technique with the simple execution of the single - breath technique through a continuous measurement of the recovery in the exhaled air . however , inertial deposition can be substantially altered in patients with airflow obstruction , due to the velocity field in partially blocked airways , and affect the deposition of particles > 200 nm.49,72 apart from the distinct particle size , the breathing pattern of adam and aida is also different . in adam , a comparable breathing pattern is used , but the flow rate during inhalation and exhalation is held constant , usually at 250 ml / s , by the subjects following a signal on a screen . because of the low airflow , inhalation normally begins at the functional residual capacity ( frc ) , or between the rv and frc , and ends at around 70%85% of the tlc . the fast and deep inhalation , as used in dl , co , could result in substantial uncertainties if it was applied in adam , with the high airflow rates leading to increased micron - sized particle losses due to inertial impaction which are harder to measure with high precision , especially in copd patients.6 in addition , deposition during inhalation and exhalation may be enhanced if a patient has an unsteady flow rate.74 it is also notable that particle losses differ between inspiration and expiration.75,76 the breathing procedure in adam , with controlled breathing flow rate , is possible to achieve by most subjects after some training , but has been reported to be challenging for some groups of patients and for children.8,39 in clinical practice , difficulties may be larger as time is more limited and as volunteers in research studies may be more approving and adaptive than patients in general . for aida , high flows during inspiration and expiration are not expected to cause difficulties , as they will increase rather than decrease the fraction of the nanoparticles that deposit in the pulmonary region ( figures 3 and 4 ) . as seen , deposition of nanoparticles ( 50100 nm ) in the pulmonary region is relatively insensitive to the breathing rate ( figure 3 ) , whereas increased deposition of larger particles in the extra - thoracic and tracheobronchial regions is evident at higher flows . a number of studies develop the theoretical framework for the relationship between pulmonary airway dimensions and recovery of monodisperse particles that deposit predominantly by gravitational settling.7,10,11,14,41,7881 two approaches were presented : the tube model , which can be used for both the airways and the acini , and the chord - length model , which is specific for the acini . the airway radius , r , is related to the recovery , r(t ) , of particles after a breath - hold as r(t)=e2vstr(1)where vs is the particle terminal settling velocity.7,78 thus , the tube radius can be calculated from the particle half - life time , t : r=2vt1/2in2. for nanoparticles ( < 0.1 m ) , the typical time for this to occur in the peripheral airspaces is expected to be < 5 seconds . 2 reveals that when the airway radius is assessed by recovery of diffusive nanoparticles , it is proportional to the square root of the half - life of the diffusional deposition process and not to the half - life itself , as when the assessment is based on deposition of gravitationally settling particles ( eq . hence , a change in the airway radii has a larger effect on the half - life of nanoparticles than of micron - sized particles . this is a notable advantage of aida , since based on this model , changes in the lung morphology will lead to more sizeable variations , which can be measured more easily . uncertainties in the analysis of airflow and particle deposition data in recovery experiments of both nanoparticles and micrometer - sized particles stem from the numerous assumptions used , including that the airways are tubular and uniform . in reality , the airways geometry is much more complex , and the physical meaning of the estimated airway radius is unclear.85 it is also well known that many lung diseases , such as pulmonary emphysema and asthma , are heterogeneously distributed in the lungs . the effects of this morphological heterogeneity on the measured recovery can not be easily addressed theoretically , especially for late stages of emphysema where the diseased parts of the lungs may be poorly ventilated . possibly , the most important difference between aida and adam is the potential for higher penetration of nanoparticles into the peripheral regions of diseased lungs . the optimal particle size for reaching the most distal parts of the acinus is a compromise between particle with minimal deposition characteristics , which can be carried to the deep lungs ( convective transport ) during inhalation , and a large displacement velocity during the subsequent breath - hold to reach remote areas . the former is achieved by large nanoparticles , while the latter increases for small nanoparticles.47,8688 based on the analysis explained , the optimal particle size range for aida is 40100 nm , but experiments and detailed model calculations , especially for diseased lungs , are necessary to confirm these theoretical predictions . in addition to the information on the airway radii obtained from t , the recovery without breath - hold as derived from the single exponential function , r0 , also reflects the lung morphology . the factor r0 depends on the initial concentration profile in the airway tubes , the time for the inhalation and exhalation and particle losses during transport of the aerosol . thus , r0 is likely to reveal either changes in the deeper parts of the lungs or abnormal particle concentration profiles , for instance , due to local turbulence or distorted airways . there is a need for techniques to assess abnormalities in the peripheral lung , not least in diagnosis of copd . the diagnosis of copd is based on long - standing cough and/or dyspnea on exertion in combination with airflow limitation after bronchodilatation , usually demonstrated by spirometry and a history of exposure to noxious inhalants.3 but as spirometry has poor sensitivity to changes in small airways , emphysema may remain unidentified if no other techniques are used.4,5 emphysema is an indicator of poor prognosis , and since the most effective treatment is to reduce harmful exposure , primarily by smoking cessation , an early diagnosis is crucial to motivate patients to stop smoking.89,90 current techniques with high sensitivity for emphysema are x - ray computed tomography ( ct ) and magnetic resonance imaging ( mri ) , where hyperpolarized and poorly soluble gases , normally helium-3 or xenon-129 , are used as contrast agents.73,9194 ct is expensive compared to spirometry and dl , co , and also entails a radiation dose . dl , co , although a sensitive indicator of abnormal lung function , is affected by blood flow , hemoglobin concentration or alterations of the alveolar membranes and is therefore not specific for emphysema.73 in the absence of airflow limitation , reduced dl , co can therefore not be taken to indicate emphysema . thus , there is a need for simple , sensitive and reliable method for diagnosing emphysema . compared to ct and mri with hyperpolarized gases , lung diagnosis with an aerosol - based method , such as aida , is likely to be less time consuming , considerably cheaper , simpler to use , and easier to interpret ( a single value rather than an image that has to be analyzed ) . corresponding to carbon monoxide molecules in dl , co , nanoparticles move by diffusion , and thus , a breathing maneuver similar to that of dl , co could be utilized . in contrast to carbon monoxide in dl , co , the uptake of nanoparticles in the lung is not affected by other variables than morphology . for smaller particles , more sophisticated methods , such as condensation particle counters or instruments based on electrometers , are needed these instruments are on the other hand more accurate than optical particle counters , which are sensitive to scattering properties ( for instance , caused by changes in humidity ) , particle shape and mal - detection from multiple particles in the measurement zone . another option is to use fast valves for sampling of specific volumes of the exhaled air before analysis.96 the use of inhaled particles for diagnosis has implications for approval and regulation of aida as a medical device . insoluble nanoparticles can be more toxic than larger particles at equal mass concentrations due to a higher total surface area.97 on the other hand , the mass concentration of nanoparticles in aida is expected to be very low . an optimal inhaled number concentration is below 100,000 particles / cm to avoid coincidence in the detection instruments and particle losses by coagulation.98,99 this concentration is comparable to normal urban background of nanoparticles , and over a single breath procedure , the deposited amount of particulate material will be insignificant compared to real - world particle exposures . the usage of nanoparticles for assessment of distal airspace morphology and detection of emphysema may have several advantages in comparison to previous aerosol - based methods with larger particles : 1 ) high breathing flow rates do not increase deposition and a breathing maneuver similar to the clinically well - established dl , co could most likely be used , which considerably would facilitate the procedure for the patient and reduces overall examination time as no training is needed prior to measurement . 2 ) a higher breathing flow rate makes it possible to inhale from the rv to tlc , and consequently , the peripheral airspace dimensions can be measured at maximum inflation which eliminates the need for determination of lung volumes for interpretation of data . 3 ) a change in airway radius has a larger effect on the measured parameter , the particle half - life time t , for nanoparticles than for micrometer - sized particles , and thus , the sensitivity is expected to increase . 4 ) interpretation of clinical data is expected to be simpler as the deposition of nanoparticles is almost exclusively determined by diffusion , while the deposition of micron - sized particles is a complex combination of settling and diffusion . 5 ) the mass concentration of the inhaled particles is likely to be reduced by two to three orders of magnitude , which may be of importance for acceptance by patients or regulatory authorities . 6 ) finally , but perhaps most importantly , the penetration of inhaled particles into diseased or poorly ventilated regions of the lung is probably superior for nanoparticles of certain sizes to micron - sized particles . based on the available experimental data and model calculations , the preferable particle size range is likely to be within 40100 nm .

acanthopagrus schlegeli , commonly known as black porgy or black sea bream , belong to the family sparidae in the order perciformes , and live in coastal waters . black porgy are estuarine fish found in shallow ( 50 m ) , sandy , or rocky coastal waters with algal growth and are widely distributed in coastal areas of korea , southern japan , south of hokkaido , ryukyu , the southeastern china sea , and taiwan ( chyung , 1977 ) . their spawning season is may - july ; they spawn on shallow gravel , sandy seabed , or on tidal flats with relatively complex seabed topography ( kim et al . , 2004 ; okamura & amaoka , 1997 ) . black porgy were an aquaculture candidate in the late 1980s and seedling production has been investigated ( lee & rho , 1987 ) . in particular , black porgy have attracted attention as a release target species and were the main species targeted for marine ranching on the jeonnam archipelago in 2002 . early life and growth ( seno , 1912 ; oshim , 1942 ) of larvae and juveniles ( mito , 1957 ) , morphological development of larvae and juveniles ( fukuhara , 1977 ) , and morphological development of larvae and behavioral patterns during early life have also been studied ( fukuhara , 1987 ) . studies in south korea include development of the digestive tract ( lee et al . , 2000 ) , early adaptation of released juveniles ( yoo et al . , 2003 ) , comparison of body components ( ji et al . , 2004 ) , early feeding patterns and body - component changes in released juveniles ( ji et al . , 2007 ) , morphological changes in the spinal cord ( park et al . , 2008 ) , and behavioral characteristics ( kang et al . , 2008 . however , no study has investigated the life cycle of a. schlegeli , except one study on the early life history conducted by kim ( 1970 ) regarding the characteristics of spawned eggs and newly hatched larvae . in this study , we investigated the early life history of a. schlegeli inhabiting the korean southern coast and determined the criteria for classifying larvae and juveniles by observing morphological development of newly hatched larvae . we harvested brood - stock for the lab experimets using an elongated mesh net in the coastal region around yeosusi dolsan ( fig . 1 ) in may 2007 and transported the fish to the resource biology laboratory of jeonnam university . eggs were obtained from sexually mature females ( measured data ) , and the eggs were artificially inseminated using the devrepy method on may 11 , 2007 . some of the hatched larva were kept in a transparent tank ( 30 40 90 cm ) to observe morphological changes from day 1 after hatching to the juvenile stage . we maintained the remaining fish in a larval rearing tank filled with filtered seawater to investigate survival rate and growth . water temperature was measured at 5 day intervals starting from the first day of the experiment . the interval was reset each month , so the first day of the new month was the beginning of the new 5-day interval . rearing water was maintained at 20.521.5c ( mean , 21.9c ) . after stabilizing the rearing water with condensed chlorella paste ( chlorella sp . ) , hatched larvae were fed rotifers ( brachionus plicatilis ) , and subsequently artemia sp . and mixed feed ( fig . morphological development at each growth stage was sketched while observing randomly harvested individuals ( n = 10 ) daily from the first day after hatching . each individual was anesthetized with ice or ms-222 ( tricaine methanesulfonate ; sandoz , basel , switzerland ) , and each body part was observed under a stereo microscope ( nikon , smz-100 ; tokyo , japan ) and a universal projector ( nikon , v-12b ) . each body part was measured to an accuracy of 0.01-mm , and the larval growth stage was defined according to okiyama ( 1988 ) . newly hatched larvae were 1.902.11 mm ( mean , 2.04 mm ) in total length ( tl ) . they carried a yolk - sac , swam at the surface with their mouth closed , the head was smaller than the trunk , and the tail was compressed . black pigment spots were densely distributed around the eyes and the center of the caudal region . a total of 2022 myomeres were detected ( 6 n 8 + 14 ) ( fig . a : newly hatched prelava , mean 2.04 mm total length ( tl ) ; b : 2 days after hatching , mean 2.82 mm in tl ; c : 34 days after hatching , mean 3.05 mm tl ; d : 56 days after hatching , mean 4.45 mm tl ; e : 89 days after hatching , mean 5.75 mm tl ; f : 1618 days after hatching , mean 7.02 mm tl . scale bars = 1.0 mm ; g : 2022 days after hatching , mean 9.09 mm tl ; h : 2325 days after hatching , mean 9.67 mm tl ; i : 3032 days after hatching , mean 15.83 mm tl ; j : 3436 days after hatching , mean 16.58 mm tl ; k : 4345 days after hatching , mean 22.50 mm tl . the larvae had grown to 2.71 - 2.94 mm tl ( mean , 2.82 mm ) on day 2 after hatching , with about two thirds of the yolk absorbed , a formed bladder and intestines , and mouth and anus open . the eyes , yolk , and oil globule were pigmented black , and the black pigment spots in the caudal region began to scatter . at this time , 2224 myomeres were detected ( 7 n 9 + 15 ) ( fig . total larval length was 2.963.24 mm ( mean , 3.05 mm , n = 10 ) on days 34 after hatching , but the yolk and oil globule had not yet completely absorbed . the eyes turned completely black , and dense black pigment spots appeared in the margins of the dorsal and ventral portions of the digestive tract , flexed regions , and ventral portion of the yolk . the dense black pigment spots that formed previously in the caudal region disappeared and were now ventrally aligned along the trunk . the number of myomeres increased to 25 - 26 ( 5 or 6 + 20 ) ( fig . total larval length was 4.324.66 mm ( mean , 4.45 mm , n = 10 ) at the post - larval stage on days 56 after hatching , with the yolk and oil globule almost absorbed . caudal fin rays ( n=68 ) were visible , and the end of the notochord began to flex . black pigment spots were scattered over the head posteriorly and appeared between the ventral myomeres forming a pigmented line ( fig . total larval length was 5.556.03 mm ( mean , 5.75 mm , n = 10 ) on days 89 after hatching . rays had formed in the pectoral fin , the bases of the pectoral and ventral fins appeared , and the number of caudal fin rays increased to 15 . black pigment spots were scattered over the head anteriorly and superiorly and became darker around the digestive tract ( fig . total larval length was 6.857.20 mm ( mean , 7.02 mm , n = 10 ) on days 16 - 18 after hatching . fins that had been membranous in the larval stage became slender and clear in the parts where the dorsal and anal fins would form . the numbers of pectoral and caudal fin rays increased to 7 and 15 , respectively , and eight rays appeared in each of the dorsal and anal fins . the end of the notochord reached complete flexion of 45. the number of black pigment spots decreased slightly dorsally and ventrally around the digestive tract and those on the snout and top of the head tended to be dispersed widely . the melanophores on the base of the anal fin disappeared and remained only ventrally on the caudal peduncle . spines that formed in the opercular area continued to grow , and new melanophores were deposited at the end of the notochord ( fig . total fish length was 8.859.25 mm ( mean , 9.09 mm , n = 10 ) on days 2022 after hatching . the membrane - shaped fins disappeared completely , and the dorsal , anal , and caudal fins took their respective forms . the dorsal fin had 10 spines and 11 rays , the anal fin had two spines and nine rays , and the caudal fin had 17 ( 8 + 9 ) rays . melanophores were deposited in the head posteriorly and on the operculum superiorly and were densely spread along the trunk midline down to half of the spinous dorsal fin . total fish length was 9.2710.01 mm ( mean , 9.67 mm , n = 10 ) on days 2325 after hatching , with an increase in body height . the pectoral fin was elongated around the center to form a rhombus shape , and the ventral fin elongated considerably . the dorsal fin formed notches between the spinous and soft parts , and the caudal fin took a slightly concave form in the center part of the posterior margin . black pigment spots were dispersed ventrally across the trunk from the occipital crest and snout to the pectoral and anal region , as well as dorsally around the region between the first and fifth spines . the post - larvae were now in the transition stage , as the number of soft rays reached the final count . the juveniles had grown to a tl of 15.3716.11 mm ( mean , 15.83 mm , n = 10 ) on days 3032 after hatching , with the spinous part of the dorsal and anal fins elongated , and the caudal fin becoming concave . each fin had reached its full ray count , with dorsal fin ix ( x ) , 11(10 ) , anal fin iii , 9 , pectoral fin 15 , and ventral fin , i 5 ; thus , completing growth into a juvenile . black pigmented spots formed stripes across the body in clusters of 5 - 7 spots ( fig . juveniles were 15.9716.83 mm tl ( mean , 16.58 mm , n = 10 ) on days 3436 after hatching . the black pigment spots around the snout darkened further and were scattered around the dorsal and anal membrane fins , and were particularly clear between the first and fourth dorsal fin spiny rays . the stripes across the trunk took clearer shapes , and the caudal peduncle manifested one or two striped patterns . the caudal fin was more deeply concave in the center , and the ventral fin further elongated . juveniles were covered with scales , and differentiation of the nostrils was completed ( fig . the juveniles were 22.1022.70 mm tl ( mean , 22.50 mm , n = 10 ) on days 4345 after hatching . they had reached sub - adult atage , as body shape and the distribution of black pigment spots and horizontal stripes ( n = 7 - 8 ) completely resembled those of adult fish ( fig . newly hatched larvae were 1.902.11 mm ( mean , 2.04 mm ) in total length ( tl ) . they carried a yolk - sac , swam at the surface with their mouth closed , the head was smaller than the trunk , and the tail was compressed . black pigment spots were densely distributed around the eyes and the center of the caudal region . a total of 2022 myomeres were detected ( 6 n 8 + 14 ) ( fig . a : newly hatched prelava , mean 2.04 mm total length ( tl ) ; b : 2 days after hatching , mean 2.82 mm in tl ; c : 34 days after hatching , mean 3.05 mm tl ; d : 56 days after hatching , mean 4.45 mm tl ; e : 89 days after hatching , mean 5.75 mm tl ; f : 1618 days after hatching , mean 7.02 mm tl . scale bars = 1.0 mm ; g : 2022 days after hatching , mean 9.09 mm tl ; h : 2325 days after hatching , mean 9.67 mm tl ; i : 3032 days after hatching , mean 15.83 mm tl ; j : 3436 days after hatching , mean 16.58 mm tl ; k : 4345 days after hatching , mean 22.50 mm tl . the larvae had grown to 2.71 - 2.94 mm tl ( mean , 2.82 mm ) on day 2 after hatching , with about two thirds of the yolk absorbed , a formed bladder and intestines , and mouth and anus open . the eyes , yolk , and oil globule were pigmented black , and the black pigment spots in the caudal region began to scatter . at this time , 2224 myomeres were detected ( 7 n 9 + 15 ) ( fig . total larval length was 2.963.24 mm ( mean , 3.05 mm , n = 10 ) on days 34 after hatching , but the yolk and oil globule had not yet completely absorbed . the eyes turned completely black , and dense black pigment spots appeared in the margins of the dorsal and ventral portions of the digestive tract , flexed regions , and ventral portion of the yolk . the dense black pigment spots that formed previously in the caudal region disappeared and were now ventrally aligned along the trunk . the number of myomeres increased to 25 - 26 ( 5 or 6 + 20 ) ( fig . total larval length was 4.324.66 mm ( mean , 4.45 mm , n = 10 ) at the post - larval stage on days 56 after hatching , with the yolk and oil globule almost absorbed . caudal fin rays ( n=68 ) were visible , and the end of the notochord began to flex . black pigment spots were scattered over the head posteriorly and appeared between the ventral myomeres forming a pigmented line ( fig . total larval length was 5.556.03 mm ( mean , 5.75 mm , n = 10 ) on days 89 after hatching . rays had formed in the pectoral fin , the bases of the pectoral and ventral fins appeared , and the number of caudal fin rays increased to 15 . black pigment spots were scattered over the head anteriorly and superiorly and became darker around the digestive tract ( fig . total larval length was 6.857.20 mm ( mean , 7.02 mm , n = 10 ) on days 16 - 18 after hatching . fins that had been membranous in the larval stage became slender and clear in the parts where the dorsal and anal fins would form . the numbers of pectoral and caudal fin rays increased to 7 and 15 , respectively , and eight rays appeared in each of the dorsal and anal fins . the end of the notochord reached complete flexion of 45. the number of black pigment spots decreased slightly dorsally and ventrally around the digestive tract and those on the snout and top of the head tended to be dispersed widely . the melanophores on the base of the anal fin disappeared and remained only ventrally on the caudal peduncle . spines that formed in the opercular area continued to grow , and new melanophores were deposited at the end of the notochord ( fig . total fish length was 8.859.25 mm ( mean , 9.09 mm , n = 10 ) on days 2022 after hatching . the membrane - shaped fins disappeared completely , and the dorsal , anal , and caudal fins took their respective forms . the dorsal fin had 10 spines and 11 rays , the anal fin had two spines and nine rays , and the caudal fin had 17 ( 8 + 9 ) rays . melanophores were deposited in the head posteriorly and on the operculum superiorly and were densely spread along the trunk midline down to half of the spinous dorsal fin . total fish length was 9.2710.01 mm ( mean , 9.67 mm , n = 10 ) on days 2325 after hatching , with an increase in body height . the pectoral fin was elongated around the center to form a rhombus shape , and the ventral fin elongated considerably . the dorsal fin formed notches between the spinous and soft parts , and the caudal fin took a slightly concave form in the center part of the posterior margin . black pigment spots were dispersed ventrally across the trunk from the occipital crest and snout to the pectoral and anal region , as well as dorsally around the region between the first and fifth spines . the post - larvae were now in the transition stage , as the number of soft rays reached the final count . the juveniles had grown to a tl of 15.3716.11 mm ( mean , 15.83 mm , n = 10 ) on days 3032 after hatching , with the spinous part of the dorsal and anal fins elongated , and the caudal fin becoming concave . each fin had reached its full ray count , with dorsal fin ix ( x ) , 11(10 ) , anal fin iii , 9 , pectoral fin 15 , and ventral fin , i 5 ; thus , completing growth into a juvenile . black pigmented spots formed stripes across the body in clusters of 5 - 7 spots ( fig . juveniles were 15.9716.83 mm tl ( mean , 16.58 mm , n = 10 ) on days 3436 after hatching . the black pigment spots around the snout darkened further and were scattered around the dorsal and anal membrane fins , and were particularly clear between the first and fourth dorsal fin spiny rays . the stripes across the trunk took clearer shapes , and the caudal peduncle manifested one or two striped patterns . the caudal fin was more deeply concave in the center , and the ventral fin further elongated . juveniles were covered with scales , and differentiation of the nostrils was completed ( fig . the juveniles were 22.1022.70 mm tl ( mean , 22.50 mm , n = 10 ) on days 4345 after hatching . they had reached sub - adult atage , as body shape and the distribution of black pigment spots and horizontal stripes ( n = 7 - 8 ) completely resembled those of adult fish ( fig . the average tl of newly hatched a. schlegeli larvae in our study was 2.04 mm ( range , 1.902.11 mm ) , which differed from that reported by kim ( 1970 ) at 1.57 mm , and fukuhara ( 1987 ) at 2.17 mm . the size of newly hatched larvae is assumed to be related to the size of the fertilized egg . the size of a. schlegeli larvae as measured in our study was similar to that of pagrus major in the same family sparidae [ 2.01 0.06 mm ( fukuhara , 1984 ) , 2.0 3.0 mm ( pyen & jo , 1982 ) ] , and acanthopagrus latus [ 1.982.06 mm ( leu & chou , 1996 ) ] . however , our larvae were smaller than those of acanthopagrus sivicolus [ 2.15 2.39 mm ( tawada , 1986 ) ] , sparus sarba [ 3.3 mm ( deane et al . , 2003 ) ] , and girella punctata [ 2.272.35 mm ( mito , 1957 ) ] ( table 1 ) , but larger than those of gymnocranius griseus [ 1.481.50 mm ( suzumiu & hioki , 1978 ) ] and oplegnathus fasciatus [ 1.591.82 mm ( koh , 1992 ) ] . comparison of prelarval characters in sparidae and other species larvae hatched with a closed mouth and anus . a. schlegeli is a species with a low degree of differentiation compared with that of cypselurus agoo agoo ( kim & park , 1987 ) , hyporhamphus sajori ( kim et al . , 1984 ) , and cololabis saira ( yusa , 1960 ) , which hatch from demersal eggs . the larval oil globule is located posterior to the yolk and anterior to the anus , which is different from the anteriorinferior positioning of plectorhinchus cinctus ( kobayashi & iwamoto , 1984 ) and gymnocranius griseus ( suzuki & hioki , 1978 ) and consistent with that of girella punctata ( mito , 1957 ) and dentex tumifrons ( oka et al . , 1956 ) . pagrus major ( han & kim , 1999 ) has an oil globule in a similar location to that of a. schlegeli , but they differ in that they hatch with the anus open . the number of larval myomeres was 2022 ( 6 n 8 + 14 ) in our study , which was the same as that observed by kim ( 1970 ) , 10 + 12 = 22 , but less than 8 + 18 = 26 of a. sivicolus ( tawada , 1986 ) , 89 + 1718 = 2527 and 9 + 1718 = 2628 of pagrus major ( han & kim , 1999 ; mito , 1963 ) , 9 + 15 = 24 of gymnocranius griseus ( suzumiu & hioki , 1978 ) , 10 + 17 = 27 of oplegnathus fasciatus ( koh , 1992 ) , and 11 + 16 = 27 of girella punctata ( mito , 1957 ) ( table 1 ) . the transition from larvae to post - larvae is determined by the yolk absorption period after hatching and is the most important factor determining the feeding plan during the fish production cycle ( hansen & moller , 1985 ) . the yolk absorption period varies depending on the species , habitat , and water temperature . in general , a. schlegeli requires about 5 days after hatching to complete yolk absorption ( kim , 1970 ) , as demonstrated in our study ( 56 days ) . a. sivicolus completes yolk absorption on day 4 after hatching ( tawada , 1986 ) , pagrus major completed yolk absorption 23 days ( fukuhara , 1984 ; pyen & jo , 1982 ) , acanthopagrus latus in 23 days ( leu & chou , 1996 ) , and sparus sarba in 7 days ( deane et al . , 2003 ) , demonstrating differences among genera and species in the same family . furthermore , girella punctata exhibits faster yolk absorption at 3 days ( mito , 1957 ) , and oplegnathus fasciatus was similar at 6 days ( koh , 1992 ) . such differences are assumed to result from accelerated yolk consumption as developmental speed and nutrient demands increase due to increased physiological activities in proportion to an increase in rearing water temperature ( heming , 1982 ) . the transition from post - larvae to juveniles is determined when all fins reach their full counts , and when band patterns resemble those of adult fish . in our experiment , juveniles began to show body shape and color similar to those of brood stock on days 3032 after hatching . in comparison , the duration for a. sivicolus was 45 days ( tawada , 1986 ) , that for oplegnathus fasciatus was 37 days ( koh , 1992 ) , and that for acanthopagrus latus was 3038 days ( leu & chou , 1996 ) . . however , sparus sarba can be easily distinguished by the number of rays on the dorsal fin ( n = 23 ) , anal fin ( n = 13 ) , and regions where black pigment spots appear , such as the caudal peduncle . a. latus can be distinguished by the lack of melanophores at the base of dorsal fin i and the angle region . a. sivicolus , a. berda , and a. australis can also be easily distinguished due to different habitats ( south of amami oshima in japan ) ( senta & kinoshita , 1985 ) . among different sparidae species , dentex tumifrons is conspicuous because of its well developed spines at the outer margin of the preopercle , and pagrus major and evynnis cardinalis have higher body heights than that of a. schlegeli ( okiyama , 1988 ) . some species show no evidence of melanophores until hatching , such as hyporhamphus sajori ( kim et al . , 1984 ) . similar to p. major ( han & kim , 1999 ) , the heart of a. schlegeli begins to beat after black pigment spots appear on the body . ( 2007 ) reported that a cluster of clear black pigment spots appears on the occipital crest at a tl of about 5.5 mm . an additional cluster occurs in the dorsal digestive tract , large melanophores form inferior to the digestive tract , and about 10 melanophores align inferiorly to the caudal margin . in the present study , black pigment spots densely developed in the occipital crest and around the digestive tract both dorsally and ventrally , and about 12 black pigment spots were aligned ventrally along the caudal margin at a mean tl of 5.75 mm ( fig . the slight difference in the black pigment spots was assumed to be due to coagulation or loss of melanophores during sampling and storage rather than differences among regional communities . a scorpaenidae species caught in china has a different number and shape of black pigment spots on the dorsal fin compared with those caught in korea and japan ( myoung et al . , 1989 ) . thus , further study on a. schlegeli is necessary to determine the differences , if any , among regional communities by investigating the life cycles of individuals caught in various regions . 4 ) . mean increase in total length ( cm ) of acanthopagrus schlegeli ( ) , chrysophrys major ( ) , and acanthopagrus latus ( ) during the rearing experiment . , present study ; , pyen & jo ( 1982 ) ; , leu and chou ( 1996 ) . a. schlegeli had a slower grow rate than that of p. major ( y = 2.817e0.053x , r = 0.942 vs. y = 2.218e0.077x , r = 0.995 ) but faster than that of a. latus ( y = 1.730e0.349x , r = 0.957 ) from day 1 after hatching ( leu & chou , 1996 ; pyen & jo , 1982 ) . taken together , these results show that a. schlegeli have a slightly slower growth rate during the early stages compared with that of other species in the same family .

newly hatched black porgy larvae ( acanthopagrus schlegeli ) swam to the surface , with the mouth and anus still closed and were 1.902.11 mm ( mean , 2.0 mm ) in total length ( tl ) . the larvae were 2.712.94 mm tl ( mean , 2.82 mm ) on day 2 after hatching . at this time , about two - thirds of the yolk was absorbed , the bladder and intestines had formed , and the mouth and anus were open . total length was 4.324.66 mm ( mean , 4.45 mm ) at the post - larval stage on days 56 after hatching , and the yolk and oil globule were almost absorbed . the end of the notochord began to flex , and 68 caudal fin rays were visible . the larvae were 15.3716.1 mm tl ( mean , 15.83 mm ) at the juvenile stage on days 3032 after hatching , and the number of rays in all fins was completely revealed .

INTRODUCTION MATERIALS AND METHODS RESULTS Larvae Post-larvae Juveniles DISCUSSION

acanthopagrus schlegeli , commonly known as black porgy or black sea bream , belong to the family sparidae in the order perciformes , and live in coastal waters . black porgy are estuarine fish found in shallow ( 50 m ) , sandy , or rocky coastal waters with algal growth and are widely distributed in coastal areas of korea , southern japan , south of hokkaido , ryukyu , the southeastern china sea , and taiwan ( chyung , 1977 ) . in particular , black porgy have attracted attention as a release target species and were the main species targeted for marine ranching on the jeonnam archipelago in 2002 . 1 ) in may 2007 and transported the fish to the resource biology laboratory of jeonnam university . eggs were obtained from sexually mature females ( measured data ) , and the eggs were artificially inseminated using the devrepy method on may 11 , 2007 . some of the hatched larva were kept in a transparent tank ( 30 40 90 cm ) to observe morphological changes from day 1 after hatching to the juvenile stage . rearing water was maintained at 20.521.5c ( mean , 21.9c ) . , hatched larvae were fed rotifers ( brachionus plicatilis ) , and subsequently artemia sp . each body part was measured to an accuracy of 0.01-mm , and the larval growth stage was defined according to okiyama ( 1988 ) . newly hatched larvae were 1.902.11 mm ( mean , 2.04 mm ) in total length ( tl ) . they carried a yolk - sac , swam at the surface with their mouth closed , the head was smaller than the trunk , and the tail was compressed . black pigment spots were densely distributed around the eyes and the center of the caudal region . a : newly hatched prelava , mean 2.04 mm total length ( tl ) ; b : 2 days after hatching , mean 2.82 mm in tl ; c : 34 days after hatching , mean 3.05 mm tl ; d : 56 days after hatching , mean 4.45 mm tl ; e : 89 days after hatching , mean 5.75 mm tl ; f : 1618 days after hatching , mean 7.02 mm tl . scale bars = 1.0 mm ; g : 2022 days after hatching , mean 9.09 mm tl ; h : 2325 days after hatching , mean 9.67 mm tl ; i : 3032 days after hatching , mean 15.83 mm tl ; j : 3436 days after hatching , mean 16.58 mm tl ; k : 4345 days after hatching , mean 22.50 mm tl . the larvae had grown to 2.71 - 2.94 mm tl ( mean , 2.82 mm ) on day 2 after hatching , with about two thirds of the yolk absorbed , a formed bladder and intestines , and mouth and anus open . the eyes , yolk , and oil globule were pigmented black , and the black pigment spots in the caudal region began to scatter . at this time , 2224 myomeres were detected ( 7 n 9 + 15 ) ( fig . total larval length was 2.963.24 mm ( mean , 3.05 mm , n = 10 ) on days 34 after hatching , but the yolk and oil globule had not yet completely absorbed . the eyes turned completely black , and dense black pigment spots appeared in the margins of the dorsal and ventral portions of the digestive tract , flexed regions , and ventral portion of the yolk . the number of myomeres increased to 25 - 26 ( 5 or 6 + 20 ) ( fig . total larval length was 4.324.66 mm ( mean , 4.45 mm , n = 10 ) at the post - larval stage on days 56 after hatching , with the yolk and oil globule almost absorbed . caudal fin rays ( n=68 ) were visible , and the end of the notochord began to flex . total larval length was 5.556.03 mm ( mean , 5.75 mm , n = 10 ) on days 89 after hatching . rays had formed in the pectoral fin , the bases of the pectoral and ventral fins appeared , and the number of caudal fin rays increased to 15 . total larval length was 6.857.20 mm ( mean , 7.02 mm , n = 10 ) on days 16 - 18 after hatching . the numbers of pectoral and caudal fin rays increased to 7 and 15 , respectively , and eight rays appeared in each of the dorsal and anal fins . the end of the notochord reached complete flexion of 45. the number of black pigment spots decreased slightly dorsally and ventrally around the digestive tract and those on the snout and top of the head tended to be dispersed widely . spines that formed in the opercular area continued to grow , and new melanophores were deposited at the end of the notochord ( fig . total fish length was 8.859.25 mm ( mean , 9.09 mm , n = 10 ) on days 2022 after hatching . the membrane - shaped fins disappeared completely , and the dorsal , anal , and caudal fins took their respective forms . the dorsal fin had 10 spines and 11 rays , the anal fin had two spines and nine rays , and the caudal fin had 17 ( 8 + 9 ) rays . melanophores were deposited in the head posteriorly and on the operculum superiorly and were densely spread along the trunk midline down to half of the spinous dorsal fin . total fish length was 9.2710.01 mm ( mean , 9.67 mm , n = 10 ) on days 2325 after hatching , with an increase in body height . the pectoral fin was elongated around the center to form a rhombus shape , and the ventral fin elongated considerably . the dorsal fin formed notches between the spinous and soft parts , and the caudal fin took a slightly concave form in the center part of the posterior margin . the post - larvae were now in the transition stage , as the number of soft rays reached the final count . the juveniles had grown to a tl of 15.3716.11 mm ( mean , 15.83 mm , n = 10 ) on days 3032 after hatching , with the spinous part of the dorsal and anal fins elongated , and the caudal fin becoming concave . each fin had reached its full ray count , with dorsal fin ix ( x ) , 11(10 ) , anal fin iii , 9 , pectoral fin 15 , and ventral fin , i 5 ; thus , completing growth into a juvenile . juveniles were 15.9716.83 mm tl ( mean , 16.58 mm , n = 10 ) on days 3436 after hatching . the black pigment spots around the snout darkened further and were scattered around the dorsal and anal membrane fins , and were particularly clear between the first and fourth dorsal fin spiny rays . the stripes across the trunk took clearer shapes , and the caudal peduncle manifested one or two striped patterns . the caudal fin was more deeply concave in the center , and the ventral fin further elongated . juveniles were covered with scales , and differentiation of the nostrils was completed ( fig . the juveniles were 22.1022.70 mm tl ( mean , 22.50 mm , n = 10 ) on days 4345 after hatching . newly hatched larvae were 1.902.11 mm ( mean , 2.04 mm ) in total length ( tl ) . they carried a yolk - sac , swam at the surface with their mouth closed , the head was smaller than the trunk , and the tail was compressed . black pigment spots were densely distributed around the eyes and the center of the caudal region . a : newly hatched prelava , mean 2.04 mm total length ( tl ) ; b : 2 days after hatching , mean 2.82 mm in tl ; c : 34 days after hatching , mean 3.05 mm tl ; d : 56 days after hatching , mean 4.45 mm tl ; e : 89 days after hatching , mean 5.75 mm tl ; f : 1618 days after hatching , mean 7.02 mm tl . scale bars = 1.0 mm ; g : 2022 days after hatching , mean 9.09 mm tl ; h : 2325 days after hatching , mean 9.67 mm tl ; i : 3032 days after hatching , mean 15.83 mm tl ; j : 3436 days after hatching , mean 16.58 mm tl ; k : 4345 days after hatching , mean 22.50 mm tl . the larvae had grown to 2.71 - 2.94 mm tl ( mean , 2.82 mm ) on day 2 after hatching , with about two thirds of the yolk absorbed , a formed bladder and intestines , and mouth and anus open . the eyes , yolk , and oil globule were pigmented black , and the black pigment spots in the caudal region began to scatter . at this time , 2224 myomeres were detected ( 7 n 9 + 15 ) ( fig . total larval length was 2.963.24 mm ( mean , 3.05 mm , n = 10 ) on days 34 after hatching , but the yolk and oil globule had not yet completely absorbed . the eyes turned completely black , and dense black pigment spots appeared in the margins of the dorsal and ventral portions of the digestive tract , flexed regions , and ventral portion of the yolk . the number of myomeres increased to 25 - 26 ( 5 or 6 + 20 ) ( fig . total larval length was 4.324.66 mm ( mean , 4.45 mm , n = 10 ) at the post - larval stage on days 56 after hatching , with the yolk and oil globule almost absorbed . caudal fin rays ( n=68 ) were visible , and the end of the notochord began to flex . total larval length was 5.556.03 mm ( mean , 5.75 mm , n = 10 ) on days 89 after hatching . rays had formed in the pectoral fin , the bases of the pectoral and ventral fins appeared , and the number of caudal fin rays increased to 15 . total larval length was 6.857.20 mm ( mean , 7.02 mm , n = 10 ) on days 16 - 18 after hatching . the numbers of pectoral and caudal fin rays increased to 7 and 15 , respectively , and eight rays appeared in each of the dorsal and anal fins . the end of the notochord reached complete flexion of 45. the number of black pigment spots decreased slightly dorsally and ventrally around the digestive tract and those on the snout and top of the head tended to be dispersed widely . spines that formed in the opercular area continued to grow , and new melanophores were deposited at the end of the notochord ( fig . total fish length was 8.859.25 mm ( mean , 9.09 mm , n = 10 ) on days 2022 after hatching . the membrane - shaped fins disappeared completely , and the dorsal , anal , and caudal fins took their respective forms . the dorsal fin had 10 spines and 11 rays , the anal fin had two spines and nine rays , and the caudal fin had 17 ( 8 + 9 ) rays . melanophores were deposited in the head posteriorly and on the operculum superiorly and were densely spread along the trunk midline down to half of the spinous dorsal fin . total fish length was 9.2710.01 mm ( mean , 9.67 mm , n = 10 ) on days 2325 after hatching , with an increase in body height . the pectoral fin was elongated around the center to form a rhombus shape , and the ventral fin elongated considerably . the dorsal fin formed notches between the spinous and soft parts , and the caudal fin took a slightly concave form in the center part of the posterior margin . the post - larvae were now in the transition stage , as the number of soft rays reached the final count . the juveniles had grown to a tl of 15.3716.11 mm ( mean , 15.83 mm , n = 10 ) on days 3032 after hatching , with the spinous part of the dorsal and anal fins elongated , and the caudal fin becoming concave . each fin had reached its full ray count , with dorsal fin ix ( x ) , 11(10 ) , anal fin iii , 9 , pectoral fin 15 , and ventral fin , i 5 ; thus , completing growth into a juvenile . juveniles were 15.9716.83 mm tl ( mean , 16.58 mm , n = 10 ) on days 3436 after hatching . the black pigment spots around the snout darkened further and were scattered around the dorsal and anal membrane fins , and were particularly clear between the first and fourth dorsal fin spiny rays . the stripes across the trunk took clearer shapes , and the caudal peduncle manifested one or two striped patterns . the caudal fin was more deeply concave in the center , and the ventral fin further elongated . juveniles were covered with scales , and differentiation of the nostrils was completed ( fig . the juveniles were 22.1022.70 mm tl ( mean , 22.50 mm , n = 10 ) on days 4345 after hatching . the average tl of newly hatched a. schlegeli larvae in our study was 2.04 mm ( range , 1.902.11 mm ) , which differed from that reported by kim ( 1970 ) at 1.57 mm , and fukuhara ( 1987 ) at 2.17 mm . the size of newly hatched larvae is assumed to be related to the size of the fertilized egg . the size of a. schlegeli larvae as measured in our study was similar to that of pagrus major in the same family sparidae [ 2.01 0.06 mm ( fukuhara , 1984 ) , 2.0 3.0 mm ( pyen & jo , 1982 ) ] , and acanthopagrus latus [ 1.982.06 mm ( leu & chou , 1996 ) ] . however , our larvae were smaller than those of acanthopagrus sivicolus [ 2.15 2.39 mm ( tawada , 1986 ) ] , sparus sarba [ 3.3 mm ( deane et al . , 2003 ) ] , and girella punctata [ 2.272.35 mm ( mito , 1957 ) ] ( table 1 ) , but larger than those of gymnocranius griseus [ 1.481.50 mm ( suzumiu & hioki , 1978 ) ] and oplegnathus fasciatus [ 1.591.82 mm ( koh , 1992 ) ] . comparison of prelarval characters in sparidae and other species larvae hatched with a closed mouth and anus . the larval oil globule is located posterior to the yolk and anterior to the anus , which is different from the anteriorinferior positioning of plectorhinchus cinctus ( kobayashi & iwamoto , 1984 ) and gymnocranius griseus ( suzuki & hioki , 1978 ) and consistent with that of girella punctata ( mito , 1957 ) and dentex tumifrons ( oka et al . pagrus major ( han & kim , 1999 ) has an oil globule in a similar location to that of a. schlegeli , but they differ in that they hatch with the anus open . the number of larval myomeres was 2022 ( 6 n 8 + 14 ) in our study , which was the same as that observed by kim ( 1970 ) , 10 + 12 = 22 , but less than 8 + 18 = 26 of a. sivicolus ( tawada , 1986 ) , 89 + 1718 = 2527 and 9 + 1718 = 2628 of pagrus major ( han & kim , 1999 ; mito , 1963 ) , 9 + 15 = 24 of gymnocranius griseus ( suzumiu & hioki , 1978 ) , 10 + 17 = 27 of oplegnathus fasciatus ( koh , 1992 ) , and 11 + 16 = 27 of girella punctata ( mito , 1957 ) ( table 1 ) . the transition from larvae to post - larvae is determined by the yolk absorption period after hatching and is the most important factor determining the feeding plan during the fish production cycle ( hansen & moller , 1985 ) . the yolk absorption period varies depending on the species , habitat , and water temperature . a. sivicolus completes yolk absorption on day 4 after hatching ( tawada , 1986 ) , pagrus major completed yolk absorption 23 days ( fukuhara , 1984 ; pyen & jo , 1982 ) , acanthopagrus latus in 23 days ( leu & chou , 1996 ) , and sparus sarba in 7 days ( deane et al . the transition from post - larvae to juveniles is determined when all fins reach their full counts , and when band patterns resemble those of adult fish . in our experiment , juveniles began to show body shape and color similar to those of brood stock on days 3032 after hatching . in comparison , the duration for a. sivicolus was 45 days ( tawada , 1986 ) , that for oplegnathus fasciatus was 37 days ( koh , 1992 ) , and that for acanthopagrus latus was 3038 days ( leu & chou , 1996 ) . however , sparus sarba can be easily distinguished by the number of rays on the dorsal fin ( n = 23 ) , anal fin ( n = 13 ) , and regions where black pigment spots appear , such as the caudal peduncle . a. latus can be distinguished by the lack of melanophores at the base of dorsal fin i and the angle region . among different sparidae species , dentex tumifrons is conspicuous because of its well developed spines at the outer margin of the preopercle , and pagrus major and evynnis cardinalis have higher body heights than that of a. schlegeli ( okiyama , 1988 ) . an additional cluster occurs in the dorsal digestive tract , large melanophores form inferior to the digestive tract , and about 10 melanophores align inferiorly to the caudal margin . in the present study , black pigment spots densely developed in the occipital crest and around the digestive tract both dorsally and ventrally , and about 12 black pigment spots were aligned ventrally along the caudal margin at a mean tl of 5.75 mm ( fig . mean increase in total length ( cm ) of acanthopagrus schlegeli ( ) , chrysophrys major ( ) , and acanthopagrus latus ( ) during the rearing experiment .

alcohol consumption is one of the most common risk factors contributing to physical and psychological ill health . there has been a progressive increase in the harmful use of alcohol with approximately 2.5 million deaths each year and has been identified as the leading risk factor for death in men between the age of 15 and 59 years . in the national household survey of alcohol and drug abuse ( 2003 ) , 21.4% were reported to be current users of alcohol ( used in the last 30 days ) . alcoholism accounts for 4% of all global deaths and 4.6% of global disability - adjusted life years in addition to significant increase in psychosocial burden in the community . according to the criteria for alcohol dependence described by the diagnostic and statistical manual of mental disorders , dependence is defined as a maladaptive pattern of alcohol use , leading to clinically significant impairment or distress , as manifested by three ( or more ) of the seven criteria , occurring at any time in the same 12-month period . the united states - food and drug administration ( fda ) approved medications are available for use in routine practice for the treatment of alcohol dependence . it is reported that ~50% of alcohol - dependent patients relapse within 3 months , despite following a treatment protocol using these approved medications . while , the pathophysiology of alcohol dependence is complex and incompletely understood at the present time , there is an accumulating evidence demonstrating combined pharmacotherapy with psychotherapy as more efficacious approach than either of the treatments alone . the treatment outcome is known to depend on several patient - related factors such as motivation , emotion , willingness to abstain , efficacy of medications , and adherence to medications . low adherence to prescribed medications is said to be one of the limitations contributing to failure in achieving abstinence and preventing relapse . the present study was designed to evaluate the pattern of use of anti - craving medications , the extent of adherence to them , factors that influence adherence and the influence of medication adherence on time to the first drink , and on relapse in drinking among alcohol - dependent patients . this was a prospective study conducted for 18 months at a tertiary care hospital following the institutional ethical review board 's approval . subjects included were clinically diagnosed as alcohol - dependent according to the international classification of diseases 10 criteria , aged 20 years , and received treatment for alcohol dependence as hospital inpatients . the study exclusion criteria were patients with concurrent major psychiatric and other serious organ system disorders , psychotic disorders induced by harmful use of other substances , and complicated withdrawal symptoms . the data on the sociodemography of the patients were collected during the hospital stay , while data on alcohol consumption and anti - craving medication use were recorded at discharge as well as during the time of follow - up at each outpatient clinic visit , i.e. , 1 , 3 , 8 , and 12 week . the treatment with anti - craving medications and necessary psychological interventions was discussed with each patient and their care takers by a psychiatric expert based on the reports of relevant investigations , along with their benefits and adverse effects before initiating treatment regimen to induce abstinence and/or prevent relapse . at the time of discharge , the patients were instructed to take their medications as advised by the psychiatric expert and each patient was provided with a drug diary to record the information on medication details they consumed , the number of missed tablets , if any , reasons for the same , and adverse effects when experienced . the adherence to medication was assessed at every follow - up visit through patients self - report , medication dairy , and simplified medication adherence questionnaire ( smaq ) , and the data on the levels of adherence ( proportion of prescribed medications taken by the patient ) and persistence ( whether the patient continued with prescribed medications ) were recorded . patients who were unable to fill the medication diary were provided assistance by the investigator on the day of the follow - up visit to update the information from the previous visit to the current follow - up . the patient follow - up was either on their scheduled clinic visit or through telephonic interview if they were unable to visit the clinic . the patients who neither visited the hospital nor were reachable for telephonic interview were considered as lost to follow - up . the assessment of anti - craving treatment effect on the drinking outcomes and factors affecting adherence to them was collected over a period of 12 weeks . anti - craving medication adherence was assessed using patient 's self - report by recording whether he / she was regular with consumption . furthermore , through the medication diary at each visit to see the entry for number of days and tablets that the patient had taken . the smaq which is a prevalidated , structured six - point questionnaire was also used to quantify medication adherence and to evaluate the extent of nonadherence , which is in use for patients with acquired immune deficiency syndrome . the six questions used in the study are given below and scoring consisted of yes = 1 and no = 0 responses from patients : 1 . response , 1 point was given and 0 point for no response . the increasing order of scores from 0 to 6 was considered as decreasing levels of adherence and was then correlated with the percentage adherence scores . smaq score of more than four was considered as nonadherent and reason for this was recorded . anti - craving medication efficacy outcome was assessed as time to the first drink and time to relapse . pattern and extent of adverse effects to anti - craving medications were classified based on organ system affected . baseline and continuous data such as age were subjected to descriptive statistical analysis and expressed as number as well as percentages . comparison of continuous variables between groups was done by independent t - test with a level of significance < 0.05 . spss version 17.0 ( ibm corporation , armonk , new york , united states ) was used to analyze the data . a total of 102 patients with a mean ( standard deviation ) age of 41.17 ( 9.8 ) years constituted the study group . the clinical characteristics of patients , their percentage wise distribution for the level of ses , and area of residence are shown in table 1 . the demographic profile of patients with alcohol dependence of the total number of 102 patients - 101 ( 99.01% ) , 98 ( 96.07% ) , 91 ( 89.21% ) , and 79 ( 77.45% ) patients completed the follow - up during 1 , 3 , 8 , and 12 week , respectively . acamprosate was prescribed as an anti - craving medication in 77 patients ( 75.49% ) both during hospital stay and at discharge with median interquartile dose range of 1332 and 1998 mg / day . the number of patients who were adherent to acamprosate was 69 ( 89.61% ) , 64 ( 83.11% ) , 54 ( 70.12% ) , and 42 ( 54.54% ) at 1 , 3 , 8 , and 12 week follow - up , respectively . this reduction in the proportion of patients adherent across time was significant ( p < 0.001 ) at the end of 12 weeks . naltrexone was prescribed in 7 ( 6.8% ) patients at a dose of 25 mg / day with 3 ( 37.50% ) patients adherent after 12 weeks . disulfiram was prescribed in 6 ( 5.88% ) patients at a dose of 125 - 500 mg / day and had 100% adherence over 12 weeks . during a 12-week follow - up period , 57 ( 55.89% ) patients reported to have taken 100% of the prescribed doses of anti - craving medications at any point of time . other medications prescribed were baclofen , topiramate , and ondansetron [ table 2 ] . the pattern of anti - craving medication use and their persistence pattern in patients with alcohol dependence the number of patients who reported for follow - up reduced significantly ( p < 0.001 ) from 70 ( 68.6% ) at the first visit to 19 ( 18.65% ) at the fourth visit . the number of patients who were reminded over telephone and also the number of telephonic reminders to each patient regarding the follow - up visit increased over time and this was statistically significant ( p < 0.001 ) . the odds ratio of patients coming for follow - up after phone call reduced from 29.0% at the first visit to 11.6% at the fourth visit and was significant [ table 3 and figure 1 ] . characteristics and rates of follow - up over 12 weeks characteristics and rates of follow - up of alcohol - dependent patients over 12 weeks . follow - up visit : visit 1 = 1 week , visit 2 = 3 week , visit 3 = 8 week , visit 4 = 12 week the analysis of adherence to anti - craving medications by patients self - report , through medication diary and smaq scores , did not show statistically significant difference in the levels of adherence [ table 4 ] . the responses to smaq exhibited an all or none phenomenon on adherence to medications , with 57 ( 55.89% ) patients completely adherent and 45 ( 44.11% ) nonadherent at the end of the fourth follow - up visit . these groups were comparable in terms of baseline characteristics such as age , marital status , prior admission for the management of alcohol dependence , anti - craving medication use in the recent past , and number of caregivers . comparison of baseline variables between patients who were adherent and nonadherent to anti - craving medications the adherent group showed significantly more number of abstinent days with lower relapse rate ( p < 0.001 ) . table 5 provides data on the influence of anti - craving medication adherence on drinking outcomes in alcohol - dependent patients . with increase in the number of days of follow - up , there was an increase in the number of patients who relapsed with a parallel decrease in the number of days for the first drink , namely , on 1 week , 6 ( 5.9% ) patients had the first drink since discharge and 4 ( 3.94% ) had relapsed ; by 3 week , 13 ( 12.74% ) patients had the first drink and 10 ( 9.80% ) had relapsed ; by 8 week , 28 ( 27.45% ) patients had the first drink and 24 ( 23.53% ) relapsed ; and by 12 week follow - up , 49 ( 48.03% ) patients had the first drink and 44 ( 43.17% ) patients relapsed [ table 5 ] . influence of anti - craving medication adherence on drinking outcomes in alcohol - dependent patients the univariate analysis for variables such as age , sex , history of prior hospitalization , marital status , and social support in terms of number of caregivers , place of origin , and ses showed no significant difference . multivariate analysis showed patients of younger age group ( odds ratio = 1.05 95% [ 1.01 - 1.09 ] ; p < 0.01 ) to be significantly less adherent to anti - craving medications . there were 97 responses with reasons for nonadherence , of which 38 ( 39.17% ) reported to have stopped anti - craving medication on their own . the response to reason for nonadherence given was that they had either improvement in condition and/or were confident of quitting alcohol or they had started to drink alcohol again and felt unnecessary to take medication . by 12 weeks , 20 ( 19.6% ) patients had experienced adverse drug reactions ( adrs ) , of which 9 ( 45% ) were on acamprosate who stopped medication due to subjectively perceived side effects [ table 6 ] . the results of the present study showed that the extent of adherence to anti - craving medications reduced significantly over time and that there was an early relapse in drinking behavior among alcohol - dependent patients . the co - existing medical conditions such as hypertension and diabetes mellitus justify the reasons for hospital admissions . since adherence to pharmacotherapy and clinical outcome of patients is well known to be influenced by major and serious comorbidities , such patients were excluded when they were uncontrolled or recognized in the beginning of the study . the pattern of anti - craving medication use was similar to that approved by the fda . the maximal use of acamprosate both as monotherapy and in combination with other medications in a proportion of patients may possibly be attributed to relatively reduced potential and/or absence of known serious interactions between acamprosate and alcohol or with other routinely prescribed medications . however , we observed that the most frequent reason for nonadherence was among those patients who were prescribed acamprosate and maximum number of the patients reported self - termination of the medication . the present study used three different tools to measure adherence to anti - craving medications . while , patient self - report as one of the methods is regarded as the most convenient method for measuring adherence , the same is known to have flaws since it is said to roughly give the dose count , but not the number of doses the patient missed and if the doses were taken at the right time . to minimize this limitation , we assessed medication adherence additionally using medication diary and smaq . the extent of adherence varied from highest with disulfiram to moderate among naltrexone and acamprosate groups . this finding may be attributed to the smaller number of patients in the present study , a selection bias in prescribing medications , and naturalistic study setting . a wide variation in treatment adherence rates ( 20 - 80% ) has been reported in studies based on the types of intervention . in previous studies , the nonadherence rates of 20 - 60% for naltrexone , 53% for acamprosate , and highest for disulfiram are reported where treatment duration ranged from 3 to 24 months . such difference in the extent of nonadherence to various anti - craving medications is said to be due to patient - related factors similar to that seen in the present study such as self - decision to stop taking medications , either due to confidence to abstain from alcohol or the decision to restart consuming alcohol , along with other emotional factors . in addition , a frequently reported barrier that we found was adrs to prescribed treatments , similar to those reported in the recent cochrane reviews . the who criteria that may influence medication adherence include socioeconomic , marital / educational status , health - care system , medical condition , treatment of co - morbidities , and role of caregivers . however , these did not show a significant influence in maintaining adherence in our study . in addition , barriers such as forgetfulness to take medications , their cost , complexity of regimens and polypharmacy were not found to contribute to nonadherence . another interesting observation was that adults and elderly > 40 years were found to be more adherent compared to patients < 30 years of age . in the indian context , this behavior may be attributed to social responsibilities of adults and elderly people compared to the influence of peer group pressure among younger alcohol dependents . given the unique set of barriers that are proposed to be involved in the intrinsic behavior of each patient , a complete understanding and assessment of factors involved in medication adherence is challenging in these types of study design . several studies have shown that medication nonadherence is the most common cause of therapeutic failure in many acute and chronic disorders . from the present study , it appears that the use of anti - craving medications induces abstinence and underscores the need to enhance treatment adherence through motivation . however , identifying and providing appropriate solutions to address individual patient 's needs can not be generalized . in addition , the adherence to medication in alcohol dependents is known to be influenced by efficacy outcome . interestingly , adherence to anti - craving medications in the present study was similar to the previous studies with a lower rate of relapse ( 30% ) compared to studies where 50% of the patients relapsed within 3 months of treatment . such lower rate of relapse in our study may be due to the combined effect of anti - craving medications that act by modifying neurotransmitters in the brain coupled with one or more nonpharmacological interventions such as counseling , support by family / caregivers , regular follow - up , and patient education . interestingly , 21.05% of the patients who were nonadherent to anti - craving medications never had the first drink and 47.36% of the patients from the same group had no relapse at the end of 12 weeks , indicating that these nonadherent and nonrelapsed patients had decided to stop taking anti - craving medications with confidence . in addition , in the present study , it was observed that disulfiram was prescribed to those who were confident of quitting alcohol , were adequately counseled regarding likely adrs , were found adherent to it at the end of 12 weeks , and also did not consume alcohol . these findings appear to support the finding that patient willingness may play a key role in achieving abstinence from alcohol dependence . the relapse rates were minimal among patients taking acamprosate raising concern over its efficacy in reducing craving effects . similar results have been reported in combine study , where acamprosate was not better than placebo . however , other randomized studies have reported acamprosate to be effective . since only very small number of patients received other anti - craving medications , their specific and synergistic beneficial effects alone or in combination with various other anti - craving medications need further evaluation . furthermore , comparing the outcomes of various treatment options used in the present study with other therapeutic approaches needs to be examined in a larger sample size to suggest suitable and appropriate treatment options for alcohol dependence . a controlled study by bucknam in soldiers with alcohol dependence examined the comparative efficacy of acamprosate , naltrexone , and topiramate over 1 year , and demonstrated a significant reduction in relapse rates among those who received topiramate . however , it is important to note that this difference may be due to maximum ( 76.3% ) use of topiramate which was supplied by the hospital free of charge as against out - of - pocket payment for topiramate in the present study . in addition a preliminary , double - blind , randomized , controlled study using baclofen ( 15 - 30 mg / day ) over 1 month as well as a separate efficacy study using high - dose baclofen ( 140 mg / day ) has demonstrated their beneficial effects in reducing craving as well as sustenance of anti - craving effects over 10 months compared with other fda - approved medications . these reports suggest the need to conduct randomized controlled trials with high - dose baclofen for alcohol dependence . a recent report by steven has shown the influence of group therapy aimed at personality traits among alcohol dependents . further , they propose that high - risk teenagers are more likely to progress to risky drinking behavior . thus , suggesting that it may be beneficial to conduct an intervention by therapists that targets specific personality traits to reduce drinking in teenagers who are at risk ; in addition , they stress its potential for extension as a halo effect to low - risk students in schools . this modality is suggested as an example of personalized prevention in psychiatry which appears to be a useful and feasible tool in a resource poor country like india . this study is one of the few that has provided a comprehensive overview of patterns of anti - craving medications prescribed in a tertiary care setup , adherence to them and their influence on drinking outcomes in india . given that abuse and/or dependence often occur at a younger age , the study with 102 patients from a tertiary care center has a fair representation of younger age sample from urban and semi - urban backgrounds . the methods used were suitable to get relevant information aimed at addressing the objectives of the study where anti - craving medication adherence rates were assessed using three different assessment tools . the data analysis was done using statistical tools which provided important information that is likely to impact the future studies on adherence to anti - craving medications . first , this was a tertiary care center study and hence , the results can not be easily extrapolated to a larger population at different levels of health - care facilities . further , the present study population is vulnerable to berksonian bias , which is a type of selection bias for admission to hospital , thus indicating a need for community - based multicenter studies to appreciate disease burden . second , the tools used to assess the extent of anti - craving medication adherence had some flaws such as overestimation through patient self - reporting and underestimating by the use of smaq . third , the telephonic contact with patients , which is not used in routine clinical practice , may have contributed to the observed estimate of medication adherence . finally , the association between nonadherence and its impact on clinical outcome , re - hospitalization , and quality of life was not assessed in this study . nonetheless , the present study has quantified anti - craving medication adherence rates to assist in the identification of determinants of adherence among alcohol - dependent patients as an important first step in fulfilling the objectives . however , more comprehensive , larger , and long - term studies are necessary to examine the adherence pattern to other anti - craving agents to evaluate the potential for their safety and improved efficacy . in addition , it is important to address the comparison between two anti - craving agents as monotherapy and/or their combinations , a careful assessment of factors that influence adherence / reasons for nonadherence , and a study among patients with physical and other mental comorbidities including the impact of nonpharmacological interventions . this study is one of the few that has provided a comprehensive overview of patterns of anti - craving medications prescribed in a tertiary care setup , adherence to them and their influence on drinking outcomes in india . given that abuse and/or dependence often occur at a younger age , the study with 102 patients from a tertiary care center has a fair representation of younger age sample from urban and semi - urban backgrounds . the methods used were suitable to get relevant information aimed at addressing the objectives of the study where anti - craving medication adherence rates were assessed using three different assessment tools . the data analysis was done using statistical tools which provided important information that is likely to impact the future studies on adherence to anti - craving medications . first , this was a tertiary care center study and hence , the results can not be easily extrapolated to a larger population at different levels of health - care facilities . further , the present study population is vulnerable to berksonian bias , which is a type of selection bias for admission to hospital , thus indicating a need for community - based multicenter studies to appreciate disease burden . second , the tools used to assess the extent of anti - craving medication adherence had some flaws such as overestimation through patient self - reporting and underestimating by the use of smaq . third , the telephonic contact with patients , which is not used in routine clinical practice , may have contributed to the observed estimate of medication adherence . finally , the association between nonadherence and its impact on clinical outcome , re - hospitalization , and quality of life was not assessed in this study . nonetheless , the present study has quantified anti - craving medication adherence rates to assist in the identification of determinants of adherence among alcohol - dependent patients as an important first step in fulfilling the objectives . however , more comprehensive , larger , and long - term studies are necessary to examine the adherence pattern to other anti - craving agents to evaluate the potential for their safety and improved efficacy . in addition , it is important to address the comparison between two anti - craving agents as monotherapy and/or their combinations , a careful assessment of factors that influence adherence / reasons for nonadherence , and a study among patients with physical and other mental comorbidities including the impact of nonpharmacological interventions . the present study identified modifiable factors such as age , motivation , and willingness as determinants that can adversely influence adherence to anti - craving agents among patients with alcohol dependence . the findings indicate the need for appropriate pharmacological as well as suitable nonpharmacological intervention to achieve improved rate of abstinence with a decreased rate of relapse in patients with alcohol dependence .

objective : to examine the factors influencing the pattern and extent of anti - craving medication adherence and drinking outcomes in alcohol - dependent patients.materials and methods : demographic data from 102 inpatients were collected at discharge from hospital . the pattern of anti - craving medication , extent of adherence , and drinking outcome was collected at 1st , 3rd , 8th , and 12th week follow - up . patients self - reported adherence , medication diary , and simplified medication adherence questionnaire were used and data were analyzed using spss.results:majority ( 99% ) were male patients with a mean age of 41.17 9.86 years and 70% belonged to middle socioeconomic status . there was a decrease in the number of patients coming for follow - up over time from 99.01% to 77.45% on day 90 . acamprosate was used in 74% and naltrexone and disulfiram in 7% of patients each . a significant reduction in adherence to acamprosate and naltrexone ( p < 0.001 ) was associated with simultaneous decrease in days to alcohol abstinence and increase in relapse rate compared to adherent group ( p < 0.001 ) . main barriers to adherence included younger age ( odds ratio = 1.05 95% [ 1.01 - 1.09 ] ; p < 0.01 ) , self - decision , emotional factors , and adverse effects.conclusions:the study demonstrated the need for safer therapeutic options along with suitable intervention at grass root level for sustenance of adherence to anti - craving medication among young adults to prevent relapse and achieve near - complete abstinence from alcohol dependence .

INTRODUCTION MATERIALS AND METHODS RESULT TABLES AND/OR FIGURES DISCUSSION Strengths of the study Limitations of the study CONCLUSION Financial support and sponsorship Conflicts of interest

there has been a progressive increase in the harmful use of alcohol with approximately 2.5 million deaths each year and has been identified as the leading risk factor for death in men between the age of 15 and 59 years . in the national household survey of alcohol and drug abuse ( 2003 ) , 21.4% were reported to be current users of alcohol ( used in the last 30 days ) . alcoholism accounts for 4% of all global deaths and 4.6% of global disability - adjusted life years in addition to significant increase in psychosocial burden in the community . according to the criteria for alcohol dependence described by the diagnostic and statistical manual of mental disorders , dependence is defined as a maladaptive pattern of alcohol use , leading to clinically significant impairment or distress , as manifested by three ( or more ) of the seven criteria , occurring at any time in the same 12-month period . the united states - food and drug administration ( fda ) approved medications are available for use in routine practice for the treatment of alcohol dependence . it is reported that ~50% of alcohol - dependent patients relapse within 3 months , despite following a treatment protocol using these approved medications . while , the pathophysiology of alcohol dependence is complex and incompletely understood at the present time , there is an accumulating evidence demonstrating combined pharmacotherapy with psychotherapy as more efficacious approach than either of the treatments alone . the treatment outcome is known to depend on several patient - related factors such as motivation , emotion , willingness to abstain , efficacy of medications , and adherence to medications . low adherence to prescribed medications is said to be one of the limitations contributing to failure in achieving abstinence and preventing relapse . the present study was designed to evaluate the pattern of use of anti - craving medications , the extent of adherence to them , factors that influence adherence and the influence of medication adherence on time to the first drink , and on relapse in drinking among alcohol - dependent patients . subjects included were clinically diagnosed as alcohol - dependent according to the international classification of diseases 10 criteria , aged 20 years , and received treatment for alcohol dependence as hospital inpatients . the study exclusion criteria were patients with concurrent major psychiatric and other serious organ system disorders , psychotic disorders induced by harmful use of other substances , and complicated withdrawal symptoms . the data on the sociodemography of the patients were collected during the hospital stay , while data on alcohol consumption and anti - craving medication use were recorded at discharge as well as during the time of follow - up at each outpatient clinic visit , i.e. , 1 , 3 , 8 , and 12 week . the treatment with anti - craving medications and necessary psychological interventions was discussed with each patient and their care takers by a psychiatric expert based on the reports of relevant investigations , along with their benefits and adverse effects before initiating treatment regimen to induce abstinence and/or prevent relapse . at the time of discharge , the patients were instructed to take their medications as advised by the psychiatric expert and each patient was provided with a drug diary to record the information on medication details they consumed , the number of missed tablets , if any , reasons for the same , and adverse effects when experienced . the adherence to medication was assessed at every follow - up visit through patients self - report , medication dairy , and simplified medication adherence questionnaire ( smaq ) , and the data on the levels of adherence ( proportion of prescribed medications taken by the patient ) and persistence ( whether the patient continued with prescribed medications ) were recorded . patients who were unable to fill the medication diary were provided assistance by the investigator on the day of the follow - up visit to update the information from the previous visit to the current follow - up . the patient follow - up was either on their scheduled clinic visit or through telephonic interview if they were unable to visit the clinic . the patients who neither visited the hospital nor were reachable for telephonic interview were considered as lost to follow - up . the assessment of anti - craving treatment effect on the drinking outcomes and factors affecting adherence to them was collected over a period of 12 weeks . anti - craving medication adherence was assessed using patient 's self - report by recording whether he / she was regular with consumption . furthermore , through the medication diary at each visit to see the entry for number of days and tablets that the patient had taken . the smaq which is a prevalidated , structured six - point questionnaire was also used to quantify medication adherence and to evaluate the extent of nonadherence , which is in use for patients with acquired immune deficiency syndrome . the six questions used in the study are given below and scoring consisted of yes = 1 and no = 0 responses from patients : 1 . the increasing order of scores from 0 to 6 was considered as decreasing levels of adherence and was then correlated with the percentage adherence scores . anti - craving medication efficacy outcome was assessed as time to the first drink and time to relapse . pattern and extent of adverse effects to anti - craving medications were classified based on organ system affected . comparison of continuous variables between groups was done by independent t - test with a level of significance < 0.05 . spss version 17.0 ( ibm corporation , armonk , new york , united states ) was used to analyze the data . a total of 102 patients with a mean ( standard deviation ) age of 41.17 ( 9.8 ) years constituted the study group . the clinical characteristics of patients , their percentage wise distribution for the level of ses , and area of residence are shown in table 1 . the demographic profile of patients with alcohol dependence of the total number of 102 patients - 101 ( 99.01% ) , 98 ( 96.07% ) , 91 ( 89.21% ) , and 79 ( 77.45% ) patients completed the follow - up during 1 , 3 , 8 , and 12 week , respectively . acamprosate was prescribed as an anti - craving medication in 77 patients ( 75.49% ) both during hospital stay and at discharge with median interquartile dose range of 1332 and 1998 mg / day . the number of patients who were adherent to acamprosate was 69 ( 89.61% ) , 64 ( 83.11% ) , 54 ( 70.12% ) , and 42 ( 54.54% ) at 1 , 3 , 8 , and 12 week follow - up , respectively . this reduction in the proportion of patients adherent across time was significant ( p < 0.001 ) at the end of 12 weeks . during a 12-week follow - up period , 57 ( 55.89% ) patients reported to have taken 100% of the prescribed doses of anti - craving medications at any point of time . other medications prescribed were baclofen , topiramate , and ondansetron [ table 2 ] . the pattern of anti - craving medication use and their persistence pattern in patients with alcohol dependence the number of patients who reported for follow - up reduced significantly ( p < 0.001 ) from 70 ( 68.6% ) at the first visit to 19 ( 18.65% ) at the fourth visit . the number of patients who were reminded over telephone and also the number of telephonic reminders to each patient regarding the follow - up visit increased over time and this was statistically significant ( p < 0.001 ) . the odds ratio of patients coming for follow - up after phone call reduced from 29.0% at the first visit to 11.6% at the fourth visit and was significant [ table 3 and figure 1 ] . characteristics and rates of follow - up over 12 weeks characteristics and rates of follow - up of alcohol - dependent patients over 12 weeks . follow - up visit : visit 1 = 1 week , visit 2 = 3 week , visit 3 = 8 week , visit 4 = 12 week the analysis of adherence to anti - craving medications by patients self - report , through medication diary and smaq scores , did not show statistically significant difference in the levels of adherence [ table 4 ] . the responses to smaq exhibited an all or none phenomenon on adherence to medications , with 57 ( 55.89% ) patients completely adherent and 45 ( 44.11% ) nonadherent at the end of the fourth follow - up visit . these groups were comparable in terms of baseline characteristics such as age , marital status , prior admission for the management of alcohol dependence , anti - craving medication use in the recent past , and number of caregivers . comparison of baseline variables between patients who were adherent and nonadherent to anti - craving medications the adherent group showed significantly more number of abstinent days with lower relapse rate ( p < 0.001 ) . table 5 provides data on the influence of anti - craving medication adherence on drinking outcomes in alcohol - dependent patients . with increase in the number of days of follow - up , there was an increase in the number of patients who relapsed with a parallel decrease in the number of days for the first drink , namely , on 1 week , 6 ( 5.9% ) patients had the first drink since discharge and 4 ( 3.94% ) had relapsed ; by 3 week , 13 ( 12.74% ) patients had the first drink and 10 ( 9.80% ) had relapsed ; by 8 week , 28 ( 27.45% ) patients had the first drink and 24 ( 23.53% ) relapsed ; and by 12 week follow - up , 49 ( 48.03% ) patients had the first drink and 44 ( 43.17% ) patients relapsed [ table 5 ] . influence of anti - craving medication adherence on drinking outcomes in alcohol - dependent patients the univariate analysis for variables such as age , sex , history of prior hospitalization , marital status , and social support in terms of number of caregivers , place of origin , and ses showed no significant difference . multivariate analysis showed patients of younger age group ( odds ratio = 1.05 95% [ 1.01 - 1.09 ] ; p < 0.01 ) to be significantly less adherent to anti - craving medications . there were 97 responses with reasons for nonadherence , of which 38 ( 39.17% ) reported to have stopped anti - craving medication on their own . by 12 weeks , 20 ( 19.6% ) patients had experienced adverse drug reactions ( adrs ) , of which 9 ( 45% ) were on acamprosate who stopped medication due to subjectively perceived side effects [ table 6 ] . the results of the present study showed that the extent of adherence to anti - craving medications reduced significantly over time and that there was an early relapse in drinking behavior among alcohol - dependent patients . since adherence to pharmacotherapy and clinical outcome of patients is well known to be influenced by major and serious comorbidities , such patients were excluded when they were uncontrolled or recognized in the beginning of the study . the pattern of anti - craving medication use was similar to that approved by the fda . the maximal use of acamprosate both as monotherapy and in combination with other medications in a proportion of patients may possibly be attributed to relatively reduced potential and/or absence of known serious interactions between acamprosate and alcohol or with other routinely prescribed medications . however , we observed that the most frequent reason for nonadherence was among those patients who were prescribed acamprosate and maximum number of the patients reported self - termination of the medication . the present study used three different tools to measure adherence to anti - craving medications . while , patient self - report as one of the methods is regarded as the most convenient method for measuring adherence , the same is known to have flaws since it is said to roughly give the dose count , but not the number of doses the patient missed and if the doses were taken at the right time . to minimize this limitation , we assessed medication adherence additionally using medication diary and smaq . the extent of adherence varied from highest with disulfiram to moderate among naltrexone and acamprosate groups . this finding may be attributed to the smaller number of patients in the present study , a selection bias in prescribing medications , and naturalistic study setting . such difference in the extent of nonadherence to various anti - craving medications is said to be due to patient - related factors similar to that seen in the present study such as self - decision to stop taking medications , either due to confidence to abstain from alcohol or the decision to restart consuming alcohol , along with other emotional factors . the who criteria that may influence medication adherence include socioeconomic , marital / educational status , health - care system , medical condition , treatment of co - morbidities , and role of caregivers . however , these did not show a significant influence in maintaining adherence in our study . in the indian context , this behavior may be attributed to social responsibilities of adults and elderly people compared to the influence of peer group pressure among younger alcohol dependents . given the unique set of barriers that are proposed to be involved in the intrinsic behavior of each patient , a complete understanding and assessment of factors involved in medication adherence is challenging in these types of study design . from the present study , it appears that the use of anti - craving medications induces abstinence and underscores the need to enhance treatment adherence through motivation . in addition , the adherence to medication in alcohol dependents is known to be influenced by efficacy outcome . interestingly , adherence to anti - craving medications in the present study was similar to the previous studies with a lower rate of relapse ( 30% ) compared to studies where 50% of the patients relapsed within 3 months of treatment . such lower rate of relapse in our study may be due to the combined effect of anti - craving medications that act by modifying neurotransmitters in the brain coupled with one or more nonpharmacological interventions such as counseling , support by family / caregivers , regular follow - up , and patient education . interestingly , 21.05% of the patients who were nonadherent to anti - craving medications never had the first drink and 47.36% of the patients from the same group had no relapse at the end of 12 weeks , indicating that these nonadherent and nonrelapsed patients had decided to stop taking anti - craving medications with confidence . in addition , in the present study , it was observed that disulfiram was prescribed to those who were confident of quitting alcohol , were adequately counseled regarding likely adrs , were found adherent to it at the end of 12 weeks , and also did not consume alcohol . these findings appear to support the finding that patient willingness may play a key role in achieving abstinence from alcohol dependence . since only very small number of patients received other anti - craving medications , their specific and synergistic beneficial effects alone or in combination with various other anti - craving medications need further evaluation . furthermore , comparing the outcomes of various treatment options used in the present study with other therapeutic approaches needs to be examined in a larger sample size to suggest suitable and appropriate treatment options for alcohol dependence . a controlled study by bucknam in soldiers with alcohol dependence examined the comparative efficacy of acamprosate , naltrexone , and topiramate over 1 year , and demonstrated a significant reduction in relapse rates among those who received topiramate . however , it is important to note that this difference may be due to maximum ( 76.3% ) use of topiramate which was supplied by the hospital free of charge as against out - of - pocket payment for topiramate in the present study . in addition a preliminary , double - blind , randomized , controlled study using baclofen ( 15 - 30 mg / day ) over 1 month as well as a separate efficacy study using high - dose baclofen ( 140 mg / day ) has demonstrated their beneficial effects in reducing craving as well as sustenance of anti - craving effects over 10 months compared with other fda - approved medications . these reports suggest the need to conduct randomized controlled trials with high - dose baclofen for alcohol dependence . this study is one of the few that has provided a comprehensive overview of patterns of anti - craving medications prescribed in a tertiary care setup , adherence to them and their influence on drinking outcomes in india . the methods used were suitable to get relevant information aimed at addressing the objectives of the study where anti - craving medication adherence rates were assessed using three different assessment tools . the data analysis was done using statistical tools which provided important information that is likely to impact the future studies on adherence to anti - craving medications . first , this was a tertiary care center study and hence , the results can not be easily extrapolated to a larger population at different levels of health - care facilities . further , the present study population is vulnerable to berksonian bias , which is a type of selection bias for admission to hospital , thus indicating a need for community - based multicenter studies to appreciate disease burden . second , the tools used to assess the extent of anti - craving medication adherence had some flaws such as overestimation through patient self - reporting and underestimating by the use of smaq . third , the telephonic contact with patients , which is not used in routine clinical practice , may have contributed to the observed estimate of medication adherence . finally , the association between nonadherence and its impact on clinical outcome , re - hospitalization , and quality of life was not assessed in this study . nonetheless , the present study has quantified anti - craving medication adherence rates to assist in the identification of determinants of adherence among alcohol - dependent patients as an important first step in fulfilling the objectives . however , more comprehensive , larger , and long - term studies are necessary to examine the adherence pattern to other anti - craving agents to evaluate the potential for their safety and improved efficacy . in addition , it is important to address the comparison between two anti - craving agents as monotherapy and/or their combinations , a careful assessment of factors that influence adherence / reasons for nonadherence , and a study among patients with physical and other mental comorbidities including the impact of nonpharmacological interventions . this study is one of the few that has provided a comprehensive overview of patterns of anti - craving medications prescribed in a tertiary care setup , adherence to them and their influence on drinking outcomes in india . given that abuse and/or dependence often occur at a younger age , the study with 102 patients from a tertiary care center has a fair representation of younger age sample from urban and semi - urban backgrounds . the methods used were suitable to get relevant information aimed at addressing the objectives of the study where anti - craving medication adherence rates were assessed using three different assessment tools . the data analysis was done using statistical tools which provided important information that is likely to impact the future studies on adherence to anti - craving medications . second , the tools used to assess the extent of anti - craving medication adherence had some flaws such as overestimation through patient self - reporting and underestimating by the use of smaq . third , the telephonic contact with patients , which is not used in routine clinical practice , may have contributed to the observed estimate of medication adherence . finally , the association between nonadherence and its impact on clinical outcome , re - hospitalization , and quality of life was not assessed in this study . nonetheless , the present study has quantified anti - craving medication adherence rates to assist in the identification of determinants of adherence among alcohol - dependent patients as an important first step in fulfilling the objectives . however , more comprehensive , larger , and long - term studies are necessary to examine the adherence pattern to other anti - craving agents to evaluate the potential for their safety and improved efficacy . in addition , it is important to address the comparison between two anti - craving agents as monotherapy and/or their combinations , a careful assessment of factors that influence adherence / reasons for nonadherence , and a study among patients with physical and other mental comorbidities including the impact of nonpharmacological interventions . the present study identified modifiable factors such as age , motivation , and willingness as determinants that can adversely influence adherence to anti - craving agents among patients with alcohol dependence . the findings indicate the need for appropriate pharmacological as well as suitable nonpharmacological intervention to achieve improved rate of abstinence with a decreased rate of relapse in patients with alcohol dependence .

many studies have suggested that over the course of the aging process , the amount of fat tends to increase and is preferentially stored in the abdominal area , while skeletal muscle mass and bone mineral density decline.14 understanding how changes in body composition , especially fat distribution , affect the risk for many chronic diseases such as cardiovascular diseases and type 2 diabetes mellitus , is a primary concern . it is well known that body mass index ( bmi ) is one of the most popular clinical parameters for estimating body composition , and it is a reliable indicator for body fat mass percentage ( bfm% ) . it is also an obesity index whose causal relationship with morbidity and mortality has been well established.57 because it can be efficiently used to determine obesity in a large - sized population , bmi is frequently used for the assessment of obesity in a clinical setting . it has been reported , however , that the distribution of body fat rather than its amount is more likely to be associated with health - related risks,8 and that abdominal obesity is an independent risk factor for raising the morbidity and mortality as such.911 this has led to an emphasis on the importance of other parameters , including the abdominal obesity index in particular , than bmi in assessing obesity . measures for abdominal obesity include waist circumference ( wc ) , waist - to - hip ratio ( whr ) , and abdominal sagittal diameter . in addition , abdominal obesity is also evaluated by measuring the amount of body fat using ultrasonography , computed tomography , and magnetic resonance imaging . of the various types of measures that are used to assess obesity , the amount of visceral body fat measured on computed tomography scans is known to best indicate abdominal obesity;1214 however , its use is somewhat restricted because of the cost and exposure to irradiation . accordingly , in an actual clinical setting , wc and whr are mainly used to assess abdominal obesity . according to several previous studies examining various parameters that are commonly used to assess abdominal obesity in a clinical setting , wc was a more reliable parameter than whr in making a diagnosis of abdominal obesity.15 in addition , these studies have also shown that wc was also the most reliable indicator in predicting the amount of visceral fat.10,16,17 body fat is redistributed with increasing age , as more of it becomes located in the abdominal cavity.14,18 the reason for this redistribution is not really known , but declining testosterone and growth hormone levels in combination with declining rates of lipolysis of visceral fat associated with aging may play a role in men.19 low testosterone levels have been shown to be associated with increased visceral fat mass in men.20 in women , the declining estrogen levels after menopause may be a critical factor . while several studies have investigated the relationship between weight change , obesity , and visceral fat with age and the risk of developing lifestyle - related diseases,2124 the effect of maintaining one s youthful body shape ( in terms of appearance ) on reducing such risks has been given significantly less attention . several recent studies have reported the difference in body shape between men and women , and they have examined age - related differences in body shape using three - dimensional body scans . it has become more and more clinically important to assess body shape based on the pattern of body fat distribution and redistribution , rather than to assess obesity simply based on an increased amount of body fat . meanwhile , several approaches have been used to objectively assess body shape from this context.2530 presumably , the assessment of body shape based on three - dimensional body scans might be unavailable in a clinical setting . therefore , it might also be mandatory to conduct studies to assess the status of body shape associated with age using clinical parameters that are currently used to assess obesity in a clinical setting . it remains problematic that in general , anthropometric measures such as bmi or whr are insufficient for the assessment of this redistribution.31 in addition , anthropometric measures have been mainly used to assess the status of obesity based on body composition up until the present . age after birth is a measure of the degree of aging in individual , this is termed as chronological age or calendar age , which is a definition of aging as a function of time . aging is characterized by a time - related decline in physiological functions and changes in morphology , and is usually assessed by the chronological age , which is defined as time elapsed since birth . chronological age fails to provide an accurate indicator of the aging process . because tissues age at different rates and because diseases vary enormously among individuals , humans become increasingly different from one another with age . but , biological age estimates the functional status of an individual in reference to his or her chronological peers on the basis of how well he or she functions in comparison with others of the same chronological age.32,33 biological age is estimated based on aging biomarkers that are subject to change depending on one s calendar age . overall , biological age is a more reliable indicator of the degree of aging and one s health status as compared to an individual s calendar age . to date , a variety of models for estimating biological age have therefore been proposed to meet relevant study objectives . to estimate the physical , biochemical , and hormonal biological ages , we have also previously proposed the models for estimating biological ages using relevant biomarkers;34 however , we did not propose a model for estimating body shape biological age using the clinical parameters associated with body composition . given this background , we conducted the current study to examine the correlations of age with bmi , bfm% , lean body mass percentage ( lbm% ) , wc , and the whr using clinical parameters associated with body composition . in addition , by using these parameters , we also proposed a model of body shape biological age that estimated a person s body shape based on their age . thus , our estimation model will help people understand the above correlations , and it will promote bidirectional communication between physicians and patients . the current study was conducted in 243,778 subjects aged between 20 and 90 years ; the subjects comprised men and women who received a general medical checkup for health promotion and disease prevention at health promotion centers at university and community hospitals during a period ranging from june 2004 to january 2011 . in the current study , exclusion criteria included the presence of uncontrolled hypertension and diabetes , and the subjects taking current medications for cardiovascular , pulmonary , hepatic , pancreatic , or renal diseases . subjects were also excluded if their reported clinical parameters exceeded the standard deviations of the normal range . to analyze the differences in clinical parameters due to aging , and to minimize the potential confounding effects of diseases or abnormal measurements , we set inclusion criteria considering both the standard deviations of parameters that were collected and the normal range which was established by the american medical association . of these clinical parameters , we excluded those which were not in agreement with the inclusion criteria . only the screened data were used for the statistical analysis . for the clinical parameters associated with obesity , we collected data such as height , weight , wc , hip circumference ( hc ) , lbm% , and bfm% . height and weight were measured using height - measuring equipment and an electronic scale while subjects wore light clothes . as for wc , the thinnest area between the inferior part of the lowest rib and the iliac crest was measured as a unit of cm when subjects were in an upright position . as for hc , the location of the greater trochanter or the widest circumference was measured as a unit of cm . this was followed by the measurement of lbm% and bfm% using multifrequency segmental bioelectrical impedance ( inbody , biospace co , ltd , seoul , korea ) . then , using the measurements of wc and hc , the whr was calculated : to identify the correlation between the clinical parameters and chronological age , a pearson s correlation analysis was performed . to propose a model for estimating body shape biological age , we performed a stepwise method in multiple regression analysis between the age and clinical parameters that had been selected by the correlation analysis . we employed multiple regression analysis with assigning variance inflation factor to evaluate multicollinearity among the parameters . statistical analysis was completed using spss 12.0 ( spss inc , chicago , il , usa ) . the current study was conducted in 243,778 subjects aged between 20 and 90 years ; the subjects comprised men and women who received a general medical checkup for health promotion and disease prevention at health promotion centers at university and community hospitals during a period ranging from june 2004 to january 2011 . in the current study , exclusion criteria included the presence of uncontrolled hypertension and diabetes , and the subjects taking current medications for cardiovascular , pulmonary , hepatic , pancreatic , or renal diseases . subjects were also excluded if their reported clinical parameters exceeded the standard deviations of the normal range . to analyze the differences in clinical parameters due to aging , and to minimize the potential confounding effects of diseases or abnormal measurements , we set inclusion criteria considering both the standard deviations of parameters that were collected and the normal range which was established by the american medical association . of these clinical parameters , we excluded those which were not in agreement with the inclusion criteria . only the screened data were used for the statistical analysis . for the clinical parameters associated with obesity , we collected data such as height , weight , wc , hip circumference ( hc ) , lbm% , and bfm% . height and weight were measured using height - measuring equipment and an electronic scale while subjects wore light clothes . as for wc , the thinnest area between the inferior part of the lowest rib and the iliac crest was measured as a unit of cm when subjects were in an upright position . as for hc , the location of the greater trochanter or the widest circumference was measured as a unit of cm . this was followed by the measurement of lbm% and bfm% using multifrequency segmental bioelectrical impedance ( inbody , biospace co , ltd , seoul , korea ) . to identify the correlation between the clinical parameters and chronological age , a pearson s correlation analysis was performed . to propose a model for estimating body shape biological age , we performed a stepwise method in multiple regression analysis between the age and clinical parameters that had been selected by the correlation analysis . we employed multiple regression analysis with assigning variance inflation factor to evaluate multicollinearity among the parameters . statistical analysis was completed using spss 12.0 ( spss inc , chicago , il , usa ) . a total of 243,778 subjects participated in the current study , of which there were 132,336 men and 110,042 women . mean age was 47.17 10.98 years in men and 46.94 11.96 years in women . in men , mean height and weight were 170.57 5.96 cm and 71.16 9.23 kg , respectively . in women , these values were 157.91 5.63 cm and 56.92 7.64 kg , respectively . in men , the clinical parameters with the highest correlation with the age included the whr ( r = 0.786 , p < 0.001 ) , hc ( r = 0.448 , p < 0.001 ) , and height ( r = 0.377 , p < 0.001 ) . in women , the clinical parameters with the highest correlation with age included the whr ( r = 0.859 , p < 0.001 ) , wc ( r = 0.580 , p < 0.001 ) , and hc ( r = 0.520 , p < 0.001 ) . all of the collected data surrounding the clinical parameters had a significant correlation with age . in men , however , only the clinical parameters associated with obesity other than bmi ( r = 0.005 , p = 0.191 ) had a significant correlation with age ( tables 2 and 3 ) . to estimate the optimal body shape biological age based on the clinical parameters associated with body composition , we performed a multiple regression analysis . in a model for estimating body shape biological age in men , the coefficient of determination ( r ) was 0.71 , and the relevant clinical parameters included the whr , wc , height , lbm% , and weight . in a model for estimating the body shape biological age in women , the coefficient of determination ( r ) was 0.76 , and the relevant clinical parameters included the whr , height , wc , lbm% , and weight ( tables 4 and 5 ) . figures 1 and 2 illustrate the correlations between biological age and chronological age in a model for estimating body shape biological age in men and women . a total of 243,778 subjects participated in the current study , of which there were 132,336 men and 110,042 women . mean age was 47.17 10.98 years in men and 46.94 11.96 years in women . in men , mean height and weight were 170.57 5.96 cm and 71.16 9.23 kg , respectively . in women , these values were 157.91 5.63 cm and 56.92 7.64 kg , respectively . in men , the clinical parameters with the highest correlation with the age included the whr ( r = 0.786 , p < 0.001 ) , hc ( r = 0.448 , p < 0.001 ) , and height ( r = 0.377 , p < 0.001 ) . in women , the clinical parameters with the highest correlation with age included the whr ( r = 0.859 , p < 0.001 ) , wc ( r = 0.580 , p < 0.001 ) , and hc ( r = 0.520 , p < 0.001 ) . all of the collected data surrounding the clinical parameters had a significant correlation with age . in men , however , only the clinical parameters associated with obesity other than bmi ( r = 0.005 , p = 0.191 ) had a significant correlation with age ( tables 2 and 3 ) . to estimate the optimal body shape biological age based on the clinical parameters associated with body composition , we performed a multiple regression analysis . in a model for estimating body shape biological age in men , the coefficient of determination ( r ) was 0.71 , and the relevant clinical parameters included the whr , wc , height , lbm% , and weight . in a model for estimating the body shape biological age in women , the coefficient of determination ( r ) was 0.76 , and the relevant clinical parameters included the whr , height , wc , lbm% , and weight ( tables 4 and 5 ) . figures 1 and 2 illustrate the correlations between biological age and chronological age in a model for estimating body shape biological age in men and women . as shown in the current study , a model for estimating body shape biological age was effective in predicting the age - related changes that occur in body shape based on clinical parameters associated with body composition ( whr , wc , height , lbm% , weight , and hc ) in approximately 71% of men and approximately 76% of women . regarding the correlation between age and the clinical parameters associated with body composition , seven parameters other than bmi for men , and all of the eight parameters for women had a significant correlation with age . of the clinical parameters that had a significant correlation with age . of the clinical parameters that had a significant correlation with age in men , height , weight , lbm% , and hc had a negative correlation with age , while bfm% , wc , and the whr had a positive correlation . in women , height and lbm% had a negative correlation with age , while weight , bmi , bfm% , wc , hc , and the whr had a positive correlation . previous studies have shown that both men and women have experienced a decrease in height due to increased age , and this was more notable in women.3543 a decrease in height due to increased age mainly originates from changes in the vertebrae ( eg , vertebral fracture , weakening of the paravertebral muscles , changes in posture , degenerative changes in the intervertebral disc , vertebral deformity , and kyphosis ) . in addition , its onset begins at the age of 45 years or so , and then it continues with increased age.36,37,43,44 it is therefore known that a decrease in height due to increased age is associated with osteoporotic fractures , and it is also an indicator of the prediction of vertebral fractures and risks of developing femoral fractures in the elderly.45,46 in the current study , there was also a significant correlation between a decrease in height and increased age . in a model for estimating body shape biological age , the degree of the correlation between the two parameters was significantly higher in women than men . bmi is widely used for nutritional assessment , obesity classification , and as a prognostic variable for mortality ; however , there are many limitations . first , bmi could potentially produce an inaccurate diagnosis of overweight and obese in some special populations such as athletes , body builders , and elderly patients . second , bmi - associated mortality in specific situations is controversial.47 in a model for estimating the body shape biological age , we included the height and weight of men and only the height of women rather than the bmi . in addition , the correlation between bmi and age reached statistical significance only in women . as shown in the current results , both men and women demonstrated a positive correlation between bfm% and age , and a negative correlation between age and lbm% . in addition , we included only the lbm% in a model for estimating biological age . previous studies have established that bfm% was increased and lbm% was decreased with increased age.48,49 an increase in bfm% due to increased age originated from a decrease in lbm% , rather than from an increase in bfm% in men . it has been reported that an increase in bfm% rather than a decrease in lbm% might play a key role in causing an increase body fat due to aging in women.50,51 regarding the distribution of body fat depending on body shape , it has been reported that abdominal obesity is a notable , independent risk factor for raising morbidity and mortality rates.911 therefore , the clinical significance of weight , bmi , and the bfm% would greatly vary depending on the types of abdominal obesity , ( ie , android or gynoid type ) . previous studies have shown that wc is the most reliable indicator for reflecting the amount of visceral fat in the assessment of abdominal obesity.10,1517 as shown in the current results , both men and women had a positive correlation between increased age and both wc and the whr ; however , hc had a negative correlation with age in men and a positive correlation with in women . according to previous studies , the whr was increased until the age of 5564 years , and then it decreased thereafter in men , though it continually increased with increased age in women.52,53 these authors noted that these findings occurred because of the redistribution of body fat due to changes in sex hormones in both men and women aged between 45 and 54 years.52,53 other studies have also shown that hormone therapy reduced the whr , thus providing more evidence for the redistribution of body fat because of hormonal changes.54,55 as shown in the current results , the whr was included in a model for estimating body shape biological age in both men and women . in addition , hc was included in this model for men , and both hc and wc were included for women . in particular , in estimating the body shape biological age , both men and women showed that the whr was the most powerful factor . previous studies have shown that wc is a more reliable indicator of predicting abdominal obesity . from a perspective that considers the differences in body shape due to aging , however , the whr is a more powerful factor , and it better reflects differences in body shape . in cross - sectional studies , the prevalence of high body weight or obesity ( bmi > 30 kg / m ) increases with age up to about age 60 and then declines.56 although this does not indicate that the excessive accumulation of body fat rarely occurs in the elderly , this does suggest that visceral obesity increasingly occurs because of the redistribution of body fat due to increased age , and this leads to changes in body shape.2530,56 accordingly , considering variation in body shape due to aging , it would be more appropriate for clinical evaluation to be based on body shape due to the increased occurrence of visceral obesity , rather than the consistent application of clinical parameters for assessing obesity . we propose that a model for estimating body shape biological age be appropriate for assessing differences in body shape due to increased age with the application of clinical parameters associated with body composition . moreover , by presenting the clinical parameters for assessing obesity that are commonly used in a clinical setting and thereby comparing the results with those obtained from age - matched controls our estimation model would be used to perform an overall analysis of the differences in body shape due to aging . furthermore , by comparing one s current body shape biological age with their calendar age ( thereby expressing whether the body shape biological age is older or younger than one s calendar age ) , our estimation model would be easily accepted by people , and it would also be of help for facilitating communication between physicians and patients . in addition , it would also be of help for providing the motives to improve the status of obesity , and for setting personal goals . in a model for assessing body shape biological age , however , we did not include the factors that might affect the clinical parameters associated with obesity , including the amount of physical activity , eating habits such as nutritional intake , and socioeconomic status , and we excluded the data of some subjects who were suspected of having diseases or conditions based on certain criteria . there were also limitations in the current study in that we did not perform a detailed analysis of the participants medical history such as a present illness , a past history , and a family history . our model for estimating body shape biological age might be a novel approach to variation in body shape that is due to aging despite several limitations . we assume that our estimation model would be used as an adjunctive measure in easily predicting differences in body shape with the use of clinical parameters that are commonly used to assess the status of obesity in a clinical setting . our results will provide baseline data for further studies to examine not only the effects of changes in body shape due to the aging process on the degree of aging in individuals , the rate of aging progression , and life expectancy , but they will also provide information surrounding correlations between body shape , disease , morbidity , and mortality .

backgroundto date , no studies have attempted to estimate body shape biological age using clinical parameters associated with body composition for the purposes of examining a person s body shape based on their age.objectivewe examined the relations between clinical parameters associated with body composition and chronological age , and proposed a model for estimating the body shape biological age.methodsthe study was conducted in 243,778 subjects aged between 20 and 90 years who received a general medical checkup at health promotion centers at university and community hospitals in korea from 2004 to 2011.resultsin men , the clinical parameters with the highest correlation to age included the waist- to - hip ratio ( r = 0.786 , p < 0.001 ) , hip circumference ( r = 0.448 , p < 0.001 ) , and height ( r = 0.377 , p < 0.001 ) . in women , the clinical parameters with the highest correlation to age include the waist - to - hip ratio ( r = 0.859 , p < 0.001 ) , waist circumference ( r = 0.580 , p < 0.001 ) , and hip circumference ( r = 0.520 , p < 0.001 ) . to estimate the optimal body shape biological age based on clinical parameters associated with body composition , we performed a multiple regression analysis . in a model estimating the body shape biological age , the coefficient of determination ( r2 ) was 0.71 in men and 0.76 in women.conclusionour model for estimating body shape biological age might be a novel approach to variation in body shape that is due to aging . we assume that our estimation model would be used as an adjunctive measure in easily predicting differences in body shape with the use of clinical parameters that are commonly used to assess the status of obesity in a clinical setting .

Introduction Methods Subjects Clinical parameters Statistical analysis Results Baseline characteristics The correlation between age and the clinical parameters associated with body composition Estimation of body shape biological age based on clinical parameters associated with body composition Discussion Conclusion

many studies have suggested that over the course of the aging process , the amount of fat tends to increase and is preferentially stored in the abdominal area , while skeletal muscle mass and bone mineral density decline.14 understanding how changes in body composition , especially fat distribution , affect the risk for many chronic diseases such as cardiovascular diseases and type 2 diabetes mellitus , is a primary concern . it is well known that body mass index ( bmi ) is one of the most popular clinical parameters for estimating body composition , and it is a reliable indicator for body fat mass percentage ( bfm% ) . it is also an obesity index whose causal relationship with morbidity and mortality has been well established.57 because it can be efficiently used to determine obesity in a large - sized population , bmi is frequently used for the assessment of obesity in a clinical setting . it has been reported , however , that the distribution of body fat rather than its amount is more likely to be associated with health - related risks,8 and that abdominal obesity is an independent risk factor for raising the morbidity and mortality as such.911 this has led to an emphasis on the importance of other parameters , including the abdominal obesity index in particular , than bmi in assessing obesity . measures for abdominal obesity include waist circumference ( wc ) , waist - to - hip ratio ( whr ) , and abdominal sagittal diameter . of the various types of measures that are used to assess obesity , the amount of visceral body fat measured on computed tomography scans is known to best indicate abdominal obesity;1214 however , its use is somewhat restricted because of the cost and exposure to irradiation . accordingly , in an actual clinical setting , wc and whr are mainly used to assess abdominal obesity . according to several previous studies examining various parameters that are commonly used to assess abdominal obesity in a clinical setting , wc was a more reliable parameter than whr in making a diagnosis of abdominal obesity.15 in addition , these studies have also shown that wc was also the most reliable indicator in predicting the amount of visceral fat.10,16,17 body fat is redistributed with increasing age , as more of it becomes located in the abdominal cavity.14,18 the reason for this redistribution is not really known , but declining testosterone and growth hormone levels in combination with declining rates of lipolysis of visceral fat associated with aging may play a role in men.19 low testosterone levels have been shown to be associated with increased visceral fat mass in men.20 in women , the declining estrogen levels after menopause may be a critical factor . while several studies have investigated the relationship between weight change , obesity , and visceral fat with age and the risk of developing lifestyle - related diseases,2124 the effect of maintaining one s youthful body shape ( in terms of appearance ) on reducing such risks has been given significantly less attention . several recent studies have reported the difference in body shape between men and women , and they have examined age - related differences in body shape using three - dimensional body scans . it has become more and more clinically important to assess body shape based on the pattern of body fat distribution and redistribution , rather than to assess obesity simply based on an increased amount of body fat . meanwhile , several approaches have been used to objectively assess body shape from this context.2530 presumably , the assessment of body shape based on three - dimensional body scans might be unavailable in a clinical setting . therefore , it might also be mandatory to conduct studies to assess the status of body shape associated with age using clinical parameters that are currently used to assess obesity in a clinical setting . it remains problematic that in general , anthropometric measures such as bmi or whr are insufficient for the assessment of this redistribution.31 in addition , anthropometric measures have been mainly used to assess the status of obesity based on body composition up until the present . age after birth is a measure of the degree of aging in individual , this is termed as chronological age or calendar age , which is a definition of aging as a function of time . aging is characterized by a time - related decline in physiological functions and changes in morphology , and is usually assessed by the chronological age , which is defined as time elapsed since birth . but , biological age estimates the functional status of an individual in reference to his or her chronological peers on the basis of how well he or she functions in comparison with others of the same chronological age.32,33 biological age is estimated based on aging biomarkers that are subject to change depending on one s calendar age . overall , biological age is a more reliable indicator of the degree of aging and one s health status as compared to an individual s calendar age . to date , a variety of models for estimating biological age have therefore been proposed to meet relevant study objectives . to estimate the physical , biochemical , and hormonal biological ages , we have also previously proposed the models for estimating biological ages using relevant biomarkers;34 however , we did not propose a model for estimating body shape biological age using the clinical parameters associated with body composition . given this background , we conducted the current study to examine the correlations of age with bmi , bfm% , lean body mass percentage ( lbm% ) , wc , and the whr using clinical parameters associated with body composition . in addition , by using these parameters , we also proposed a model of body shape biological age that estimated a person s body shape based on their age . thus , our estimation model will help people understand the above correlations , and it will promote bidirectional communication between physicians and patients . the current study was conducted in 243,778 subjects aged between 20 and 90 years ; the subjects comprised men and women who received a general medical checkup for health promotion and disease prevention at health promotion centers at university and community hospitals during a period ranging from june 2004 to january 2011 . in the current study , exclusion criteria included the presence of uncontrolled hypertension and diabetes , and the subjects taking current medications for cardiovascular , pulmonary , hepatic , pancreatic , or renal diseases . subjects were also excluded if their reported clinical parameters exceeded the standard deviations of the normal range . to analyze the differences in clinical parameters due to aging , and to minimize the potential confounding effects of diseases or abnormal measurements , we set inclusion criteria considering both the standard deviations of parameters that were collected and the normal range which was established by the american medical association . of these clinical parameters , we excluded those which were not in agreement with the inclusion criteria . for the clinical parameters associated with obesity , we collected data such as height , weight , wc , hip circumference ( hc ) , lbm% , and bfm% . as for hc , the location of the greater trochanter or the widest circumference was measured as a unit of cm . then , using the measurements of wc and hc , the whr was calculated : to identify the correlation between the clinical parameters and chronological age , a pearson s correlation analysis was performed . to propose a model for estimating body shape biological age , we performed a stepwise method in multiple regression analysis between the age and clinical parameters that had been selected by the correlation analysis . we employed multiple regression analysis with assigning variance inflation factor to evaluate multicollinearity among the parameters . the current study was conducted in 243,778 subjects aged between 20 and 90 years ; the subjects comprised men and women who received a general medical checkup for health promotion and disease prevention at health promotion centers at university and community hospitals during a period ranging from june 2004 to january 2011 . in the current study , exclusion criteria included the presence of uncontrolled hypertension and diabetes , and the subjects taking current medications for cardiovascular , pulmonary , hepatic , pancreatic , or renal diseases . to analyze the differences in clinical parameters due to aging , and to minimize the potential confounding effects of diseases or abnormal measurements , we set inclusion criteria considering both the standard deviations of parameters that were collected and the normal range which was established by the american medical association . of these clinical parameters , we excluded those which were not in agreement with the inclusion criteria . for the clinical parameters associated with obesity , we collected data such as height , weight , wc , hip circumference ( hc ) , lbm% , and bfm% . to identify the correlation between the clinical parameters and chronological age , a pearson s correlation analysis was performed . to propose a model for estimating body shape biological age , we performed a stepwise method in multiple regression analysis between the age and clinical parameters that had been selected by the correlation analysis . we employed multiple regression analysis with assigning variance inflation factor to evaluate multicollinearity among the parameters . a total of 243,778 subjects participated in the current study , of which there were 132,336 men and 110,042 women . mean age was 47.17 10.98 years in men and 46.94 11.96 years in women . in men , mean height and weight were 170.57 5.96 cm and 71.16 9.23 kg , respectively . in women , these values were 157.91 5.63 cm and 56.92 7.64 kg , respectively . in men , the clinical parameters with the highest correlation with the age included the whr ( r = 0.786 , p < 0.001 ) , hc ( r = 0.448 , p < 0.001 ) , and height ( r = 0.377 , p < 0.001 ) . in women , the clinical parameters with the highest correlation with age included the whr ( r = 0.859 , p < 0.001 ) , wc ( r = 0.580 , p < 0.001 ) , and hc ( r = 0.520 , p < 0.001 ) . all of the collected data surrounding the clinical parameters had a significant correlation with age . in men , however , only the clinical parameters associated with obesity other than bmi ( r = 0.005 , p = 0.191 ) had a significant correlation with age ( tables 2 and 3 ) . to estimate the optimal body shape biological age based on the clinical parameters associated with body composition , we performed a multiple regression analysis . in a model for estimating body shape biological age in men , the coefficient of determination ( r ) was 0.71 , and the relevant clinical parameters included the whr , wc , height , lbm% , and weight . in a model for estimating the body shape biological age in women , the coefficient of determination ( r ) was 0.76 , and the relevant clinical parameters included the whr , height , wc , lbm% , and weight ( tables 4 and 5 ) . figures 1 and 2 illustrate the correlations between biological age and chronological age in a model for estimating body shape biological age in men and women . a total of 243,778 subjects participated in the current study , of which there were 132,336 men and 110,042 women . mean age was 47.17 10.98 years in men and 46.94 11.96 years in women . in men , mean height and weight were 170.57 5.96 cm and 71.16 9.23 kg , respectively . in women , these values were 157.91 5.63 cm and 56.92 7.64 kg , respectively . in men , the clinical parameters with the highest correlation with the age included the whr ( r = 0.786 , p < 0.001 ) , hc ( r = 0.448 , p < 0.001 ) , and height ( r = 0.377 , p < 0.001 ) . in women , the clinical parameters with the highest correlation with age included the whr ( r = 0.859 , p < 0.001 ) , wc ( r = 0.580 , p < 0.001 ) , and hc ( r = 0.520 , p < 0.001 ) . all of the collected data surrounding the clinical parameters had a significant correlation with age . in men , however , only the clinical parameters associated with obesity other than bmi ( r = 0.005 , p = 0.191 ) had a significant correlation with age ( tables 2 and 3 ) . to estimate the optimal body shape biological age based on the clinical parameters associated with body composition , we performed a multiple regression analysis . in a model for estimating body shape biological age in men , the coefficient of determination ( r ) was 0.71 , and the relevant clinical parameters included the whr , wc , height , lbm% , and weight . in a model for estimating the body shape biological age in women , the coefficient of determination ( r ) was 0.76 , and the relevant clinical parameters included the whr , height , wc , lbm% , and weight ( tables 4 and 5 ) . figures 1 and 2 illustrate the correlations between biological age and chronological age in a model for estimating body shape biological age in men and women . as shown in the current study , a model for estimating body shape biological age was effective in predicting the age - related changes that occur in body shape based on clinical parameters associated with body composition ( whr , wc , height , lbm% , weight , and hc ) in approximately 71% of men and approximately 76% of women . regarding the correlation between age and the clinical parameters associated with body composition , seven parameters other than bmi for men , and all of the eight parameters for women had a significant correlation with age . of the clinical parameters that had a significant correlation with age . of the clinical parameters that had a significant correlation with age in men , height , weight , lbm% , and hc had a negative correlation with age , while bfm% , wc , and the whr had a positive correlation . in women , height and lbm% had a negative correlation with age , while weight , bmi , bfm% , wc , hc , and the whr had a positive correlation . previous studies have shown that both men and women have experienced a decrease in height due to increased age , and this was more notable in women.3543 a decrease in height due to increased age mainly originates from changes in the vertebrae ( eg , vertebral fracture , weakening of the paravertebral muscles , changes in posture , degenerative changes in the intervertebral disc , vertebral deformity , and kyphosis ) . in addition , its onset begins at the age of 45 years or so , and then it continues with increased age.36,37,43,44 it is therefore known that a decrease in height due to increased age is associated with osteoporotic fractures , and it is also an indicator of the prediction of vertebral fractures and risks of developing femoral fractures in the elderly.45,46 in the current study , there was also a significant correlation between a decrease in height and increased age . in a model for estimating body shape biological age , the degree of the correlation between the two parameters was significantly higher in women than men . second , bmi - associated mortality in specific situations is controversial.47 in a model for estimating the body shape biological age , we included the height and weight of men and only the height of women rather than the bmi . in addition , the correlation between bmi and age reached statistical significance only in women . as shown in the current results , both men and women demonstrated a positive correlation between bfm% and age , and a negative correlation between age and lbm% . in addition , we included only the lbm% in a model for estimating biological age . previous studies have established that bfm% was increased and lbm% was decreased with increased age.48,49 an increase in bfm% due to increased age originated from a decrease in lbm% , rather than from an increase in bfm% in men . it has been reported that an increase in bfm% rather than a decrease in lbm% might play a key role in causing an increase body fat due to aging in women.50,51 regarding the distribution of body fat depending on body shape , it has been reported that abdominal obesity is a notable , independent risk factor for raising morbidity and mortality rates.911 therefore , the clinical significance of weight , bmi , and the bfm% would greatly vary depending on the types of abdominal obesity , ( ie , android or gynoid type ) . previous studies have shown that wc is the most reliable indicator for reflecting the amount of visceral fat in the assessment of abdominal obesity.10,1517 as shown in the current results , both men and women had a positive correlation between increased age and both wc and the whr ; however , hc had a negative correlation with age in men and a positive correlation with in women . according to previous studies , the whr was increased until the age of 5564 years , and then it decreased thereafter in men , though it continually increased with increased age in women.52,53 these authors noted that these findings occurred because of the redistribution of body fat due to changes in sex hormones in both men and women aged between 45 and 54 years.52,53 other studies have also shown that hormone therapy reduced the whr , thus providing more evidence for the redistribution of body fat because of hormonal changes.54,55 as shown in the current results , the whr was included in a model for estimating body shape biological age in both men and women . in addition , hc was included in this model for men , and both hc and wc were included for women . in particular , in estimating the body shape biological age , both men and women showed that the whr was the most powerful factor . from a perspective that considers the differences in body shape due to aging , however , the whr is a more powerful factor , and it better reflects differences in body shape . in cross - sectional studies , the prevalence of high body weight or obesity ( bmi > 30 kg / m ) increases with age up to about age 60 and then declines.56 although this does not indicate that the excessive accumulation of body fat rarely occurs in the elderly , this does suggest that visceral obesity increasingly occurs because of the redistribution of body fat due to increased age , and this leads to changes in body shape.2530,56 accordingly , considering variation in body shape due to aging , it would be more appropriate for clinical evaluation to be based on body shape due to the increased occurrence of visceral obesity , rather than the consistent application of clinical parameters for assessing obesity . we propose that a model for estimating body shape biological age be appropriate for assessing differences in body shape due to increased age with the application of clinical parameters associated with body composition . moreover , by presenting the clinical parameters for assessing obesity that are commonly used in a clinical setting and thereby comparing the results with those obtained from age - matched controls our estimation model would be used to perform an overall analysis of the differences in body shape due to aging . furthermore , by comparing one s current body shape biological age with their calendar age ( thereby expressing whether the body shape biological age is older or younger than one s calendar age ) , our estimation model would be easily accepted by people , and it would also be of help for facilitating communication between physicians and patients . in addition , it would also be of help for providing the motives to improve the status of obesity , and for setting personal goals . in a model for assessing body shape biological age , however , we did not include the factors that might affect the clinical parameters associated with obesity , including the amount of physical activity , eating habits such as nutritional intake , and socioeconomic status , and we excluded the data of some subjects who were suspected of having diseases or conditions based on certain criteria . our model for estimating body shape biological age might be a novel approach to variation in body shape that is due to aging despite several limitations . we assume that our estimation model would be used as an adjunctive measure in easily predicting differences in body shape with the use of clinical parameters that are commonly used to assess the status of obesity in a clinical setting . our results will provide baseline data for further studies to examine not only the effects of changes in body shape due to the aging process on the degree of aging in individuals , the rate of aging progression , and life expectancy , but they will also provide information surrounding correlations between body shape , disease , morbidity , and mortality .